InteractiveFly: GeneBrief

Fmr1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - Fmr1

Synonyms - dfxr

Cytological map position - 85F11--12

Function - RNA-binding protein

Keywords - translational repression, synaptic structure, a model for Fragile X syndrome,

Symbol - Fmr1

FlyBase ID: FBgn0028734

Genetic map position -

Classification - KH domain, S1 RNA binding domain

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Burguete, A. S., Almeida, S., Gao, F. B., Kalb, R., Akins, M. R. and Bonini, N. M. (2015). GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function. Elife 4. PubMed ID: 26650351
Microsatellite expansions are the leading cause of numerous neurodegenerative disorders. This study demonstrates that GGGGCC and CAG microsatellite repeat RNAs associated with C9orf72 in ALS/FTD and with polyglutamine diseases, respectively, localize to neuritic granules that undergo active transport into distal neuritic segments. In cultured mammalian spinal cord neurons, the presence of neuritic GGGGCC repeat RNA correlates with neuronal branching defects and the repeat RNA localizes to granules that label with FMRP, a transport granule component. Using a Drosophila GGGGCC expansion disease model, this study characterized dendritic branching defects that are modulated by FMRP and Orb2. The human orthologues of these modifiers are misregulated in induced pluripotent stem cell-differentiated neurons from GGGGCC expansion carriers. These data suggest that expanded repeat RNAs interact with the mRNA transport and translation machinery, causing transport granule dysfunction. This could be a novel mechanism contributing to the neuronal defects associated with C9orf72 and other microsatellite expansion diseases.

Jiang, F., Lu, F., Li, P., Liu, W., Zhao, L., Wang, Q., Cao, X., Zhang, L. and Zhang, Y. Q. (2016). Drosophila homolog of FMRP maintains genome integrity by interacting with Piwi. J Genet Genomics 43: 11-24. PubMed ID: 26842990
Fragile X syndrome (FraX), the most common form of inherited mental retardation, is caused by the absence of the evolutionally conserved fragile X mental retardation protein (FMRP). While neuronal functions of FMRP have been intensively studied for the last two decades, its role in non-neuronal cells remains poorly understood. Piwi, a key component of the Piwi-interacting RNA (piRNA) pathway, plays an essential role in germline development. This study report that similar to piwi, dfmr1, the Drosophila homolog of human FMR1, is required for transposon suppression in the germlines. Genetic analyses showed that dfmr1 and piwi act synergistically in heterochromatic silencing, and in inhibiting the differentiation of primordial germline cells and transposon expression. Northern analyses showed that roo piRNA expression levels are reduced in dfmr1 mutant ovaries, suggesting a role of dfmr1 in piRNA biogenesis. Biochemical analysis demonstrated a physical interaction between dFMRP and Piwi via their N-termini. Taken together, it is proposed that dFMRP cooperates with Piwi in maintaining genome integrity by regulating heterochromatic silencing in somatic cells and suppressing transposon activity via the piRNA pathway in germlines.

McMahon, A. C., Rahman, R., Jin, H., Shen, J. L., Fieldsend, A., Luo, W. and Rosbash, M. (2016). TRIBE: Hijacking an RNA-editing enzyme to identify cell-specific targets of RNA-binding proteins. Cell 165: 742-753. PubMed ID: 27040499
RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of an RBP are imperfect and not amenable to examining small numbers of cells. TRIBE (targets of RNA-binding proteins identified by editing) technique that couples an RBP to the catalytic domain of the Drosophila RNA-editing enzyme ADAR and expresses the fusion protein in vivo, was developed to address these issues. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. TRIBE was used to identify the targets of three RBPs (Hrp48, dFMR1, and NonA). TRIBE compares favorably to other methods, including CLIP, and this study has identified RBP targets from as little as 150 specific fly neurons. TRIBE can be performed without an antibody and in small numbers of specific cells.
O'Connor, et al. (2017). A Drosophila model of Fragile X syndrome exhibits defects in phagocytosis by innate immune cells. J Cell Biol 216(3): 595-605. PubMed ID: 28223318
Fragile X syndrome, the most common known monogenic cause of autism, results from the loss of FMR1, a conserved, ubiquitously expressed RNA-binding protein. Recent evidence suggests that Fragile X syndrome and other types of autism are associated with immune system defects. This study found that Drosophila melanogaster Fmr1 mutants exhibit increased sensitivity to bacterial infection and decreased phagocytosis of bacteria by systemic immune cells. Using tissue-specific RNAi-mediated knockdown, Fmr1 was shown to play a cell-autonomous role in the phagocytosis of bacteria. Fmr1 mutants also exhibit delays in two processes that require phagocytosis by glial cells, the immune cells in the brain: neuronal clearance after injury in adults and the development of the mushroom body, a brain structure required for learning and memory. Delayed neuronal clearance is associated with reduced recruitment of activated glia to the site of injury. These results suggest a previously unrecognized role for Fmr1 in regulating the activation of phagocytic immune cells both in the body and the brain.
Bienkowski, R. S., Banerjee, A., Rounds, J. C., Rha, J., Omotade, O. F., Gross, C., Morris, K. J., Leung, S. W., Pak, C., Jones, S. K., Santoro, M. R., Warren, S. T., Zheng, J. Q., Bassell, G. J., Corbett, A. H. and Moberg, K. H. (2017). The conserved, disease-associated RNA binding protein dNab2 interacts with the Fragile X Protein ortholog in Drosophila neurons. Cell Rep 20(6): 1372-1384. PubMed ID: 28793261
The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. This study presents a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.
Doll, C. A., Vita, D. J. and Broadie, K. (2017). Fragile X mental retardation protein requirements in activity-dependent critical period neural circuit refinement. Curr Biol 27(15): 2318-2330.e2313. PubMed ID: 28756946
Activity-dependent synaptic remodeling occurs during early-use critical periods, when naive juveniles experience sensory input. Fragile X mental retardation protein (FMRP) sculpts synaptic refinement in an activity sensor mechanism based on sensory cues, with FMRP loss causing the most common heritable autism spectrum disorder (ASD), fragile X syndrome (FXS). In the well-mapped Drosophila olfactory circuitry, projection neurons (PNs) relay peripheral sensory information to the central brain mushroom body (MB) learning/memory center. FMRP-null PNs reduce synaptic branching and enlarge boutons, with ultrastructural and synaptic reconstitution MB connectivity defects. Critical period activity modulation via odorant stimuli, optogenetics, and transgenic tetanus toxin neurotransmission block show that elevated PN activity phenocopies FMRP-null defects, whereas PN silencing causes opposing changes. FMRP-null PNs lose activity-dependent synaptic modulation, with impairments restricted to the critical period. It is concluded that FMRP is absolutely required for experience-dependent changes in synaptic connectivity during the developmental critical period of neural circuit optimization for sensory input.
Kennedy, T. and Broadie, K. (2017). Fragile X Mental Retardation Protein restricts small dye iontophoresis entry into central neurons. J Neurosci. 37(41): 9844-9858. PubMed ID: 28887386
Fragile X Mental Retardation Protein (FMRP) loss causes Fragile X syndrome (FXS), a major disorder characterized by autism, intellectual disability, hyperactivity and seizures. FMRP is both an RNA- and channel-binding regulator, with critical roles in neural circuit formation and function. However, it remains unclear how these FMRP activities relate to each other and how dysfunction in their absence underlies FXS neurological symptoms. In testing circuit level defects in the Drosophila FXS model, a completely unexpected and highly robust neuronal dye iontophoresis phenotype was discovered in the well-mapped Giant Fiber (GF) circuit. Controlled dye injection into the GF Interneuron (GFI) results in a dramatic increase in dye uptake in neurons lacking FMRP. Transgenic wildtype FMRP reintroduction rescues the mutant defect, demonstrating a specific FMRP requirement. This phenotype affects only small dyes, but is independent of dye charge polarity. Surprisingly, the elevated dye iontophoresis persists in shaking B mutants that eliminate gap junctions and dye coupling among GF circuit neurons. Therefore a wide range of manipulations was used to investigate the dye uptake defect, including timed injection series, pharmacology and ion replacement, and optogenetic activity studies. The results show FMRP strongly limits the rate of dye entry via a cytosolic mechanism. This study reveals an unexpected new phenotype in a physical property of central neurons lacking FMRP that could underlie aspects of FXS disruption of neural function.
Bienkowski, R. S., Banerjee, A., Rounds, J. C., Rha, J., Omotade, O. F., Gross, C., Morris, K. J., Leung, S. W., Pak, C., Jones, S. K., Santoro, M. R., Warren, S. T., Zheng, J. Q., Bassell, G. J., Corbett, A. H. and Moberg, K. H. (2017). The conserved, disease-associated RNA binding protein dNab2 interacts with the Fragile X Protein ortholog in Drosophila neurons. Cell Rep 20(6): 1372-1384. PubMed ID: 28793261
The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. This study presents a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.
Luhur, A., Buddika, K., Ariyapala, I. S., Chen, S. and Sokol, N. S. (2017). Opposing post-transcriptional control of InR by FMRP and LIN-28 adjusts stem cell-based tissue growth. Cell Rep 21(10): 2671-2677. PubMed ID: 29212015
Although the intrinsic mechanisms that control whether stem cells divide symmetrically or asymmetrically underlie tissue growth and homeostasis, they remain poorly defined. This study reports that the RNA-binding protein fragile X mental retardation protein (FMRP) limits the symmetric division, and resulting expansion, of the stem cell population during adaptive intestinal growth in Drosophila. The elevated insulin sensitivity that FMRP-deficient progenitor cells display contributes to their accelerated expansion, which is suppressed by the depletion of insulin-signaling components. This FMRP activity is mediated solely via a second conserved RNA-binding protein, LIN-28, known to boost insulin signaling in stem cells. Via LIN-28, FMRP controls progenitor cell behavior by post-transcriptionally repressing the level of Insulin receptor (InR). This study identifies the stem cell-based mechanism by which FMRP controls tissue adaptation, and it raises the possibility that defective adaptive growth underlies the accelerated growth, gastrointestinal, and other symptoms that affect fragile X syndrome patients.
Weisz, E. D., Towheed, A., Monyak, R. E., Toth, M. S., Wallace, D. C. and Jongens, T. A. (2017). Loss of Drosophila FMRP leads to alterations in energy metabolism and mitochondrial function. Hum Mol Genet 27(1): 95-106. PubMed ID: 29106525
Fragile X Syndrome (FXS), the most prevalent form of inherited intellectual disability and the foremost monogenetic cause of autism, is caused by loss of expression of the FMR1 gene. This study shows that dfmr1 modulates the global metabolome in Drosophila. Despite a previous discovery of increased brain insulin signaling, the results indicate that dfmr1 mutants have reduced carbohydrate and lipid stores and are hypersensitive to starvation stress. The observed metabolic deficits cannot be explained by feeding behavior, as it is reported that dfmr1 mutants are hyperphagic. Rather, the data identify dfmr1 as a regulator of mitochondrial function. Under supersaturating conditions, dfmr1 mutant mitochondria have significantly increased maximum electron transport system (ETS) capacity. Moreover, electron micrographs of indirect flight muscle reveal striking morphological changes in the dfmr1 mutant mitochondria. Taken together, these results illustrate the importance of dfmr1 for proper maintenance of nutrient homeostasis and mitochondrial function.

Mammalian Fragile X mental retardation gene (FMR1) encodes an RNA binding protein that acts as a negative translational regulator. A Drosophila fragile X syndrome model has been developed using loss-of-function mutants and overexpression of the FMR1 homolog (Fmr1). Drosophila Fmr1 nulls display enlarged synaptic terminals, whereas neuronal overexpression results in fewer and larger synaptic boutons. Synaptic structural defects are accompanied by altered neurotransmission, with synapse type-specific regulation in central and peripheral synapses. These phenotypes mimic those observed in mutants of microtubule-associated Futsch. Immunoprecipitation of Drosophila Fmr1 shows association with Futsch mRNA, and Western analyses demonstrate that Fmr1 inversely regulates Futsch expression. Fmr1;futsch double mutants restore normal synaptic structure and function. It is proposed that Fmr1 acts as a translational repressor of Futsch to regulate microtubule-dependent synaptic growth and function (Zhang, 2001).

Fragile X syndrome (FraX) is the most common inherited disease causing mental retardation. The defect is a trinucleotide CGG expansion in the regulatory region of fragile X mental retardation 1 (FMR1), causing transcriptional silencing and loss of the gene product, FMRP (Verkerk, 1991; Verheij, 1993). FMRP is widely expressed in fetal and adult tissues, with pronounced expression in brain and testis where major symptoms are manifested (Devys, 1993). FMRP is predominantly in the cytoplasm with occasional nuclear staining (Devys, 1993; Verheij, 1993). FMRP contains nuclear localization (NLS) and export (NES) signals (Eberhart, 1996), suggesting that it functions as a nucleo-cytoplasmic shuttle protein. FMRP contains three RNA binding domains: two K homology (KH) domains and one RGG box (Ashley, 1993a; Siomi, 1993). FMRP binds ~4% of human fetal brain mRNA in vitro, but the targets are largely unknown, except its own mRNA and myelin basic protein mRNA (Ashley, 1993a; Brown, 1998). FMRP associates with polyribosomes (Khandjian, 1996; Tamanini, 1996; Feng, 1997a) and functions as a negative translational regulator (Laggerbauer, 2001; Li, 2001; Schaeffer, 2001) (Zhang, 2001).

FraX neurological pathogenesis has attracted intensive analysis. Cerebral cortical autopsies from FraX patients show abnormal neuronal dendritic spine morphology, postulated to be associated with synaptic immaturity (Hinton, 1991, Irwin, 2001). In FMR1 knockout mice, longer and denser dendritic spines are observed, consistent with the human phenotype (Comery, 1997; Nimchinsky, 2001). FMRP is observed at synapses in the developing rat brain (Weiler, 1997) and is present in mouse brain synaptosomes (Feng, 1997b; Tamanini, 1997). Furthermore, FMRP mRNA associates with translational complexes in synaptic subcellular fractions, and the expression of FMRP is increased within minutes of glutamate receptor stimulation, suggesting that FMRP acts as a synaptic activity-dependent translational regulator (Weiler, 1997; Jin, 2000). These different lines of evidence suggest that the underlying mechanism of mental retardation in FraX patients is the result of defective synapse development or function (Zhang, 2001).

A Drosophila FraX model has been generated to specifically address the hypothesis that FMRP regulates synaptic development and function. Wan (2000) identified the Drosophila homolog of FMR1. Drosophila Fmr1 (Drosophila fragile X related) has been mutated and its roles in synaptic development and function has been assayed in two model systems in Drosophila: the eye and the neuromuscular junction (NMJ). The level of Fmr1 protein has been shown to regulate both synaptic structure and function. The Fmr1 synaptic phenotypes mimic defects observed in mutants with altered levels of Futsch, a microtubule-associated protein with homology to mammalian MAP1B. Fmr1 associates with Futsch mRNA and negatively regulates Futsch expression. Most importantly, a Fmr1;futsch double mutant restores the Fmr1 synaptic structural and functional defects in the eye and NMJ. These results suggest that Fmr1 is acting as a translational repressor of Futsch to regulate the synaptic microtubule cytoskeleton and that Futsch misregulation is sufficient to explain both synaptic structure and function defects characterizing the Drosophila FraX model (Zhang, 2001).

It is now clear that Drosophila contains a single, functionally conserved member of the FMR1 family, Fmr1 (Wan, 2000 and Zhang, 2001), compared to the three related genes present in mammals. The molecular characteristics, cellular and subcellular expression pattern, and functions of Drosophila Fmr1 and mammalian FMRP show extensive parallels. Most importantly, Fmr1 mutant phenotypes are consistent with the synaptic defects associated with human FraX patients and FMR1 knockout mice. These observations suggest Drosophila is an attractive genetic system to model FraX (Zhang, 2001).

At a gross level, lack of Drosophila Fmr1 and mammalian FMRP have similar consequences. In both cases the gene is not essential; null mutants are adult viable with a normal developmental time course. Behaviorally, both Drosophila and mammalian mutants show locomotory deficits. Although a direct comparison cannot be drawn between flight defects in the Fmr1 mutants and movement abnormalities in FraX patients, it is interesting to note that both display impaired motor control. FraX patients have visuospatial defects and Drosophila Fmr1 mutants show decreased photoreceptor function in the retina. All of these common defects can be readily explained by impaired synaptic development or function (Zhang, 2001).

Recent studies indicate that mammalian FMRP is present at synapses and regulates synaptic structure (Comery, 1997; Feng, 1997b; Tamanini, 1997; Weiler, 1997; Nimchinsky, 2001). Similarly, Drosophila Fmr1 is highly expressed in both pre- and post-synaptic neurons, as well as in postsynaptic muscles, and regulates synaptic structure. Overgrowth of dendritic spines, sites of synaptic input, is a diagnostic characteristic in FraX patients (Hinton, 1991; Irwin, 2001) and also is the primary phenotype of FMR1 knockout mice (Comery 1997; Nimchinsky, 2001), suggesting a common synaptic basis of the disease. Similarly in the Fmr1 null, NMJ synaptic terminals are overgrown, containing more arboreal branches and more synaptic boutons. It is not presently known whether human patients and FMR1 knockout mice show similar NMJ defects. In addition, it has been found that Fmr1 overexpression has the opposite and complementary consequence of inhibiting synaptic growth and arborization. Thus, synaptic growth, branching, and bouton differentiation are negatively regulated proportional to Fmr1 levels (Zhang, 2001).

Fmr1 is also a key regulator of synaptic function. Different functional/chemical classes of synapses respond differently to Fmr1 misregulation. In the eye, histaminergic photoreceptor neurotransmission is equally impaired by either loss or overexpression of Fmr1, demonstrating that a precise level of the protein is required to maintain synaptic function. At the peripheral glutamateric NMJ, in contrast, neurotransmission is strikingly enhanced by either loss or overexpression of Fmr1. The role of Fmr1 is primarily presynaptic, mediating synaptic vesicle fusion probability. It is not currently known why the polarity of Fmr1 regulation differs between these central and peripheral synapses (Zhang, 2001).

Taken together, these results strongly support a Fmr1/FMRP synaptic function: Fmr1 and FMRP are similarly expressed in pre/postsynaptic cells, play a conserved role in dendritic spine/synapse structural regulation, and Fmr1, at least, is required for differential regulation of synaptic neurotransmission. It is suggested that the FMRP family plays a conserved role in synaptic development and function, which likely underlies the behavioral and developmental symptoms of FraX patients (Zhang, 2001).

The expression of FMRP is increased locally following glutamate receptor stimulation, suggesting that FMRP acts as a synaptic activity-dependent translational regulator (Weiler, 1997; Jin, 2000). Recent evidence has shown that FMRP is a negative translational regulator (Laggerbauer, 2001; Li, 2001; Schaeffer, 2001). Given these studies, it is hypothesized that Fmr1 may act as a translational repressor mediating the coupled regulation of synaptic structure and function. Several lines of evidence suggested that Futsch, a microtubule-associated MAP1B homolog, may be a target for Fmr1 translational regulation in the Drosophila nervous system. Futsch is required for dendritic and axonal development, as well as for synaptic growth. Moreover, futsch mutants alter Drosophila NMJ architecture in a fashion similar to Fmr1 NOE (neuronal overexpression of Fmr1) animals. Misregulation of the microtubule-based synaptic cytoskeleton appears to be a likely candidate for the coupled structural and functional defects observed in Fmr1 mutants (Zhang, 2001).

Evidence is presented that Fmr1 negatively regulates Futsch expression. (1) Fmr1 associates with Futsch mRNA. This interaction is specific, since Fmr1 fails to bind other targets such as alpha-tubulin mRNAs and the interaction is missing in Fmr1 null mutants. (2) In Fmr1 null mutants, Futsch protein level in the nervous system is increased and Fmr1 neuronal overexpression causes Futsch expression to be reduced. These results show that the level of Futsch in the nervous system is inversely regulated by the level of Fmr1. Taken together, the biochemical association between Fmr1 protein and Futsch mRNA and the inverse regulation of Futsch expression by Fmr1 strongly support a hypothesis that Fmr1 acts as a negative regulator of Futsch translation (Zhang, 2001).

Futsch appears to be the major target for Fmr1 in the regulation of synaptic structure and function. Structurally, futsch hypomorphs display fewer and enlarged NMJ synaptic boutons with dispersed, punctate anti-Futsch immunoreactivity, a phenotype indistinguishable from that caused by overexpression of Fmr in dfxrNOE. However, futschNOE causes synaptic overgrowth, a phenotype similar to Fmr1 null mutants. Functionally, all four genotypes (loss and overexpression of either Fmr1 or Futsch) enhance neurotransmission at the larval NMJ, and all four genotypes impair neurotransmission in the adult eye. Thus, the expression alterations of Futsch are sufficient to explain the synaptic phenotypes of Fmr1 mutants (Zhang, 2001).

The most conclusive experimental result is the suppression of Fmr1 synaptic phenotypes by the Fmr1;futsch double mutants. The double mutant develops normal synaptic architecture, including the normal number of arboreal branches and synaptic boutons. Strikingly, the double mutant reduces NMJ transmission to suppress the peripheral synaptic phenotype, while at the same time it increases photoreceptor transmission to suppress the central synaptic phenotype. Based on these results, it is proposed that the major function of Fmr1 is the negative regulation of Futsch in the nervous system, which in turn regulates microtubule-dependent synaptic structure and function. Of course, it remains probable that Fmr1 is translationally regulating multiple proteins. However, the Futsch misregulation is sufficient to explain the synaptic phenotypes in Fmr1 mutants and, by extrapolation, possibly the mental retardation of FraX patients (Zhang, 2001).

Fragile X protein controls neural stem cell proliferation in the Drosophila brain

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function for Fragile X protein (FMRP), an RNA-binding protein thought to regulate synaptic plasticity by controlling the localization and translation of specific mRNAs. FMRP is required to control the proliferation of the germline in Drosophila. To determine whether FMRP is also required for proliferation during brain development, the distribution of cell cycle markers was examined in dFmr1 brains compared with wild-type throughout larval development. The results indicate that the loss of dFmr1 leads to a significant increase in the number of mitotic neuroblasts (NB) and BrdU incorporation in the brain, consistent with the notion that FMRP controls proliferation during neurogenesis. Developmental studies suggest that FMRP also inhibits neuroblast exit from quiescence in early larval brains, as indicated by misexpression of Cyclin E. Live imaging experiments indicate that by the third instar larval stage, the length of the cell cycle is unaffected, although more cells are found in S and G2/M in dFmr1 brains compared with wild-type. To determine the role of FMRP in neuroblast division and differentiation, MARCM approaches were used in the developing larval brain; single dFmr1 NBs generate significantly more neurons than controls. The results demonstrate that FMRP is required during brain development to control the exit from quiescence and proliferative capacity of NB as well as neuron production, which may provide insights into the autistic component of FXS (Callan, 2010).

To determine the role of FMRP during early neurogenesis in vivo, loss of function and clonal approaches were used in the Drosophila larval brain. This study shows that whole dFmr1 mutant brains from late third instar larvae exhibit altered cell cycle profiles, with more cells found in S and G2/M at the expense of G1. These cell cycle defects indicate that FMRP is necessary for correct cell cycle progression in neural stem cells. Developmental studies coupled with live imaging experiments indicate that FMRP controls the exit from quiescence and proliferative capacity of larval brain NB. This is the first evidence that FMRP controls the exit from quiescence of neural progenitors in the developing brain. Such developmental defects could lead to significant problems in neural connections that are dependent on precise timing for proper function (Callan, 2010).

Clonal analyses provide further support to these findings, by showing that dFmr1 mutant NBs produce an increased number of neurons, which persist in the adult brain. In the future, it will be interesting to determine whether some neuronal populations are more sensitive to loss of FMRP than others and what are the functional consequences of having supranumerary neurons. Taken together, the results show that FMRP is required cell-autonomously for neurogenesis in vivo and are consistent with recent reports indicating that neural stem cell proliferation is increased and that the density of intermediate progenitors and pyramidal cells is increased in the early postnatal cortex of FMR1 KO mice. In addition, the work suggest a mechanism involving the control of the G1/S transition point possibly through the regulation of CycE expression in NB during early larval brain development (Callan, 2010).

The data reveal a novel and surprising role for FMRP during brain development. In the young first instars, there are comparable numbers of Miranda/CycE expressing NB in the mutant compared with wild-type but only 6 h later (6-12 h ALH), there is a shift, with more dFmr1 mutant cells expressing Miranda/CycE compared with wild-type. The coexpression of the neuroblast marker Miranda and the G1/S transition marker CycE indicates that these additional cells correspond to NB exiting the G0 quiescence phase and entering the cell cycle. Based on these results, it is proposed that FMRP controls the timing of neuroblast exit from quiescence. It remains to be seen whether CycE is a direct target or whether FMRP acts through other factors such as E2F, Rb or the SCF/cullin complex. Interestingly, recent work in mice has shown that FMRP plays a role in adult neurogenesis by regulating the expression of cell cycle regulators such as Cyclin D and CDK4 (Callan, 2010).

While no significant change was found in the number of mitotic cells in early larval stages, at the onset of the third instar larval stage, a slight increase was observed in PH3 positive cells in dFmr1 mutant brains compared with wild-type. In late third instar larval brains, however, the difference in PH3 positive NB became statistically significant, which could be accounted for by seemingly opposite scenarios whereby the mutant NB either divide faster, or are progressing through mitosis at a slower pace. It remains to be determined what mRNA targets mediate FMRP's role in the timing of mitotic events. Given that dFmr1 mutant NB generate significantly larger lineages containing more neurons and that the live imaging experiments showed no change in the total length of the cell cycle of third instars, a model is proposed whereby FMRP controls neuroblast proliferation during early brain development by regulating the timing of reentry into the cell cycle. This is supported by developmental studies showing misexpression of CycE, which suggests a premature exit from quiescence due to the loss of dFmr1. Clonal analyses indicate that dFmr1 mutant NB produce on average 15-16 more neurons than wild-type by the end of third instar. This correlates with the mutant neuroblast completing approximately eight more divisions than its wild-type counterparts during larval development (every two neurons are the result of a single neuroblast division). By the end of the larval stage, however, the proliferative activity of dFmr1 NB is comparable to that of their wild-type counterparts, which supports the notion that these additional cell cycles are restricted to the early stages of brain development (Callan, 2010).

Neural stem cell proliferation and differentiation are the basis for generating the correct number of neurons and support cells in the developing nervous system. The Drosophila larval brain NB has emerged as a premiere model for neural stem cells that have been successfully used to elucidate the molecular mechanisms underlying stem cell renewal and differentiation in the brain. Larval type I NBs give rise to only neurons as opposed to both neurons and glia, while type II NB give rise to both, as is the case with mammalian neural stem cells. Despite some differences, it is clear that Drosophila NBs follow general stem cell principles and utilize conserved pathways. Given the powerful genetic tools available in the fly, including the ability to study individual neuroblast lineages, the clues obtained from the fly are likely to provide useful insights into mammalian brain development and the mechanisms for RNA regulation in neural stem cells (Callan, 2010).

Clonal analyses demonstrate that dFmr1 mutant NB generate more neurons than their wild-type counterparts. These findings are consistent with previous in vitro studies using neurospheres as well as a recent study using the FMR1 KO mouse, both of which reported the presence of supranumerary neurons in the absence of FMRP. Recently, the loss of FMRP from adult neural progenitors was shown to generate more glial cells at the expense of neurons. The differences in neuronal numbers between this study and that with the mouse may reflect inherent differences between the fly and mouse models. Alternatively, FMRP has a developmental component and exerts distinct modes of regulation on neural stem cell proliferation and differentiation during the various stages of brain development. Supporting this notion is a study that found more neurons at the expense of glia in embryonically derived neurospheres lacking FMRP (Callan, 2010).

Quantification of the Elav positive cells within clones suggests that dFmr1 NB generate an estimated 16% more neurons. With approximately 15,000 neurons present in the larval brain and considering that each brain lobe is shaped as a sphere, a 16% increase in neurons is predicted to amount to a 1.13 change in the area occupied by the brain when mounted on a microscope slide (1.13 = ratio between mutant and wild-type surface area of two spheres that differ by 16% in volume). While such a small increase in brain size may go unnoticed by the experimentalist, inter-neuronal connections and circuit formation are likely to be more sensitive. The presence of wiring defects would suggest that the loss of FMRP affects brain development earlier than previously thought and may account for the autistic component of FXS (Callan, 2010).

Finally, the finding that FMRP regulates the timing of neuroblast reentry into the cell cycle is particularly interesting, as this critical aspect of brain development remains poorly understood. Whole brain analyses suggest that FMRP may control the exit from quiescence by regulating the expression of CycE. These results are consistent with previous findings that the loss of dFmr1 leads to CycE misexpression in the fly ovary. Another interesting parallel to the oogenesis study is that loss of FMRP leads to both an increase and a delay in proliferation. In the larval brain, an increase in proliferation was detected during early larval stages, followed by a slowdown towards the end of the larval life. This suggests the presence of compensatory mechanisms that alleviate the consequences due to loss of FMRP in the brain and provide an explanation for the lack of an obvious effect on brain size in dFmr1 mutants (Callan, 2010).

Previous studies have shown that neuroblast exit from quiescence is controlled in part by the glycoprotein Anachronism (Ana), which is secreted by the surrounding glial cells. Ana's role in neuroblast proliferation suggests that stem cell proliferation in the brain is controlled by its microenvironment. Whether FMRP is required in glial cells remains to be elucidated. In further support of this notion, a recent study has implicated both Branchless and Hedgehog signaling pathways in NB' exit from quiescence. It will be interesting to see whether FMRP cooperates with these genes or utilizes distinct mechanisms to regulate neural stem cell quiescence during brain development (Callan, 2010).

The Drosophila fragile X mental retardation protein participates in the piRNA pathway

RNA metabolism controls multiple biological processes, and a specific class of small RNAs, called piRNAs, act as genome guardians by silencing the expression of transposons and repetitive sequences in the gonads. Defects in the piRNA pathway affect genome integrity and fertility. The possible implications in physiopathological mechanisms of human diseases have made the piRNA pathway the object of intense investigation, and recent work suggests that there is a role for this pathway in somatic processes including synaptic plasticity. The RNA-binding fragile X mental retardation protein (FMRP, also known as FMR1) controls translation and its loss triggers the most frequent syndromic form of mental retardation as well as gonadal defects in humans. This study demonstrates for the first time that germline, as well as somatic expression, of Drosophila Fmr1 (denoted dFmr1), the Drosophila ortholog of FMRP, are necessary in a pathway mediated by piRNAs. Moreover, dFmr1 interacts genetically and biochemically with Aubergine, an Argonaute protein and a key player in this pathway. These data provide novel perspectives for understanding the phenotypes observed in Fragile X patients and support the view that piRNAs might be at work in the nervous system (Bozzetti, 2015).

dFmr1 is a translational regulator and its role in the miRNA pathway is widely accepted. This study provides several lines of evidence that dFmr1 can be considered as a ‘bona fide’ member of the piRNA pathway that keeps repetitive sequences and transposons silenced. First, dFmr1 mutant testes display crystalline aggregates, as do other mutants of the piRNA pathway. Second, the levels of cry (Suppressor of Stellate)-specific and transposon-specific piRNAs dramatically decrease in dFmr1 mutant testes. Third, as a consequence of this decrease, the Ste RNA is produced and, in addition, transposons are expressed at higher levels than in wt animals. Fourth, dFmr1 mutant animals display fertility defects, a phenotype shown by several mutations affecting the piRNA pathway. The fact that earlier screens did not identify dFmr1 as a member of the somatic piRNA pathway could be due to the heterogeneous phenotypes observed with the somatic transposons (this study) and/or to the material used for those assays. The crySte system thus proves very efficient for identifying new members of this important pathway (Bozzetti, 2015).

The movement of transposable elements is one of the molecular causes of DNA instability and sterility. Considering that human patients mutant for FMRP also display defects in male and female gonads, it will be interesting to characterize the activity of transposons and repetitive sequences in the gonads of mice or humans that are mutant for the FMRP pathway, although there might be no observable defects in mammals because they express three members of the FMRP family versus the single ortholog in fly. Finally, mutations affecting the piRNA pathway might also induce gonadal defects in humans (Bozzetti, 2015).

Until now, the members of the piRNA pathway controlling the crySte interaction, including Aub, have been described as being required in the male germline. Surprisingly, the conditional dFmr1 rescue and KD experiments demonstrate that dFmr1 controls the piRNA pathway both in the germline and in the somatic cells of the gonad, which raises questions as to the somatic contribution of other members of the piRNA pathway in the male gonad. The phenotypes induced by somatic Aub expression also suggest that the hub expresses one or more AGO proteins that are involved in the somatic piRNA-mediated Ste silencing and that interact with dFmr1; however, the only other protein of the Piwi clade present in the somatic tissue, Piwi, does not participate in Ste silencing. Based on preliminary data, this study proposes that AGO1 might be one such protein. First, AGO1/+ testes display Ste-made crystals, as do testes expressing UAS-AGO1 RNAi driven by the upd-Gal4 driver. Second, aubsting rescues the AGO1-mediated crystal phenotype. Third, AGO1 and dFmr1 interact biochemically and are known to interact genetically in the ovaries to control germline stem cell maintenance, as well as in the nervous system, where they modulate synaptic plasticity. Taken together, these data suggest that AGO1 contributes to the piRNA pathway that controls the cry–Ste system in the somatic part of the gonad (Bozzetti, 2015).

The finding that Aub somatic expression affects the NMJ and counteracts the AGO1 loss of function phenotype is also unexpected. Recent work has documented the activation of piRNA pathway in the nervous system in flies, mice, humans and molluscs and it has been proposed that synaptic plasticity, cognitive functions and neurodegeneration might involve the control of genome stability, even though the precise mode of action and impact of this pathway are not completely understood. Because Aub is not required in the larval somatic tissues, its ectopic expression could affect the NMJ by replacing AGO1 in its known role on the miRNA pathway. However, AGO1 might also affect the NMJ through the piRNA pathway, much in the same way as AGO1 loss of function affects a piRNA pathway in the gonad. Even though AGO1 has been previously described as being exclusively involved in the miRNA pathway, some degree of overlapping between different RNAi pathways has been recently described: (1) the double-stranded-RNA-binding protein Loquacious (Loqs) is involved in the miRNA pathway and in the endogenous siRNA pathway, (2) AGO1 and AGO2 can compete for binding with miRNAs, and (3) ectopic expression of Aub in the soma competes for the siRNAs pathway mediated by AGO2. In addition, miRNAs have been demonstrated to have a role on easi-RNA biogenesis in plants. In a similar manner, AGO1 could act on piRNAs through its activity on the miRNA pathway. Although future studies will clarify the connection between AGO1 and the piRNA pathway, the present data provide novel perspectives in the field and could have a broad relevance to diseases affecting cognitive functions (Bozzetti, 2015).

Expression, genetic and biochemical data indicate that Aub and dFmr1 interact directly. dFmr1 has been proposed to bind specific cargo RNAs and the human FMRP binds small RNA, in addition to mRNAs. Similarly, the Aub–dFmr1 interaction might allow the targeting of piRNAs to the transcripts of repetitive sequences and transposable elements, dFmr1 providing the molecular link between small RNAs and AGO proteins of the RISC (Bozzetti, 2015).

The Aub and dFmr1 proteins colocalize and likely interact in the piRNA pathway in a specific stage of testis development and also have additional functions that are independent from each other. Typically, dFmr1 accumulates at high levels in more differentiated cells of the testis, where Aub is not detectable, likely accounting for the axoneme phenotype described in dFmr1 testes. In the future, it will be interesting to analyze whether the other genes involved in the piRNA pathway in testis are also required at specific stages, as also recently found in the ovary (Bozzetti, 2015).

Finally, FMRP proteins work in numerous molecular networks, show complex structural features (TUDOR, KH, NLS, NES RGG domains) and are characterized by widespread expression and subcellular localization (cytoplasm, nucleus, axons, dendrites, P bodies), providing versatile platforms that control mRNA and small RNA metabolism (e.g. translation, degradation and transport). Understanding whether FMRP proteins interact with other members of the piRNA pathway, whether this interaction is modulated physiologically and how does the interaction with this pathway compare with that observed with other AGO proteins will clarify the role and mode of action this family of proteins in small RNA biogenesis and metabolism (Bozzetti, 2015).

The biogenesis of the piRNAs requires two pathways. The primary pathway involves Piwi and predominantly occurs in the somatic tissues. The ping-pong pathway involves Aub, as well as AGO3, and predominantly occurs in the germline, where Aub is thought to bind an antisense piRNA, to cleave the sense transcript from an active transposon and to produce a sense piRNA that is loaded onto AGO3. The AGO3–piRNA complex binds complementary transcripts from the piRNA cluster, producing the so-called secondary piRNAs by an amplification loop. Although the piRNA pathways have emerged as a very important tool to understand the role of RNA metabolism in physiological and pathological conditions, the relationship and interactions among the involved proteins are not simple to interpret, mostly because not all the players have been characterized. Moreover, recent data support the hypothesis that the somatic and the germline piRNA pathways share components: for example, shutdown (shu), vreteno (vret) and armitage (arm) affect primary as well as ping-pong pathways in ovaries. Results from this study call for a role of dFmr1 in both piRNA pathways at least in testes. Based on the alignment of the human, mouse and fly FMRP family members, dFmr1 might participate in piRNA biogenesis as a Tudor domain (TDRD) containing protein (Bozzetti, 2015).

TDRDs are regions of about 60 amino acids that were first identified in a Drosophila protein called Tudor. In the recent years, the requirement of TDRD proteins in piRNA biogenesis and metabolism has become evident. Typically, the founding member of the family, Tudor, binds AGO proteins and helps them interact with specific piRNAs. Among the different TDRD proteins, fs(1)Yb works in the primary pathway; Krimper, Tejas, Qin/Kumo, and PAPI work in the ping-pong pathway; and Vret works in both systems. PAPI, the only TDRD protein that has a modular structure closely related to dFmr1 (two KH domains and one TDRD), interacts with the di-methylated arginine residues of AGO3 and controls the ping-pong cycle in the nuage. At least during the early stages of testis development, dFmr1 might interact with Aub in a similar way. Given that TDRDs are involved in the interactions between proteins and in the formation of ribonucleoprotein complexes, future studies will assess whether RNAs mediate the Aub–dFmr1 interaction (Bozzetti, 2015).

In conclusion, the discovery of dFmr1 as a player in the piRNA pathway highlights the importance of the fly model. Data from this study also adds a new perspective to understanding the role and mode of action of this protein family and the physiopathological mechanisms underlying the Fragile X syndrome (Bozzetti, 2015).


Characterization of Fmr1 RNA-binding activity and ability to dimerize

Previous studies have demonstrated that mammalian FMR1 has RNA-binding activity that is conferred by the two KH domains and the RGG box. In an RNA homopolymer binding assay, in vitro-translated, 35S-labeled hFMR1 protein shows strong binding to poly(G), weaker but significant binding to poly(U), and no detectable binding to poly(C) and poly(A). Under the same binding conditions, the same RNA homopolymer binding profile was observed for Fmr1. The conservation of the RNA-binding activity between Fmr1 and hFMR1 lends further support to the conclusion that Fmr1 is functionally related to the vertebrate FMR1/FXR proteins. To assess whether the KH domains of Fmr1 confer its RNA-binding capability, point mutations predicted to inactivate the function of either KH domain were engineered into the Drosophila Fmr1 cDNA by site-directed mutagenesis. A codon for a highly conserved isoleucine residue within each of the KH domains was mutated to a codon for asparagine (I244N or I307N). These KH domain mutations are analogous to a mutation identified in the FMR1 protein of a fragile X patient, and such analogous mutations in human FMR1 have been shown to impair RNA binding in vitro. Furthermore, solution structure data of these KH domains predict that these substitutions will disrupt an alpha-helix structure within the KH domain. RNA homopolymer binding assays with these mutant forms of Fmr1 show that either mutation impairs the ability of Fmr1 to bind poly(U) in vitro as is observed for the analogous hFMR1 mutants (Wan, 2000).

Another biochemical feature of hFMR1 and hFXR proteins is their capacity to form heteromers with other FMR1/FXR proteins. To determine if Fmr1 has a similar capacity, in vitro binding experiments were performed to test the interaction of purified recombinant GST-Fmr1 with Fmr1 and with hFMR1 and hFXR produced by in vitro transcription and translation. GST-Fmr1 binds with the highest avidity to hFMR1, less well to hFXR2, and very weakly to hFXR1 and to itself. In reciprocal experiments in which hFMR1, hFXR1, and hFXR2 were immobilized as GST fusion proteins, the same profiles of relative binding avidity were observed. The order of preference of binding of Fmr1 suggests that each of the FMR1/FXR proteins has a characteristic selectivity of protein-protein (Wan, 2000).

Targets of Activity

FMRP is a negative translational regulator (Laggerbauer, 2001; Li, 2001; Schaeffer, 2001). What potential targets for Fmr1 could explain its regulation of synaptic structure and transmission in the eye and NMJ? Futsch, the Drosophila homolog of the mammalian microtubule-associated protein MAP1B, regulates the microtubule cytoskeleton to mediate dendritic, axonal, and synaptic growth. Hypomorphic futsch mutants display a distinctive NMJ morphology phenotype similar to dfxrNOE (neuronal overexpression of Fmr1), i.e., fewer and larger synaptic boutons. Moreover, microtubules play an essential role in the transport and subsequent regulation of synaptic vesicle dynamics underlying neurotransmission. Based on these lines of reasoning, immunoprecipitation and Western analyses were performed to determine whether Futsch expression is regulated by Fmr1 (Zhang, 2001).

First, tests were carried out for a physical interaction between Fmr1 protein and futsch mRNA. Immunoprecipitation analyses were performed using a monoclonal anti-Fmr1 to identify mRNAs that associate with the protein: (1) as a positive control, RT-PCR shows that the protein complex immunoprecipitated by anti-Fmr1 contains Fmr1 mRNA, in agreement with previous findings with mammalian FMRP (Ashley, 1993a; Brown, 1998); (2) futsch mRNA is also present in the immunoprecipitated protein complex. As a negative control, it was shown that anti-Fmr1 immunoprecipitation does not pull down alpha-tubulin mRNA, which is highly expressed in brain. Subsequent sequencing of the RT-PCR products confirms the association of Fmr1 and futsch mRNA. These results demonstrate that Fmr1 protein specifically binds futsch mRNA and may regulate Futsch expression at a translational level (Zhang, 2001).

Quantitative Western analyses have shown that Futsch expression in the nervous system is inversely correlated with Fmr1 expression. Initially, observed alterations were observed in the distribution and intensity of Futsch immunoreactivity in the nervous systems of Fmr1 mutants. To quantify these changes in Futsch expression, Western analyses was performed on adult head extracts from Fmr1 mutants and dfxrNOE lines. Futsch protein levels are significantly decreased to an average of 78.2% ± 2.5% of control levels in dfxrNOE. In Fmr1 null mutants, in contrast, Futsch is altered in the opposite direction, displaying an increase to 208% ± 32.4% of control levels. These results demonstrate that Fmr1 negatively regulates the expression of Futsch in the nervous system. The negative regulation of Futsch by Fmr1, together with the binding of Fmr1 with futsch mRNA as well as reports of a role of FMRP in translational regulation, suggests that Fmr1 acts as a negative translational regulator of Futsch (Zhang, 2001).

Futsch regulates microtubules at the Drosophila NMJ (Roos, 2000). Therefore, it was hypothesized that Fmr1-dependent Futsch regulation might mediate the control of synaptic structure and function, explaining the synaptic dysfunction observed in Fmr1 mutants. Since Fmr1 null mutants elevate Futsch expression, this model predicts that Futsch overexpression should mimic Fmr1 synaptic phenotypes. Similarly, Fmr1 overexpression decreases Futsch expression, so futsch hypomorph mutants should display dfxrNOE phenotypes. Finally, double mutants of Fmr1;futsch would be predicted to rescue Fmr1 synaptic structure and function phenotypes. These predictions were tested in futsch mutants, futsch transgenic NOE lines, and Fmr1;futsch double mutants by assaying synaptic structure and neurotransmission at both the larval NMJ and adult eye (Zhang, 2001).

Consistent with the hypothesis, futschNOE causes an NMJ overgrowth phenotype similar to Fmr1 null mutants, with increased synaptic area, branching, and bouton number. Also consistent with the hypothesis, hypomorphic futsch mutants display reduced NMJ growth and enlarged synaptic boutons, similar to dfxrNOE. Therefore, a Fmr1;futsch double mutant (Fmr1 null allele 50M; futsch hypomorph allele N94) and NMJ structure were assayed. The double mutant forms a structurally normal NMJ indistinguishable from wild-type with regard to synaptic branching and bouton number. These results demonstrate that upregulation of Futsch is sufficient to explain the synaptic structural defects caused by Fmr1 loss-of-function (Zhang, 2001).

Functional assays of neurotransmission in the eye and NMJ were performed with futsch hypomorphs and NOE lines to determine whether the regulation fits predictions from the model. Consistent with the hypothesis, both futsch hypomorphs and futschNOE elevate neurotransmission at the NMJ. Also consistent with the hypothesis, both futsch hypomorphs and futschNOE significantly reduce photoreceptor neurotransmission. It is particularly striking that either loss or overexpression of both Fmr1 and futsch have identical effects on synaptic transmission, i.e., elevated at the NMJ and suppressed in the eye (Zhang, 2001).

These observations suggest that precise regulation of Futsch levels by Fmr1 is required to properly maintain transmission in central and peripheral synapses. If the defects observed in Fmr1 mutants are due to the upregulation of Futsch levels, then one should be able to bring the levels of Futsch down to compensate by combining Fmr1 nulls with futsch hypomorphs in the same genome. Double mutants of Fmr1;futsch were generated and synaptic transmission at the NMJ and in the eye was assayed. A remarkable suppression of synaptic defects observed in Fmr1 mutants was seen. In the NMJ, the double mutant reduces transmission to levels indistinguishable from controls, whereas in the eye, the double mutant increases neurotransmission to wild-type levels. This genetic suppression provides convincing evidence that Fmr1 regulates synaptic mechanisms entirely through its regulation of Futsch. Taken together, this study strongly supports the hypothesis that Fmr1 acts as a translational repressor of Futsch to regulate microtubule dynamics and thereby control synaptic structure and function (Zhang, 2001).

Fmr1 associates with RISCs, RNAi effector complexes

RNA interference (RNAi) is a flexible gene silencing mechanism that responds to double-stranded RNA by suppressing homologous genes. This study reports the characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs (miRNAs). Two putative RNA-binding proteins, the Drosophila homolog of the mammalian fragile X mental retardation protein (FMRP), Fmr1, and VIG (Vasa intronic gene), are identified through their association with RISC. FMRP, the product of the human fragile X locus, regulates the expression of numerous mRNAs via an unknown mechanism. The possibility that Fmr1, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease (Caudy, 2002).

Biochemical purification of the RNAi effector nuclease, RISC, revealed the Drosophila Argonaute-2 (Ago-2) protein as a core component of this complex (Hammond. 2001). In an effort to identify additional RISC components, large-scale biochemical purification was performed following both RISC activity and Ago-2 protein by Western blotting. After five purification steps, a number of additional proteins also consistently co-purified. Among those were two RNA-binding proteins, VIG and Fmr1. Each protein was identified in multiple independent preparations of purified RISC (Caudy, 2002).

The VIG protein is encoded from within an intron of the Vasa gene. This protein has no recognizable protein domains, other than an RGG box, a motif that is known to bind RNA. VIG is an evolutionarily conserved protein, with homologs in C. elegans, Arabidopsis, mammals, and Schizosaccharomyces pombe. Little is known regarding the function of this protein family. However, a human homolog of VIG, PAI-RBP-1, was originally identified as a protein with affinity for an AU-rich element in the 3'-UTR of the plasminogen activator inhibitor (PAI) RNA, which regulates its stability (Caudy, 2002).

A human ortholog of Fmr1, FMRP, is encoded from a locus on the X chromosome that is epigenetically silenced in Fragile X syndrome. FMRP is expressed not only in neuronal cells but also in numerous other tissues, and Fragile X patients display additional phenotypes, including macroorchidism. The human genome also encodes two additional FMRP homologs, FXR1 and FXR2. Fragile X family members each contain two copies of a KH domain (hnRNP K homology) and an RGG box. All three of these domains have been proposed to bind RNA (Caudy, 2002).

To test the association of VIG and Fmr1 with RISCs, epitope-tagged proteins were expressed in Drosophila S2 cells. Notably, both Vig and Fmr1 co-fractionate with Ago-2 and RISC activity in an ~500-kD complex, as predicted if all three were components of RISC. Both RISC and Fmr1 pellet in a high-speed centrifugation and can be extracted from the P100 pellet by treatment with 0.4 M KOAc. Essentially, all expressed Fmr1 co-fractionates with RISC through subsequent ion exchange columns. In accord with this hypothesis, immunoprecipitation of Fmr1 results in co-precipitation of Ago-2 protein. To test whether the interaction is bridged by binding separately to a common mRNA, immunoprecipitations were treated with RNAse A. Interaction between Ago-2 and Fmr1 is resistant to treatment of the immunoprecipitates with RNAse A up to concentrations of 10 mg/mL. Similarly, Ago-2 was also recovered in immunoprecipitations of epitope-tagged VIG, and VIG and Fmr1 were present in mutual immunoprecipitations (Caudy, 2002).

The presence of VIG and Fmr1 in RISCs would predict that these proteins would also interact with siRNAs, another established component of RISCs. To test this possibility, Drosophila S2 cells were co-transfected with 32P-labeled dsRNA triggers of ~500 nt in length and either tagged VIG or Fmr1. In each case, immunoprecipitation selectively recovered ~21 nt siRNAs, even though these are a minor component in analyses of total RNA extracted from cells transfected with labeled silencing triggers (Caudy, 2002).

To examine the requirement for Fmr1 and VIG in RNAi, RNAi was used to reduce the expression of each of these gene products in S2 cells. This approach has been used previously to test the dependency of RNAi on Dicer and Argonaute family members in Drosophila cells, mammalian cells, and C. elegans and as a means to identify components of the RNAi machinery in worms. Pretreatment of cells with dsRNAs covering the coding sequences of either Fmr1 or VIG partially impair silencing. In the former case, expression returned to 50% of the unsuppressed control, whereas in the latter case, luciferase was de-repressed to ~70% of the control. The partial effect of suppressing Fmr1 can be interpreted in several ways. First, Fmr1 could be an accessory factor for RISCs, affecting efficiency of the complex but not being absolutely required for RNAi. Second, Fmr1 could be essential to only a subset of RISCs, whereas other (perhaps KH domain) proteins replace Fmr1 in alternative versions of RISCs. Third, Fmr1 might not be required for RNAi per se; however, loss of Fmr1 could affect the long-term stability of some RISC complexes. Another Argonaute family member expressed in S2 cells, Ago-1, appears to have little effect on the efficiency of RNAi in S2 cells. Ago-1 can be biochemically separated from active RISCs. This study by no means excludes a role for Ago-1 in RNAi since previous genetic studies have indicated that Ago-1 is required for efficient RNAi during embryogenesis (Caudy, 2002).

The possibility that a sequence-specific nuclease activity might co-immunoprecipitate with either of these components was tested. Immunoprecipitates of Fmr1 substantially degraded the cognate but not the noncognate substrate. In the case of Fmr1, >80% of the homologous substrate was degraded in 1 h of incubation (Caudy, 2002).

The majority of Fragle X mutations occur in the 5'-UTR of the gene and represent a trinucleotide repeat expansion that is thought to act by targeting FMR1 for epigenetic silencing. However, one point mutation in the FMRP protein itself has also been linked to the disease (I304N). This hypomorphic mutation in the human protein causes defects in ribosome association and RNA binding. An analogous mutation was constructed in the second KH domain of the Drosophila protein, I307N, and it was asked whether this mutation affects association with the RNAi machinery. Biochemical fractionation showed a significant alteration in the ability of the mutant Fmr1 to associate with RISC and Ago-2, with ~30% of the mutant protein shifting from the ribosome pellet (where Ago-2 localizes) to a soluble cytoplasmic form. This fraction is completely devoid of Ago-2, and Ago-2 cannot be found in association with cytoplasmic Fmr1 (Caudy, 2002).

The specific biochemical role of the human protein FMRP is not known; however, numerous studies suggest that it regulates the expression of a large number of genes at the level of protein synthesis. Recent studies of Dicer function and of Argonaute family members in C. elegans have revealed that endogenously encoded small hairpin RNAs (generically, miRNAs) regulate the expression of protein coding genes via pathways related to RNAi. The presence of Fmr1 in RISCs suggests the hypothesis that FMRP or FMRP family members may regulate gene expression via a RISC-like complex (Caudy, 2002).

Little is known of the effector complexes that contain miRNAs. The current study indicates that siRNAs and miRNAs are distinguished to some extent and preferentially, although not exclusively, assembled into separate, functional effector RNPs. A recent report has shown association of miRNAs in human cells with a 15S complex containing Gemin3, Gemin4, and eIF2c, a member of the Argonaute protein family. Gemin3 is a DEAD-box family RNA helicase, and a helicase has previously been predicted as an essential activator of siRNA-containing complexes in Drosophila. Also, in C. elegans two DExH/D box helicases, one of which is Dicer, associate with Rde-4 and are required for RNAi. Gemin4 lacks both recognizable protein motifs and an identifiable homolog in the Drosophila genome. Thus, the relationship between the previously characterized miRNP complex and RISC remains unclear; this led to further characterization of the association between miRNAs and RISCs (Caudy, 2002).

A significant portion of two miRNAs, mir2b and mir13a, in Drosophila S2 cells, are found in association with polyribosomes, as would be predicted for small RNAs that regulate protein synthesis. Furthermore, a functional connection between siRNAs and ribosome association has been made in trypanosomes. miRNAs can be extracted from a high-speed pellet from S2 cell extracts (which contains polysomes) using buffers containing elevated salt concentrations. Extracted material was fractionated by size-exclusion chromatography, and the behavior of miRNAs and siRNAs was compared with that of known RISC subunits. The majority of siRNAs co-fractionate with Ago-2 and with VIG and Fmr1. In contrast, the majority of miRNAs fractionate in an ~250-kD complex, although ~20% of miRNAs are present in fractions containing RISC. However, the possibility cannot be ruled out that this represents a tail of the 250-kD peak, given the nonquantitative nature of Western blotting. Considering the established role of Fmr1 in the regulation of endogenous protein-coding genes, the possibility that miRNAs might be present in Fmr1-containing complexes was examined. Immunoprecipitates of Fmr1 were prepared from column fractions using appropriate affinity resins. Notably, mir2b was detected in Fmr1 immunoprecipitates from transfected cells. Similarly, VIG could be used to immunoprecipitate mir2B directly from extracts (Caudy, 2002).

The forgoing data suggest that miRNAs associate with RISCs that also contain Fmr1 and VIG. This is consistent with recent data suggesting that miRNAs can direct cleavage by RISC, given appropriate homologous substrates. However, the observation of an ~250-kD peak containing the majority of miRNAs raised questions regarding the composition of these smaller complexes. Another Argonaute family member, Ago-1, showed co-fractionation of this protein with the major miRNA peak both on sizing columns and subsequently on ion exchange columns. To test the association of Ago-1 with miRNAs, immunoprecipitation experiments were performed with an epitope-tagged Ago-1 protein. Both mir2b and mir13a could be detected in association with Ago-1. Furthermore, Ago-1 immunoprecipitates also contained siRNAs. Reciprocal immunoprecipitations and Western blots indicate that Fmr1 and Vig do not detectably associate with Ago-1 (Caudy, 2002).

The forgoing data raises the possibility that siRNAs and miRNAs assemble into separate, functional effector RNPs. Recently, it has been shown that Ago-1 is required for efficient interference with endogenous genes in Drosophila embryos. Notably, Ago-1 complexes in S2 cells are similar in size to the active effector complexes, RISC*, that have previously been detected in embryo lysates. The failure of this study to detect substantial RISC activity in Ago-1 complexes and the failure to detect a substantial dependence on Ago-1 for RNAi in S2 cells could reflect developmental differences in the utilization of Ago family members or experimental differences in the assays used to detect RISCs, namely measuring loss of substrate (in this case) versus endonucleolytic cleavage as in the other studies (Caudy, 2002).

The identification of the Drosophila homolog of the fragile X protein as a RISC subunit suggests that disruptions in RNAi-related pathways may contribute to human disease. The fragile X protein has previously been implicated in the regulation of gene expression at the level of protein synthesis. In Drosophila, a bona fide Fmr1 regulatory target has been identified as Futsch, a Map1B homolog. Indeed, Fmr1 and Futsch are epistatic with respect to the neuronal defects of Fmr1-mutant flies, although they are not epistatic with respect to circadian defects. Many miRNAs in Drosophila show complementarity to common sequence motifs that negatively regulate gene expression at the posttranscriptional level. These K boxes, Brd boxes, and GY boxes are present in numerous mRNAs. Notably, a K box and a GY box are found in the 3'-UTR of Futsch. However, a sequenced miRNA has not been definitively linked to suppression of Futch expression. In humans, FMRP has been isolated as a component of large cytoplasmic mRNA complexes and, through this association, has been implicated in the regulation of numerous mRNAs. None of the several proteins known to bind FMRP has been implicated in RNAi. However, there are many possible resolutions to this seeming contradiction. First, FMRP could function through several completely independent mechanisms. Second, FMRP could be an accessory factor for (rather than an integral component of) RISCs, using RISCs for identifying FMRP's cognate targets that may be regulated via mechanisms that involve its interaction with other proteins. This would be consistent with the partial effects of Fmr1 depletions on RNAi efficiency in S2 cells (Caudy, 2002).

The precise mechanisms through which FMRP is delivered to its regulatory targets is unclear. Selection of random RNA oligonucleotides has provided evidence that FMRP binds G-quartet structures that lie adjacent to short RNA helices. This interaction required only the RGG motif and did not involve the KH domain. Thus, it was proposed that FMRP recognizes and regulates G-quartet-containing mRNAs. This study, however, proposes an alternative mechanism of regulation by fragile X protein. The results are consistent with FMRP1 being targeted to its substrates as part of RNAi complexes, which are guided by miRNAs. Recognition of G-quartets might be an important determinant of further specificity or might be critical for translational regulation. It is further proposed that FMRP might be one of many distinct protein subunits that join RISCs, depending on the tissue, subcellular localization, and the developmental stage. In this way, RISCs could function as a flexible platform on which might be constructed a variety of regulatory machines that alter gene expression at the level of mRNA metabolism, at the level of protein synthesis, and at the level of genome structure (Caudy, 2002).

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins

In Drosophila, Fmr1 binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. A Fmr1-associated complex has been isolated that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The Fmr1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. Dmp68 is also required for efficient RNAi. Fmr1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that Fmr1 is part of the RNAi-related apparatus. These findings suggest a model in which the RNAi and Frm1-mediated translational control pathways intersect in Drosophila. The findings also raise the possibility that defects in an RNAi-related machinery may cause human disease (Ishizuka, 2002).

The identification of AGO2 as a Fmr1-interacting protein is particularly striking. AGO2 is a member of the Argonaute gene family and is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNAi in Drosophila. Therefore, the finding that Fmr1 forms a complex in vivo with AGO2 suggests that Fmr1 may function in RNAi. To test this, RNAi was used to suppress the endogenous proteins, much as had been done previously to establish a role for AGO2 in RNAi. Suppression of ribosomal proteins L5 and L11 with specific dsRNAs made S2 cells so sick that their roles in RNAi could not be assessed. However, treatment of S2 cells with dsRNAs homologous to AGO2, Fmr1, or Dmp68 markedly reduces the levels of these proteins. The ability of these cells to carry out RNAi was tested by transfection with enhanced green fluorescent protein (EGFP) expression plasmid in combination with an EGFP dsRNA. Suppression of AGO2 expression correlates with a pronounced reduction in the ability of cells to silence the reporter EGFP by RNAi. Interestingly, RNAi targeting Dmp68 results in inhibition of RNAi in S2 cells. These results suggest that the DEAD-box helicase Dmp68 not only interacts with Fmr1 but is also required for efficient RNAi in S2 cells. Dmp68 is a Drosophila ortholog of human p68, which has been demonstrated to unwind short but not long dsRNAs in an ATP-dependent manner. It is concluded that at least two of the Fmr1-interacting proteins, AGO2 and Dmp68, are required for RNAi in cultured Drosophila S2 cells. In contrast, depletion of Fmr1 did not appear to affect the EGFP silencing. Therefore, although Fmr1 interacts strongly with AGO2 and Dmp68 in vivo, Fmr1 does not appear to be essential for efficient RNAi (Ishizuka, 2002).

Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs. These small RNAs (siRNAs and miRNAs) are thought to be incorporated into silencing complexes that mediate mRNA destruction during RNAi and translational control during development, respectively. Therefore, it is suggested that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. AGO2 and Dmp68 are essential for RNAi in Drosophila. However, Fmr1 appears to be a translation repressor. Because Fmr1 interacts with AGO2 and Dmp68 in vivo, it was of interest to examine whether Fmr1 is also present in an AGO2- and/or Dmp68-associated complex. To do this, TAP-tagged AGO2 (AGO2-TAP) or Dmp68 (Dmp68-TAP) were expressed in S2 cells. Cytoplasmic lysate of the cells expressing AGO2-TAP or Dmp68-TAP was prepared and subjected to TAP purifications. In reciprocal assays, endogenous Fmr1 and AGO2 were found to associate with each other. In addition, endogenous AGO2 was copurified with AGO2-TAP. Endogenous Fmr1 and AGO2 were also found to be present in the Dmp68-associated complex. Because AGO2 can be coimmunoprecipitated with Dicer, which initiates RNAi by processing dsRNA silencing triggers into siRNAs, and also processing miRNA precursors into mature miRNAs, the possibility was considered that Fmr1 might also interact physically with Dicer. Indeed, endogenous Dicer can be copurified not only with AGO2-TAP but also with Fmr1-TAP, and it was also shown that Fmr1 remains associated with AGO2 after RNAi induction. It is well established that siRNAs associate with AGO2 during RNAi in S2 cells. Therefore, these results indicate that Fmr1 may be a part of RISC. Finally, analogous to the human AGO2 ortholog (eIF2C2)-associated complex that contains a DEAD-box type RNA helicase and miRNAs, it was of interest to test whether miRNAs are also found in AGO2- and/or Fmr1-associated complexes. RNA molecules copurified with AGO2-TAP or Fmr1-TAP were recovered, dissolved on a 15% polyacrylamide denaturing gel, and subjected to Northern blot analysis. A known miRNA, miR-2b, in Drosophila S2 cells could be detected both in the AGO2- and Fmr1-associated complexes. Together, these data show that Fmr1 is present in a complex with components of RNAi and miRNAs in cultured Drosophila S2 cells (Ishizuka, 2002).

Fmr1 is thought to have important roles in the translation of some specific mRNAs such as futsch mRNA. Although it is unclear how Fmr1 regulates translation of such mRNAs, these findings may hold some clues. Because Fmr1 interacts with ribosomal L5/5S rRNA and L11, all of which are located at the top of the 60S ribosomal subunit, it is likely that this interaction brings Fmr1 to the 60S ribosomal subunit. Therefore, the association of Fmr1 with the 60S ribosomal subunit through direct interactions with ribosomal L5/5S rRNA and L11 may inhibit translation by preventing the assembly of initiation complexes or by giving rise to structural change of the ribosomes, which, in turn, influences a step(s) after translation initiation. Alternatively, because 5S rRNA is the only known RNA species that binds ribosomal proteins, including L5, and forms a 5S RNP before it is incorporated into the ribosomes, Fmr1 may interact with the cytoplasmic nonribosome-associated 5S RNP, which, in turn, influences the formation of the mature 60S ribosomal subunit. It is interesting to note in this context that only about half of the 5S rRNA molecules in mammalian cells are associated with the 60S ribosomal subunit and that although 5S rRNA enhances ribosomal activity, it is not absolutely essential for it (Ishizuka, 2002).

Fmr1 is present in a complex isolated from S2 cells, which also contains AGO2 and Dicer. AGO2 and Dicer are essential components of RNAi. The interaction between Fmr1 and AGO2 remains constant before and after RNAi induction, suggesting that Fmr1 is part of RISC during RNAi. However, there is no evidence to support the notion that RISC formation is induced by treatment of S2 cells with dsRNA. As one of the functions of the RNAi apparatus is to silence transposons and repetitive sequences residing naturally in the Drosophila genome, these cells are therefore likely to be full of pre-formed RISC complexes, irrespective of dsRNA treatment. Therefore, it is possible that Fmr1 is part of the pre-formed RISC complexes and remains to be part of the active RISC after ATP-dependent siRNA unwinding (Ishizuka, 2002).

The involvement of another Fmr1-interacting protein, Dmp68, in RNAi further suggests the close association of Fmr1 with RNAi. The p68 RNA helicase was first identified by cross-reaction with a monoclonal antibody that was originally raised against SV40 large T antigen two decades ago. The helicase plays important roles in cell proliferation and organ maturation and belongs to a large family of highly evolutionarily conserved proteins, the so-called DEAD-box family of putative ATPases and helicases. Recent studies have revealed that several RNA helicases, including mut6, SDE3, mut14 , drh-1, and spindle-E are required for RNAi and related posttranscriptional gene silencing (PTGS) pathways. Dmp68 is similar to, but clearly not an ortholog of these proteins. Therefore, Dmp68 is a novel component of RNAi. Because ATP-dependent unwinding of the siRNA duplex remodels the RISC to generate an active RISC in the RNAi pathway, Dmp68 may mediate the unwinding process. It is also conceivable that Dmp68 may be involved in downstream events such as target RNA recognition, as an RNA chaperone or an RNPase (Ishizuka, 2002).

The connection that has been established between Fmr1, components of RNAi, miRNAs, and the general translation machinery is of considerable significance because they provide intriguing clues and possible connections to the function of Fmr1 and the pathways with which it may intersect. Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs. For example, both the foreign dsRNAs that trigger RNAi and the endogenous miRNA precursors that function in development are processed into small RNAs by Dicer. Members of the Argonaute gene family are also involved in both the siRNA and miRNA pathways. In C. elegans, Dicer, the dsRNA-binding protein RDE-4, and a conserved DExH-box RNA helicase (DRH-1) are in a complex with RDE-1, an AGO2 ortholog. Furthermore, the human AGO2 ortholog, eIF2C2, is in a complex, the miRNP, that contains the DEAD-box RNA helicase Gem3. Therefore, Argonaute proteins appear to be in a complex that contains an RNA helicase(s), Dicer and small guide RNAs, and function in a variety of homology-dependent mechanisms that involve base-pairing between small guide RNAs and target mRNAs. The findings that Fmr1 interacts with AGO2, Dmp68, Dicer, miRNAs, and the general translation machinery, provide a means to link RNAi enzymes to translational control pathways, and are also consistent with the fact that the RISC nuclease fractionates with ribosomes (Ishizuka, 2002).

It appears that Fmr1 is important for translational control, possibly mediated by RNAi-related components and miRNAs. Although recent studies have identified a list of mRNAs that are potential FMRP targets, these results further suggest a model in which FMRP may not directly bind its mRNA targets but rather it is targeted to its mRNA substrates as part of RNAi-related apparatus, which are guided by miRNAs. How then might FMRP regulate translation of its mRNA targets? In the case of lin-4, a prototype of miRNAs, its mRNA targets (lin-14 and lin-28) are translationally repressed yet remain associated with polyribosomes, suggesting a block at a step after translation initiation. FMRP may form an miRNP complex on its mRNA targets and the association of this complex with ribosomal L5/5S rRNA and L11 may inhibit translation at one or more postinitiation steps, including elongation, termination, or the release of functional protein as discussed above. Finally, it is proposed that fragile X syndrome may be the result of protein synthesis abnormality caused by a defect(s) in an RNAi-related apparatus (Ishizuka, 2002).

Fmr1 interactor CYFIP/Sra-1 controls neuronal connectivity in Drosophila and links the Rac1 GTPase pathway to the Fragile X protein

Neuronal plasticity requires actin cytoskeleton remodeling and local protein translation in response to extracellular signals. Rho GTPase pathways control actin reorganization, while the fragile X mental retardation protein (FMRP) regulates the synthesis of specific proteins. Mutations affecting either pathway produce neuronal connectivity defects in model organisms and mental retardation in humans. CYFIP, the fly ortholog of vertebrate FMRP interactors CYFIP1 and CYFIP2, is specifically expressed in the nervous system. CYFIP mutations affect axons and synapses, much like mutations in Drosophila Fmr1 and in Drosophila Rho GTPase Rac1. CYFIP interacts biochemically and genetically with Fmr1 and Rac1. Finally, CYFIP acts as a Rac1 effector that antagonizes Fmr1 function, providing a bridge between signal-dependent cytoskeleton remodeling and translation (Schenck, 2003).

fragile X-related regulates the mRNA level of Pickpocket1

Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 (FMR1) gene. How FMR1 affects the function of the central and peripheral nervous systems is still unclear. FMR1 is an RNA binding protein that associates with a small percentage of total mRNAs in vivo. It remains largely unknown what proteins encoded by mRNAs in the FMR1-messenger ribonuclear protein (mRNP) complex are most relevant to the affected physiological processes. Loss-of-function mutations in the Drosophila fragile X-related (dfmr1) gene, which is highly homologous to the human fmr1 gene, decrease the duration and percentage of time that crawling larvae spend on linear locomotion. Overexpression of DFMR1 in multiple dendritic (MD) sensory neurons increases the time percentage and duration of linear locomotion; this phenotype is similar to that caused by reduced expression of the MD neuron subtype-specific degenerin/epithelial sodium channel (DEG/ENaC) family protein Pickpocket1 (PPK1). Genetic analyses indicate that PPK1 is a key component downstream of DFMR1 in controlling the crawling behavior of Drosophila larvae. DFMR1 and ppk1 mRNA are present in the same mRNP complex in vivo and can directly bind to each other in vitro. DFMR1 downregulates the level of ppk1 mRNA in vivo, and this regulatory process also involves Argonaute2 (Ago2), a key component in the RNA interference pathway. These studies identify ppk1 mRNA as a physiologically relevant in vivo target of DFMR1. The finding that the level of ppk1 mRNA is regulated by DFMR1 and Ago2 reveals a genetic pathway that controls sensory input-modulated locomotion behavior (Xu, 2004).
To study subtle behavioral defects caused by dfmr1 mutations, the crawling behavior of wild-type and mutant larvae were examined at the wandering stage with DIAS software in an environment devoid of cues for chemotaxis and phototaxis; because larvae were placed on a flat surface, as verified by a level, there were presumably no cues for geotaxis. The larval crawling behavior is relatively simple and stereotyped and can be separated into two phases: linear locomotion and nonlocomotive turning. As shown by analysis of the centroid paths of crawling larvae, wild-type and dfmr14 mutant larvae have different crawling patterns. Analysis at higher magnification showed that the wild-type larvae had longer linear paths and made fewer turns than dfmr14 mutants (Xu, 2004).

To quantify differences between wild-type and dfmr14 mutant larvae, the direction change was determined, defined as the absolute value of the difference in direction from one frame to the next. Computer analysis of crawling routes indicated that, during 1.5 min of recording (150 data points), a representative dfmr14 mutant larva had 17 data points with direction changes larger than 60°, and a representative wild-type larva had only eight such points (Xu, 2004).

Because the number of direction changes varied substantially among larvae of a given genotype, a large number of larvae were recorded and analyzed to quantify the difference at the population level. The average direction change for a larva is the mean value of all the data points. More dfmr14/dfmr14 mutants than wild-type larvae exhibited an average direction change greater than 20°. dfmr14/dmr14 mutant larvae showed a greater average direction change than wild-type larvae. No significant differences were seen between dfmr14/+ heterozygous larvae and wild-type larvae. To confirm that the abnormal crawling behavior was caused by dfmr1 mutations, mutant larvae were examined that had a combination of different dfmr1 loss-of-function alleles that were independently generated from the genetic screen. Larvae with different dfmr1 alleles all exhibited similar crawling behaviors. In addition, the alteration in direction change could be rescued by introducing a genomic DNA fragment containing the wild-type dfmr1 gene, indicating that dfmr1 was indeed responsible for the observed phenotype in mutant larvae (Xu, 2004).

To further characterize the alterations in the crawling pattern of dfmr1 mutant larvae, 'linear locomotion' was defined as the time period during which at least five consecutive frames showed a direction change that was smaller than 20°. Wild-type larvae spent a greater percentage of time on linear locomotion than dfmr14 mutant larvae. Mutant larvae with different dfmr1 alleles showed a phenotype similar to that of dfmr14 mutant larvae. In addition, the average duration of linear locomotion of dfmr14 mutant larvae was shorter than that of wild-type larvae. Both alterations were rescued by the introduction of a genomic DNA fragment containing the wild-type dfmr1 gene. These findings indicate that dfmr1 mutations significantly alter the crawling pattern of wandering larvae (Xu, 2004).

DFMR1 has been shown to play a role in the proper development of higher-order fine dendritic processes of MD sensory neurons in the PNS of Drosophila larvae. Most MD neurons elaborate extensive dendritic arbors just underneath the epidermis in each segment. To test whether MD neurons modulate crawling behavior, DFMR1 was expressed in MD neurons under the control of Gal4 109(2)80, which drives target gene expression in all MD neurons and less than 100 central neurons. This manipulation decreased the average direction change and increased the time percentage and duration of linear locomotion; together, these create a phenotype opposite that of dfmr1 mutant larvae. Furthermore, expression of normal DFMR1 protein by the same Gal4 driver in a dfmr1 mutant background rescued the defects in crawling behavior. These results are consistent with the notion that changes in DFMR1 activity in MD neurons affect the crawling behavior of Drosophila larvae (Xu, 2004).

To uncover the molecular mechanism underlying DFMR1 function, the association between DFMR1 and several mRNAs that encode known channel molecules was examined. A monoclonal antibody against DFMR1 was used to immunoprecipitate DFMR1-mRNP complexes and reverse transcription-polymerase chain reaction (RT-PCR) was used to demonstrate the presence or absence of a particular mRNA in the complexes. The mRNA encoding PPK1, an MD neuron subtype-specific member of the DEG/ENaC family, was associated with DFMR1 in vivo. Lysates from dfmr14 mutant larvae served as negative controls. No association was detected in the same immunoprecipitation experiment between DFMR1 and the mRNAs encoding Hyperkinetic (HK) and Shaker, both of which have been implicated in larval crawling behavior. To further investigate the association between DFMR1 and ppk1 mRNA, GST-DFMR1 fusion protein was purified and it was found that ppk1 mRNA could bind in vitro directly to GST-DFMR1 but not GST alone. To demonstrate the binding specificity, a competition binding experiment was performed. The affinity of DFMR1 for ppk1 mRNA was at least one order of magnitude higher than that for a control mRNA. These findings demonstrate that DFMR1 is associated with ppk1 mRNA in vivo and that they can directly bind to each other at least in vitro (Xu, 2004).

The level of ppk1 mRNA is regulated by DFMR1 in MD sensory neurons. dfmr1 mutant larvae have been shown to have slightly more higher-order dendritic branches of sensory neurons than wild-type larvae. It seems that ppk1 mRNA and dendrite development are independently regulated by DFMR1, because overexpression of Rac1 in MD neurons increased dendritic branching without affecting the level of ppk1 mRNA and locomotion behavior. Recent studies demonstrate that DFMR1 associates with microRNAs and proteins involved in the RNAi pathway. Although it was reported that DFMR1 could affect the efficiency of RNAi in vitro, it was found that DFMR1, unlike Ago2, is not required for efficient RNAi-mediated degradation of ppk1 mRNA in vivo. Deletion mutations were generated in ago2 and it was found that Ago2 also regulates the ppk1 mRNA level in a manner similar to DFMR1. Genetic-interaction studies further support the notion that the two molecules work in the same genetic pathway to control ppk1 mRNA level. A GST-Ago2 fusion protein was purified and it was found that Ago2 itself does not bind to ppk1 mRNA. These findings suggest a model in which DFMR1 binds to ppk1 mRNA and recruits Ago2 and presumably other components to regulate the level of ppk1 mRNA, which in turn modulates larval locomotion behavior. Interestingly, overexpression of PPK1 by itself does not affect locomotion behavior, possibly due to the fact that PPK1 is only a subunit of a functional channel. Therefore, other channel subunits or downstream components may be coordinately regulated by DFMR1. The association of DFMR1 and Ago1 has also been reported (Jin, 2004). Argonaute family proteins are involved not only in RNAi pathways but also in microRNA-mediated gene regulation. These studies provide strong evidence that sensory-channel molecules can be regulated by components associated with microRNA pathways (Xu, 2004).

Although DFMR1 is known to be an RNA binding protein, the RNAs that associate with it in vivo have not been systematically identified. Recent studies suggest that a small percentage of mRNAs are associated with FMR1-mRNA complexes in mouse brain and cell lines. Since DFMR1 and human FMR1 share a high degree of sequence homology and are likely to function similarly, DFMR1 might also regulate multiple mRNAs in Drosophila. It remains a major challenge to identify the key mRNA targets that mediate DFMR1's effect on a particular physiological pathway. In this study, by using both biochemical and genetic approaches, it has been shown that regulation of ppk1 mRNA by DFMR1 contributes to MD sensory neuron-modulated larval crawling behavior (Xu, 2004).

Because DEG/ENaC superfamily proteins are highly conserved through evolution, it would be of great interest to determine which DEG/ENaC channel molecules in mammals are also regulated by FMR1. Further understanding of the functional alterations in the sensory pathway caused by DFMR1/FMR1 mutations may provide deeper insights into the mechanisms underlying fragile X syndrome in humans (Xu, 2004).

Fragile X protein functions with Lgl and the PAR complex in flies and mice

Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss of function for the Fragile X Mental Retardation 1 gene (FMR1). FMR1 protein (FMRP) has specific mRNA targets and is thought to be involved in their transport to subsynaptic sites as well as translation regulation. A saturating genetic screen of the Drosophila autosomal genome was used to identify functional partners of dFmr1. Nineteen mutations were recovered in the tumor suppressor lethal (2) giant larvae (dlgl) gene and 90 mutations at other loci. dlgl encodes a cytoskeletal protein involved in cellular polarity and cytoplasmic transport and is regulated by the PAR complex through phosphorylation. Direct evidence is provided for a Fmrp/Lgl/mRNA complex, which functions in neural development in flies and is developmentally regulated in mice. The data suggest that Lgl may regulate Fmrp/mRNA sorting, transport, and anchoring via the PAR complex (Zarnescu, 2005).

This study reports the identification of Lgl as a functional partner of the Fragile X protein, Fmrp. Lgl forms a large macromolecular complex with Fmrp, which is developmentally regulated and modulates the architecture of the neuromuscular junction in the fly. At the cellular level, Lgl and Fmrp are temporally and spatially coexpressed during development and colocalize in granules in the soma and the developing neurites of mouse cultured cells. Fractionation experiments show that Fmrp and Lgl comigrate with Golgi membrane-associated complexes. Furthermore, the Fmrp/Lgl complex contains a subset of mRNAs and interacts physically and genetically with the PAR complex, an essential component of the cellular polarization pathway. These results suggest that Lgl functions with Fmrp to regulate a subset of target mRNAs during synaptic development and/or function. It is proposed that Lgl may regulate Fmrp/mRNA containing RNPs by (1) sorting at the Golgi, (2) transport in neurites, and (3) anchoring at specific membrane domains, such as subsynaptic sites. The neurite transport function may involve molecular motors such as myosin II and kinesin, previously shown to associate with Lgl and Fmrp, respectively. The anchoring mechanism may involve the PAR complex, which has a demonstrated role in defining membrane domains and has recently been shown to generate asymmetry in the C. elegans embryo by stabilizing RNPs at the posterior pole (Zarnescu, 2005).

The data demonstrate that Fmrp and Lgl form a functional complex in living neurons, and this is conserved in flies and mice. In the mouse brain, mFmrp associates with mLgl preferentially at a time of increased synaptogenesis, demonstrating a developmentally regulated interaction between Lgl and Fmrp. The data suggest that dLgl acts to regulate a subset of dFmr1-associated mRNAs with some encoding circadian regulated molecules (CG3348 and CG9681) and some encoding secreted or transmembrane proteins (CG6136, CG4101, CG9681) among others. dLgl also associates with mRNA independent of dFmr1; thus, it is formally possible that dLgl interacts with other RNA binding proteins, which remain to be determined (Zarnescu, 2005).

Fmrp has been implicated in the translational regulation of specific target mRNAs, perhaps via the RNAi pathway. Also, it has been proposed that Fmrp is involved in the transport and localization of mRNAs: cellular fractionation and immunolocalization data revealed the association of Fmrp-containing complexes with molecular motors such as kinesin and myosin V. Lgl functions in cellular polarity via regulating myosin motor activity and/or vesicle transport and has been shown to regulate polarized delivery by sorting at the Golgi. Taken together, these concepts suggest that Lgl may act as a scaffold for Fmrp granules, possibly at the Golgi, and perhaps aids in carrying specific mRNA targets to sites of locally controlled translation (such as subsynaptic sites) (Zarnescu, 2005).

It is also possible that Lgl anchors Fmrp at specific membrane domains such as synapses, perhaps via the PAR complex. Lgl function is regulated by the PAR complex, specifically via phosphorylation by aPKC-zeta and by direct binding to PAR6. The genetic interaction data suggest that PAR6 and Baz antagonize dFmr1 function, which is in accordance with previously published work showing that the PAR complex inhibits dlgl and with data that demonstrate that dlgl functions cooperatively with dFmr1. aPKC-zeta was shown to antagonize most dLgl functions with the exception of its role in regulating neuroblast apical size and the data are be consistent with such reports. Loss of function for aPKC-zeta suppresses gain of function sev:dFmr1 as well as the loss-of-function phenotype of dFmr1 at the NMJs. Taken together, these data suggest a dynamic relationship between the various members of the complex. One possible interpretation of the results is that aPKC/PAR can act on Fmrp directly or via Lgl depending on the developmental and/or cellular context. Furthermore, this is consistent with sucrose fractionation experiments, which suggest the existence of at least two complexes comprising dFmr1/PAR proteins and dFmr1/dLgl/PAR proteins (Zarnescu, 2005).

The PAR complex not only functions in cell polarity, but also at the synapse, where it is believed to function in synaptic tagging. Synaptic tags have been proposed to transiently mark a synapse after activation in a way that will translate the local events into persistent functional changes (such as long-term depression), processes in which Fmrp is also thought to act. Thus, the Fmrp/Lgl/PAR complex may act in synaptic plasticity linking synaptic input to the remodeling of the cytoskeleton and mediating required translational changes (Zarnescu, 2005).

The Drosophila fragile X protein functions as a negative regulator in the orb autoregulatory pathway

Translational regulation of maternal mRNAs in distinct temporal and spatial patterns underlies many key decisions in developing eggs and embryos. In Drosophila, Orb is responsible for mediating the translational activation of mRNAs localized within the developing oocyte. Orb is a germline-specific RNA binding protein and is one of the founding members of the CPEB family of translational regulators. Orb associates with the Drosophila Fragile X Mental Retardation (dFMR1) protein as part of a ribonucleoprotein complex that controls the localized translation of mRNAs in developing egg chambers. One of the key orb regulatory targets is orb mRNA, and this autoregulatory activity is critical for ensuring that Orb protein is expressed at high levels in the oocyte. dFMR1 functions as a negative regulator in the orb autoregulatory circuit, downregulating orb mRNA translation (Costam, 2005).

To identify factors that could function in regulating Orb activity and/or localization proteins that are physically associated with Orb in vivo were sought. For this purpose ovarian extracts were immunoprecipitated with Orb antibodies. Besides Orb, the immunoprecipitates contained a complex set of proteins that were absent in the control immunoprecipitates. Attention was focused on four larger proteins because they are present in nearly the same yield as Orb (as judged by staining) and are well resolved from other polypetides. The association of these four proteins with Orb is RNase resistant; they are observed not only when an RNase inhibitor is present, but also when the immunoprecipitates are treated with RNase or when the immunoprecipitation is done in the presence of RNase (Costam, 2005).

The four proteins were identified by mass spectroscopy. The largest 150 kd species corresponds to Lingerer. Lingerer is expressed at high levels in the nervous system, imaginal discs, and gonads. It is required for viability and has been implicated in sexual behavior. The 120 kd species is CG18811-PA. Nothing is known about its function in flies; however, related proteins called Caprins have been identified in vertebrates including humans and are thought to function in cell proliferation. The species migrating slightly more slowly than Orb is Rasputin (Rin). Rin is the fly homolog of the RasGAP SH3 binding protein (G3BP), and it is thought to regulate RNA metabolism in response to Ras signaling. Finally, the smallest protein, which is the subject of this study, corresponds to the Drosophila Fragile X Mental Retardation protein (dFMRP) (Costam, 2005).

Though dFMR1 is a KH domain RNA binding protein, it is associated with Orb in an RNase resistant complex. Although this could indicate that Orb and dFMR1 complex with each other through (direct or indirect) protein-protein interactions, an inability to produce soluble recombinant Orb has precluded testing for direct interactions in vitro. In addition, it should be noted that the fact that the Orb-dFMR1 complex in ovarian extracts is RNase resistant does not exclude the possibility that Orb and dFMR1 coassemble into mRNPs because the two proteins recognize sequences in the same RNA species. That there must be some type of target specificity in Orb-dFMR1 complex assembly is suggested by both Western and confocal analysis. Though dFMR1 appears to be present in Orb immunoprecipitations in near molar yield, Western blots indicate that only a subfraction of the total dFMR1 in ovaries is in an immunoprecipitable complex with Orb. One reason for this is that all of the somatic cells in the ovary have dFMR1, whereas they do not contain Orb. However, even within the germline not all of the dFMR1 is associated with Orb. For example, in the germarium dFMR1 is found in stem cells and the mitotic cysts, whereas Orb is not readily detected until the 16-cell cyst is formed. Even after the 16-cell cysts are formed and begin to develop, a significant fraction of the germline dFMR1 is localized in nurse cells, and with the exception of perinuclear particles, this protein does not seem to be associated with Orb. In contrast, much of the dFMR1 protein present in the oocyte appears to be specifically associated with Orb. Moreover, it is in the oocyte where Orb activity is known to be required. In previtellogenic chambers, dFMR1 and Orb colocalize around the oocyte nucleus, concentrating most heavily at the posterior pole. After the onset of vitellogenesis, Orb and dFMR1 colocalize along most of the oocyte cortex except at the very posterior pole where there is a relatively low level of dFMR1 and a high level of Orb (Costam, 2005).

An important question is whether the Orb-dFMR1 association in the oocyte (and in the perinuclear particles in the nurse cells) has regulatory consequences. Several lines of evidence argue that it does. (1) dfmr1 interacts genetically with orb. orb is weakly haploinsufficient and a significant fraction of the eggs laid by females heterozygous for strong loss of function mutations such as orb343 have D-V polarity defects indicative of a failure in the grk-EGFR signaling pathway. This haploinsufficiency is enhanced by transgenes, like HD19G, that express orb 3′ UTR sequences fused to heterologous LacZ protein coding sequence. The HD19G mRNAs compete with the endogenous orb mRNA for Orb protein binding and further compromise the orb positive autoregulatory circuit. D-V polarity defects of HD19G orb343/+ females can be suppressed by reducing the dose of dfmr1 in half, whereas they can be enhanced by adding an extra dfmr1 gene. Genetic interactions between dfmr1 and orb are also seen in the absence of the HD19G transgene. Moreover, in this case the D-V polarity defects seen at 18°C for females heterozygous for orb343 or for another allele, orbdec, can be almost completely suppressed by eliminating dfmr1 altogether. (2) As would be expected if dfmr1 exerts its effects on D-V polarity by modulating the orb autoregulatory circuit, it was found that dfmr1 negatively regulates Orb protein expression. Consistent with the suppression of D-V polarity defects, Orb accumulation in HD19G orb343/+ females can be increased by reducing the dose of dfmr1. Conversely, as would be expected from the finding that excess dFMR1 enhances the frequency of D-V polarity defects, increasing the dose of dfmr1 decreases Orb expression. (3) The effects of dfmr1 on Orb accumulation are not restricted to circumstances in which orb autoregulation is partially compromised. It is also seen in females that are wild-type for orb. In this case, eliminating dfmr1 leads to the overexpression of Orb protein. (4) orb interacts genetically with dfmr1. Two independent orb mutations dominantly suppress the excess germ cell phenotype seen in egg chambers from dfmr1 mutant females. The fact that this phenotype can be suppressed by reducing the orb gene dose would suggest that it arises, either directly or indirectly, from the overproduction of Orb protein. (5) While the inhibitory effects of dfmr1 on Orb expression cannot be unambiguously attributed to the particular subfraction of the dFMR1 protein that is complexed specifically with Orb, the data indicate that dfmr1 activity is required in the germline in order to regulate Orb expression (Costam, 2005).

Based on the pattern of Orb accumulation when dfmr1 activity is reduced, it would appear that dFMR1 is initially required to repress the translation of orb mRNAs while they are in transit from the nurse cells to the oocyte. If dFMR1 action is direct in the nurse cells, it presumably associates with orb message soon after it is synthesized in the nurse cell nuclei and acts to represses translation. In this respect, it may be of interest that dFMR1-Orb particles are observed around the edge of the nurse cell nuclei. These perinuclear particles resemble the sponge bodies that are thought to be involved in assembling newly synthesized mRNAs into translationally dormant mRNPs so that they can be transported from the nurse cells to the oocyte. Although dFMR1 clearly functions to block Orb expression in nurse cells, the fact that the amount of Orb in nurse cells in the absence of dfmr1 activity is still much lower than it is in the oocyte argues that there must be other factors besides dFMR1 that inhibit the translation of orb mRNA while it is in transit. As observed in the nurse cells, Orb accumulation is upregulated in dfmr13 oocytes. Orb expression is also upregulated in the oocytes of HD19G orb343/+ egg chambers when dfmr1 activity is reduced, whereas it is repressed when dfmr1 activity is increased. The effects of dfmr1 on Orb expression in the oocyte suggest that it functions to attenuate the orb positive autoregulatory feedback loop; however, the fact that Orb levels do not become excessive in the mutant oocytes indicates that there must be other mechanisms to prevent Orb over accumulation (Costam, 2005).

Unlike the nurse cells, most of the dFMR1 in the oocyte colocalizes with Orb. Consequently, it would be reasonable to suppose that dFMR1 modulates the orb autoregulatory circuit in the oocyte through its association with Orb complexes that contain orb mRNA. As noted above, while Orb-dFMR1 complexes in ovary extracts were found ot be RNase resistant, it is suspected that complex assembly may depend upon the presence of recognition sequences for each protein in the mRNA. In the case of orb mRNA, previous studies indicate that Orb interacts with several sequences in the 3′ UTR in vivo and in extracts. While the 3′ UTR does not have sequences that match the dFMR1 consensus, there are three recognition motifs in the 5′ UTR, at the beginning of the protein coding sequence. Thus, a plausible idea is that these 5′ and 3′ recognition motifs serve to recruit dFMR1 and Orb into the orb mRNA RNPs. That the dFMR1 recognition motifs may, in fact, be important for dFMR1 regulation is suggested by the behavior of the HD19G mRNA, which contains all of the known Orb target sequences in the orb 3′ UTR but does not have the dFMR1 motifs. The HD19G mRNA mimics the endogenous orb mRNA with respect to its dependence on orb activity for both localization within the oocyte and translational activation. However, HD19G mRNA responds differently from the endogenous orb message to changes in dfmr1 activity (Costam, 2005).

Seemingly similar specificities are evident in the requirements for orb and dfmr1 activity in regulating fs(1)K10 and osk mRNA translation. Both of these mRNAs have Orb target sequences in their 3′ UTRs, whereas only fs(1)K10 has potential dFMR1 recognition motifs. Whereas both fs(1)K10 and osk depend upon orb activity for proper localization and translation, the expression of Fs(1)K10 is upregulated in the absence of dfmr1 activity, while there is no apparent effect on Osk. Because Orb is overexpressed in dfmr1 mutants, one explanation for the upregulation of Fs(1)K10 is that it is an indirect consequence of excess orb activity. However, since Osk levels are unaltered, the idea is favored that dfmr1 functions to inhibit orb-mediated activation of fs(1)K10 mRNA translation but does not have a role in osk regulation. In this respect, it may be significant that Orb specifically promotes the translation of osk mRNA localized at the posterior pole in vitellogenic stage egg chambers. The posterior pole differs from elsewhere along the cortex in that there seems to be much less dFMR1 associated with Orb. Presumably other factors, such as Bruno and another KH domain RNA protein, Bicaudal-C, would function in repressing osk translation and counteracting orb activation. Much like dFMR1, these proteins coimmunoprecipitate with Orb and are necessary for proper repression of osk translation (Costam, 2005).

An intriguing question is whether the connection between Orb and dFMR1 in fly ovaries is relevant to the neurological phenotypes induced by inactivation of the fragile X gene in humans and mice. It is interesting in this regard that recent studies have implicated both CPEBs and FMRPs as key players not only in CNS development but also in learning and memory. Moreover, much as is observed in the fly germline, CPEBs and FMRPs appear to function antagonistically in regulating the localized translation of specific target mRNAs. If the activity and/or expression of mammalian CPEBs is negatively regulated by FMRPs, as is the case for Orb in flies, then it seems possible that the loss of FMRP activity may lead to excess CPEB activity in the nervous system and perturb the proper spatial or temporal regulation of translation (Costam, 2005).

Fragile X mental retardation protein controls trailer hitch expression and cleavage furrow formation in Drosophila embryos

During the cleavage stage of animal embryogenesis, cell numbers increase dramatically without growth, and a shift from maternal to zygotic genetic control occurs called the midblastula transition. Although these processes are fundamental to animal development, the molecular mechanisms controlling them are poorly understood. This study demonstrates that Drosophila fragile X mental retardation protein (dFMRP) is required for cleavage furrow formation and functions within dynamic cytoplasmic ribonucleoprotein (RNP) bodies during the midblastula transition. dFMRP is observed to colocalize with the cytoplasmic RNP body components Maternal expression at 31B (ME31B) and Trailer Hitch (TRAL) in a punctate pattern throughout the cytoplasm of cleavage-stage embryos. Complementary biochemistry demonstrates that dFMRP does not associate with polyribosomes, consistent with their reported exclusion from many cytoplasmic RNP bodies. By using a conditional mutation in small bristles (sbr), which encodes an mRNA nuclear export factor, to disrupt the normal cytoplasmic accumulation of zygotic transcripts at the midblastula transition, the formation of giant dFMRP/TRAL-associated structures was observed, suggesting that dFMRP and TRAL dynamically regulate RNA metabolism at the midblastula transition. Furthermore, dFMRP associates with endogenous tral mRNA and is required for normal TRAL protein expression and localization, revealing it as a previously undescribed target of dFMRP control. It was also shown genetically that tral itself is required for cleavage furrow formation. Together, these data suggest that in cleavage-stage Drosophila embryos, dFMRP affects protein expression by controlling the availability and/or competency of specific transcripts to be translated (Monzo, 2006).

The data suggest that in cleavage-stage Drosophila embryos, dFMRP affects translational initiation of specific mRNA molecules within cytoplasmic RNP bodies by controlling their availability and/or modulating their competency to be translated. dFMRP does not measurably associate with polyribosomes under a wide range of conditions in cleavage-stage Drosophila extracts, similar to results obtained for Drosophila S2 cells, but in contrast to reports in other systems. Instead, dFMRP colocalize and cosediment was observed with TRAL and ME31B, known components of translationally quiescent cytoplasmic RNP bodies. Although dAGO2 cosediments with polyribosomes in cleavage-stage embryo extracts and could directly suppress translational elongation or termination, a similar role for dFMRP is unlikely. In fact, there is no indication that endogenous dFMRP directly interacts with dAGO2 in cleavage-stage Drosophila embryos, in contrast to their observed association in Drosophila S2 cell extracts. This discrepancy could result from a fundamental difference in RNA metabolism between S2 cells and cleavage-stage embryos undergoing the MBT (Monzo, 2006).

tral mRNA represents a previously undescribed in vivo target of dFMRP regulation. Although there is no direct evidence that dFMRP and TRAL form a stable complex in cleavage-stage embryos in vitro, dFMRP activity is clearly required for normal TRAL protein expression in vivo. Mislocalization of TRAL protein but not ME31B in both fmr1- and sbrts148 mutant embryos suggests that a specific functional relationship exists between dFMRP and TRAL. In fmr1- embryos, TRAL protein levels also are reduced 2-fold, indicating that TRAL does not simply get redistributed into abnormal structures, its rate of synthesis and/or degradation must also be affected. The co-IP of tral mRNA with dFMRP from WT embryo extracts demonstrates that dFMRP and tral mRNA form a stable RNP complex and suggests that dFMRP is involved in tral mRNA metabolism. Although it has not yet been determined whether dFMRP directly binds tral mRNA, this analysis of the tral mRNA sequence, by using the fast RNA motif/pattern searcher RNABOB identified a single G-quartet stem-loop structure within the tral 3' UTR, a motif that FMRP can bind with high affinity. Regardless of whether dFMRP binds tral mRNA directly, dFMRP could control the assembly of a translationally competent tral mRNP complex and/or its localized delivery for translation. The transient association of tral mRNA with cytoplasmic RNP bodies in a translationally quiescent state might be required for dFMRP to promote the assembly of a translationally competent tral mRNP. Alternatively, the restricted translation of tral mRNA, controlled by dFMRP-dependent localized release from cytoplasmic RNP bodies, might promote the normal assembly of a functional TRAL RNP complex. In either case, lower steady-state TRAL protein levels resulting from decreased synthesis and/or increased degradation in fmr1- embryos could be related to abnormal TRAL RNP complex assembly, observed as large structures by IF. Interestingly, the higher steady-state level of tral mRNA observed in fmr1- embryo extracts is reminiscent of the increased levels of another dFMRP target, pickpocket mRNA, observed in fmr1- embryo extracts and may reflect a common feature of dFMRP mRNA processing (Monzo, 2006).

In conclusion, it is believed that a system of cytoplasmic RNP bodies exists in cleavage-stage embryos that associates with maternal and zygotic mRNAs to mediate their degradation or processing for subsequent release for translation during the MBT. A large proportion of these cytoplasmic RNP bodies contain dFMRP. It is likely that the cleavage furrow formation defect observed in fmr1- mutants is the result of disrupting TRAL function. Indeed, tral- embryos have a cellularization phenotype that resembles that of fmr1- embryos. A similar requirement has also been found for the Caenorhabditis elegans homolog of tral, car-1, in cleavage furrow formation. However, as with Fragile X syndrome, it is possible that the altered expression of many targets is responsible for the full fmr1- cellularization phenotype (Monzo, 2006).

Bicaudal-D regulates fragile X mental retardation protein levels, motility, and function during neuronal morphogenesis

The expression of the RNA-binding factor Fragile X mental retardation protein (FMRP) is disrupted in the most common inherited form of cognitive deficiency in humans. FMRP controls neuronal morphogenesis by mediating the translational regulation and localization of a large number of mRNA targets, and these functions are closely associated with transport of FMRP complexes within neurites by microtubule-based motors. However, the mechanisms that link FMRP to motors and regulate its transport are poorly understood. This study shows that FMRP is complexed with Bicaudal-D (BicD) through a domain in the latter protein that mediates linkage of cargoes with the minus-end-directed motor dynein. In Drosophila the motility and, surprisingly, levels of FMRP protein are dramatically reduced in BicD mutant neurons, leading to a paucity of FMRP within processes. Functional evidence is provided that BicD and FMRP cooperate to control dendritic morphogenesis in the larval nervous system. These findings open new perspectives for understanding localized mRNA functions in neurons (Bianco, 2010).

BicD proteins (BicD in Drosophila and BicD1 and BicD2 in mammals) play roles in the transport of a subset of cargoes by the minus-end-directed microtubule motor dynein. The N-terminal two-thirds of BicD interact with dynein and its accessory complex dynactin, and the C-terminal third (the C-terminal domain [CTD]) mediates mutually exclusive association with different cargoes. The best-characterized roles of BicD proteins are in the bidirectional transport of Golgi vesicles and a subset of asymmetrically localized Drosophila mRNAs, which are mediated by binding of the CTD to the membrane-associated G protein Rab6 and the RNA-binding protein Egalitarian (Egl), respectively. The interactions of the BicD CTD with both proteins are inhibited by the K730M substitution in the Drosophila BicD protein, which is a null mutation in vivo. K730M does not, however, inhibit binding of the BicD CTD to other copies of BicD, indicating that it specifically effects association of BicD with motor cargoes (Bianco, 2010).

In an attempt to elucidate the basis of linkage of other cargoes to dynein, a GST pull-down from fly embryonic extracts was performed with the Drosophila BicD CTD (amino acids 536-782) and an equivalent K730M mutant protein as a specificity control. Mass spectrometry revealed that a protein of 80-85 kDa reproducibly recruited only by the wild-type CTD was Drosophila FMRP (27 unique peptides), and this was confirmed by western blotting. Endogenous BicD and FMRP were specifically coimmunoprecipitated from Drosophila embryonic extracts. Unlike known Egl-interacting proteins, FMRP was not coimmunoprecipitated with a GFP-tagged Egl protein. This finding, together with the observation that binding of both Egl and FMRP to BicD is impaired by the K730M mutation, suggests that BicD:FMRP complexes are largely, or completely, distinct from BicD:Egl complexes (Bianco, 2010).

The ability to detect FMRP in CTD pull-downs and BicD immunoprecipitations from extracts was abolished by treatment with RNase. In contrast, the complex of Egl with BicD was not sensitive to RNase treatment. Thus, the stable association of BicD and FMRP in extracts is dependent on RNA. Nonetheless, a weak interaction of the BicD CTD with a subfragment of FMRP (aa 220-618) was found in yeast two-hybrid assays. This interaction was specific, as shown by the fact that it was disrupted by the K730M mutation within the BicD CTD. These findings raise the possibility of a direct contact of BicD and FMRP in vivo that is stabilized by the association of FMRP with RNA targets and possibly other RNA-associated proteins (Bianco, 2010).

These results suggest that BicD could be a functional interactor of FMRP in vivo. Subsequent studies therefore focused on neurons, where FMRP plays a prominent role. As previously observed, endogenous FMRP is enriched in puncta within the cell body and neurites of Drosophila primary neurons cultured from larval brains. Endogenous BicD was also found in puncta in these cells, but these were much more frequent than those containing FMRP. Although there was overlap of a subset of FMRP puncta with BicD puncta, the widespread distribution of BicD precluded a meaningful interpretation about the extent of complex formation of BicD and FMRP in fixed primary neurons (Bianco, 2010).

Therefore neuronal cultures were established from brains of transgenic larvae expressing FMRP::GFP and BicD::mCherry and time-lapse microscopy was used to assay for cotransport of puncta containing both proteins. These fluorescent fusion proteins retain function and account for ~20% and 50% of the levels of total FMRP and BicD proteins, respectively, in transgenic larval brain extracts (Bianco, 2010).

Both BicD::mCherry and FMRP::GFP were widely distributed in the cytoplasm of the primary neurons, but bidirectionally transported FMRP::GFP puncta were found in all cells and 92.4% ± 3.2% of them were cotransported with a puncta of BicD:mCherry. Thus, FMRP and BicD can be contained within the same motile transport complexes in neurons. The motility of FMRP::GFP in these experiments will be described in more detail below. Only 77.2% ± 4.6% of motile BicD::mCherry puncta were cotransported with a puncta of FMRP::GFP (155 particles in 20 cells), indicating that BicD may transport additional cargoes in these cells and/or that a subset of BicD::mCherry complexes may contain only nonfluorescent, endogenous FMRP (Bianco, 2010).

Next whether BicD has a functional role in FMRP:motor complexes in neurons was explored by assessing the subcellular localization of FMRP in third instar BicD mutant larvae. Because the high expression of FMRP expression in neighboring nonneuronal cells obfuscates the distribution of the endogenous protein in thin neuronal processes, UAS-FMRP::GFP was expressed at low levels by using a panneuronal GAL4 driver. In neurons of zygotic BicD null mutant larvae, which also lack detectable maternal BicD protein, the amount of FMRP::GFP within the neurites was greatly reduced compared to wild-type. Surprisingly, there was also a much weaker FMRP::GFP signal in the cell body of BicD mutant neurons relative to wild-type. Western blotting of third instar larval brain extracts confirmed a striking reduction in levels of both FMRP::GFP and endogenous FMRP in the absence of BicD (Bianco, 2010).

Strong mutations in genes encoding the dynein and kinesin-1 motor proteins, which should inhibit microtubule-based FMRP transport in Drosophila, did not alter the amount of FMRP. These findings, together with observations from interfering with dynactin function, suggest that the reduction in FMRP protein levels in BicD mutants is caused by a specific role of BicD rather than an indirect consequence of inefficient FMRP transport (Bianco, 2010).

Levels of the Fmr1 mRNA, which encodes FMRP, were indistinguishable in wild-type and BicD mutant brain extracts, as revealed by quantitative RT-PCR. Thus, the requirement for BicD in maintaining FMRP protein levels is not associated with RNA decay or transcription. Further evidence against a defect in Fmr1 transcription in BicD mutants is provided by the strong reduction in the levels of the GFP-tagged FMRP protein, which is transcribed under the control of yeast-derived UAS promoter elements. The FMRP::GFP transgene also lacks the untranslated sequences from the Fmr1 gene, revealing that BicD’s regulation of FMRP protein amount is mediated through the Fmr1 coding sequence. BicD may therefore influence FMRP protein stability through an unknown mechanism. However, it currently cannot be rule out that BicD regulates the translation of FMRP; at least in mammals, the coding sequence of Fmr1 mRNA contains a translational control element, which negatively regulates protein production by binding FMRP. Distinguishing between these and other possibilities will require long-term studies. Interestingly, the underlying mechanism appears to be restricted to certain cell types as shown by the fact that FMRP levels in cultured Drosophila D-Mel cells (a derivative of S2 cells) were not reduced by RNAi-mediated depletion of BicD (Bianco, 2010).

To investigate whether BicD also has a role in controlling FMRP motility, the distribution of residual FMRP::GFP was examined in BicD mutant primary cultured larval neurons. There was a strong decrease in the proportion of FMRP::GFP particles that localized to neurites in BicD mutants compared to wild-type, with FMRP particles also less likely to reach the most distal regions of the mutant processes. The changes in FMRP distribution are unlikely to result from differences in cellular morphology or general effects on trafficking processes because the length and complexity of neurites, as well as the distribution of mitochondria, was comparable in BicD mutant and wild-type neurons (Bianco, 2010).

To test directly whether BicD is required for FMRP motility, time-lapse imaging of FMRP::GFP particles was performed in cultured larval neurons. FMRP particles in wild-type neurons were usually stationary during several minutes of filming, but some occasionally underwent periods of rapid, directed movement. Motile particles in the processes exhibited persistent motion both toward and away from the cell body, with some particles rapidly switching directions. There was no overall bias in the length of directed, continuous movements (run lengths) toward and away from the cell body, consistent with a completely mixed microtubule polarity in both neurites and the soma (Bianco, 2010).

Consistent with BicD’s well-characterized role in dynein/ dynactin-mediated transport, inhibition of dynactin function by neuron-specific expression of a dominant-negative version of the p150Glued subunit (DGlued) strongly reduced the motility of FMRP puncta and their localization into neuronal processes. Despite the strong difference in efficiency of FMRP transport, the amounts of FMRP were indistinguishable between DGlued and wild-type extracts. This observation provides further evidence that the role of BicD in regulating FMRP protein levels is not due to a general effect of inhibiting transport (Bianco, 2010).

BicD is complexed with dynein and the plus end motor kinesin-1 on at least some bidirectional cargoes, and a kinesin-1 family member associates with FMRP complexes and contributes to their transport in mammalian neurons. In Drosophila primary neurons with a strong kinesin-1 heavy chain mutant genotype (Khc17/27), there was a striking alteration of FMRP appearance compared to wild-type cells, with discrete particles not detectable above the diffuse cytoplasmic signal. This observation, which is reminiscent of the reduced size of a kinesin-1 mRNP cargo in Drosophila oocytes, raises the possibility that both dynein and kinesin-1 cooperate in FMRP transport in Drosophila neurons (Bianco, 2010).

BicD may have a direct role as a constituent of FMRP:motor complexes. Alternatively, reduced levels of FMRP in BicD mutants might have an indirect effect by reducing the probability of FMRP encountering other transport factors. To attempt to discriminate between these possibilities, advantage was taken of the observation that overexpression of BicD, even to a very large extent, does not alter the total amount of FMRP. This presumably reflects wild-type levels of BicD being nonlimiting for the function in controlling FMRP levels (Bianco, 2010).

2-fold overexpression of BicD (tagged with mCherry) dramatically increased the run lengths and net displacements of motile FMRP::GFP particles in cultured neurons, compared to the wild-type. Run lengths in processes were similar for movements both toward and away from the cell body upon BicD overexpression. Nonetheless, there was increased targeting of FMRP into distal processes compared to wild-type. Once again, this presumably reflects the ability of long-distance, unbiased bidirectional transport to aid cargo spreading. These results demonstrate that BicD is able to control motility and subcellular localization of FMRP independently from the role in regulating overall levels of the protein (Bianco, 2010).

The results of quantification of particle motility, together with observations that (1) FMRP is recruited by means of the domain of BicD involved in linking cargoes to dynein and (2) FMRP colocalizes in motile particles with BicD in vivo, provides strong evidence that BicD plays a direct role in FMRP:motor complexes. In the case of other cargoes studied, BicD is not obligatory for their linkage to motor complexes but increases their travel distances significantly. The residual directed transport of FMRP particles in BicD mutant neurons suggests that BicD may play a similar stimulatory role in this context. Other components of FMRP-containing transport particles presumably also contribute to linkage with motor proteins (Bianco, 2010).

The functional significance of the BicD:FMRP interaction in dendrites was explored by focusing on the role of FMRP in dendritic morphogenesis. The well-characterized model system for dendritic development in the Drosophila third instar larva, the dorsal class IV dendritic arborization (da) neuron ddaC, was explored (Bianco, 2010).

Dorsal ddaC neurons within zygotic BicD mutant larvae had a much less extensively branched dendritic arbor than wildtype cells. A similar inhibition of the dendritic branching program was observed in three different zygotic Fmr1 null mutant genotypes. Intermediate terminal branching defects were also found in ddaC neurons heterozygous for Fmr1D50M. This phenotype, which could be suppressed by the FMRP::GFP transgene, underscores the importance of correct FMRP protein levels for neuronal morphogenesis (Bianco, 2010).

These results demonstrate that both BicD and FMRP are required for efficient branching of the dendritic arbor in dorsal ddaC neurons. Interestingly, FMRP negatively regulates dendritic elaboration in mushroom body neurons in adult brains. It has also previously been reported that mutating Fmr1 increases branching of ventral da neurons, although effects on specific classes of neurons within the cluster have not been reported. The differential requirements for Fmr1 in controlling the morphology of different neuronal cells is consistent with previous findings. It has been showed that Fmr1 mutations cause overextended axons in LNv cells but a failure of axon extension in DC neurons. Cell type-specific effects of FMRP on neuronal morphogenesis may reflect differences in the repertoire of its mRNA targets (Bianco, 2010).

BicD overexpression specifically in class IV da neurons significantly increased the number of dendritic branches in the distal regions of arbors in dorsal ddaC neurons compared to wild-type. This result, together with the diminished branching in BicD mutant neurons, reveals a correlation between the amount of available BicD and the degree of arborization of ddaC and that BicD can function autonomously within neurons to control this process (Bianco, 2010).

Strikingly, the ability of overexpressed BicD to augment dendritic branching of ddaC appears to be due predominantly to its interaction with FMRP, as evidenced by a strong suppression of the BicD overexpression phenotype in Fmr1 null mutants, with neuronal morphology not significantly different to the Fmr1 mutant alone. Because BicD overexpression does not alter FMRP protein levels, increased branching is likely to be influenced by BicD’s ability to control FMRP motility. Live cell imaging revealed that BicD promotes long-distance bidirectional transport of FMRP complexes on microtubules, thereby facilitating the exploration of neuronal processes. Such a mechanism may increase the probability of encounters of these complexes with factors that activate translation of associated mRNAs, which in some contexts could be responsive to local signaling. Nonetheless, the reduction of overall FMRP protein levels is highly likely to contribute to BicD loss-of-function phenotypes in da neurons, as potentially is the altered transport of FMRP-independent cargoes (Bianco, 2010).

Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein

Loss of FMR1 gene function results in fragile X syndrome, the most common heritable form of intellectual disability. The protein encoded by this locus (FMRP) is an RNA-binding protein that is thought to primarily act as a translational regulator; however, recent studies have implicated FMRP in other mechanisms of gene regulation. This study found that the Drosophila fragile X homolog (dFMR1) biochemically interacts with the adenosine-to-inosine RNA-editing enzyme dADAR. Adar and Fmr1 mutant larvae exhibit distinct morphological neuromuscular junction (NMJ) defects. Epistasis experiments based on these phenotypic differences revealed that Adar acts downstream of Fmr1 and that dFMR1 modulates dADAR activity. Furthermore, sequence analyses revealed that a loss or overexpression of dFMR1 affects editing efficiency on certain dADAR targets with defined roles in synaptic transmission. These results link dFMR1 with the RNA-editing pathway and suggest that proper NMJ synaptic architecture requires modulation of dADAR activity by dFMR1 (Bhogal, 2011).

Genetic and molecular findings lead to a proposal that modulation of dADAR activity by dFMR1 is important for NMJ synaptic architecture. The epistatic relationship of these two genes, the requirement of RNA editing by dADAR for normal NMJ morphology, and the genetic suppression of the Fmr1 loss-of-function NMJ defects all support a model in which dFMR1 affects the editing activity of dADAR. Molecular analyses of dADAR substrates support this prediction, since both loss and overexpression of dFMR1 was found to result in changes in editing efficiency in several dADAR-dependent editing sites. Although the changes in editing observed in Fmr1 mutant whole larvae were not large (for example, a ~15% change in editing was observed for lap), they are statistically significant, and it is proposed that these changes would likely be larger if analyses could be performed using mRNA prepared from isolated neurons or synapses rather than whole larvae. In addition, despite a few transcripts that were highly edited throughout development, dADAR function during developmental stages is relatively low compared with its high editing activity in pupal and adult stages. Thus, a larger effect of dFMR1 levels on dADAR substrates with lower efficiency editing sites cannot be ruled out (Bhogal, 2011).

In considering how dFMR1 affects editing, an important clue might come from the fact that both dFMR1 and dADAR are RNA-binding proteins that associate with secondary and higher order RNA structures. FMRP can bind to two separate complex RNA structures that are believed to allow for specificity of FMRP-associated transcripts: the RGG domain in the C terminus of FMRP protein interacts with an intramolecular G quartet stem loop RNA structure, whereas the KH2 domain associates with a complex tertiary kissing complex RNA structure. Similarly, the dADAR family of proteins contains several double-stranded RNA-binding domains and requires duplex RNA structures to identify, bind to and function on its target RNAs. The RNA structure required for dADAR activity, however, can vary from a simple hairpin to complex pseudoknot structures (Bhogal, 2011).

In addition, immunoprecipitation experiments indicate that dFMR1 and dADAR associate on common RNA targets. The dADAR-dFMR1 biochemical interaction was reduced through both a decrease in the amount of RNA in lysates using RNase A as well as by mutating the KH domains of dFMR1, suggesting that the ability for dFMR1 to bind to RNA is important for its association with dADAR. Molecular analyses of lap<.i> and <>Caα1D in the dFMR1 RNA-binding mutants further support this theory, although differential effects were observed with respect to the two transcripts analyzed. It is possible that dFMR1 associates with these two particular transcripts via different RNA-binding motifs. Although the analogous I307N mutation in mammals reduces FMRP's ability to associate with both poly(U)-rich sequences and large RNP complexes, FMRP can still bind to RNA, including transcripts containing G-quartet structures, through an intact RGG RNA-binding motif. Thus, it is proposed that the I244N and I307N mutations in dFMR1 reduce particular dFMR1-dADAR complexes associating with certain edited transcripts while concurrently enriching for dFMR1-dADAR complexes associating with the dFMR1 RGG box. Further studies delving into the importance of each RNA-binding domain in both dFMR1 and dADAR will give more insight into the biochemical and functional interaction between these two proteins (Bhogal, 2011).

On the basis of these results, it is predicted that dFMR1 and dADAR can associate in a common complex and converge on similar RNA substrates. Because the effect that dFMR1 has on the editing efficiency is context dependent, it is proposed that the association of dFMR1 with dADAR has no net positive or negative effect on the editing activity of dADAR, but instead maintains a balance of dADAR activity. At sites that demonstrate enhanced editing in the presence of dFMR1, dFMR1 could promote editing by either recruiting dADAR to the site via its own RNA-binding activity, or it could help form and/or stabilize RNA structures that create a site for editing by dADAR. At sites that are negatively affected by the presence of dFMR1, it is proposed that the RNA binding activity of dFMR1 interferes with the formation of a substrate for dADAR (Bhogal, 2011).

This analyses revealed several transcripts whose level of editing is regulated by the interaction between dFMR1 and dADAR; however, at this time, it is not known how many such transcripts are important for the proper formation of the NMJ. Although many identified dADAR targets encode for proteins that function in synaptic transmission at the NMJ34 and mutations in several dADAR substrates (for example, syt1, lap and unc-13) affect NMJ synaptic architecture and/or function, how editing is affecting the function of most of these gene products remains unknown. It is also important to note that a role for dFMR1 in translational regulation is already proposed to be important for proper NMJ development through its genetic interaction with the microtuble-associated protein homolog Futsch. Collectively, these studies suggest that both dADAR and dFMR1 have multifaceted roles at the NMJ (Bhogal, 2011).

In summary, this study found that dFMR1 physically and genetically interacts with dADAR-dependent RNA editing. This is the first report of a disease-associated protein that associates with and modulates A-to-I RNA editing. In addition, these findings reveal a previously unknown function for FMRP with respect to neuronal architecture and expand FMRP's predicted role as a translational regulator. Understanding all of the mechanisms by which FMRP functions to regulate synaptic development and function is essential to better understand the pathogenesis of the FXS symptoms, and consequently can lead to effective therapeutic treatments for people afflicted with this disease (Bhogal, 2011).

In vivo neuronal function of the fragile X mental retardation protein is regulated by phosphorylation

Fragile X syndrome (FXS), caused by loss of the Fragile X Mental Retardation 1 (FMR1) gene product (FMRP), is the most common heritable cause of intellectual disability and autism spectrum disorders. It has been long hypothesized that the phosphorylation of serine 500 (S500) in human FMRP controls its function as an RNA-binding translational repressor. To test this hypothesis in vivo, neuronally targeted expression of three human FMR1 transgenes, including wild-type (hFMR1), dephosphomimetic (S500A-hFMR1) and phosphomimetic (S500D-hFMR1), was employed in the Drosophila FXS disease model to investigate phosphorylation requirements. At the molecular level, dfmr1 null mutants exhibit elevated brain protein levels due to loss of translational repressor activity. This defect is rescued for an individual target protein and across the population of brain proteins by the phosphomimetic, whereas the dephosphomimetic phenocopies the null condition. At the cellular level, dfmr1 null synapse architecture exhibits increased area, branching and bouton number. The phosphomimetic fully rescues these synaptogenesis defects, whereas the dephosphomimetic provides no rescue. The presence of Futsch-positive (microtubule-associated protein 1B) supernumerary microtubule loops is elevated in dfmr1 null synapses. The human phosphomimetic restores normal Futsch loops, whereas the dephosphomimetic provides no activity. At the behavioral level, dfmr1 null mutants exhibit strongly impaired olfactory associative learning. The human phosphomimetic targeted only to the brain-learning center restores normal learning ability, whereas the dephosphomimetic provides absolutely no rescue. It is concluded that human FMRP S500 phosphorylation is necessary for its in vivo function as a neuronal translational repressor and regulator of synaptic architecture, and for the manifestation of FMRP-dependent learning behavior (Coffee, 2012).

FXS is caused solely by the loss of human FMRP. It has been widely hypothesized that the phosphorylation state of S500 acts as a 'switch' to transition human FMRP from an inactive to active state. This hypothesis predicts that human FMRP that cannot be phosphorylated will remain functionally inactive, equivalent to full protein loss, whereas a constitutively phosphorylated protein will be constantly active, but this has never been tested in vivo. To test this hypothesis, both a phosphomimetic (S500D-hFMR1) and a dephosphomimetic (S500A-hFMR1) were expressed in the well-characterized Drosophila FXS model (dfmr1 null mutant). Then functional in vivo rescue of a diverse range of null mutant phenotypes was tested. Specifically, core molecular and cellular phenotypes were assayed in diverse circuits in the neuromusculature and brain, as well as the core behavioral defect of learning impairment. The findings show that the phosphorylation of the S500 residue of human FMRP is necessary for protein function as a regulator of translation and modulator of synaptic connectivity, which, in turn, lays the foundation for normal behavioral output. The phosphomimetic, S500D-hFMR1, provides activity that restores normal function at all levels, to closely mimic the wild-type state. Since the phosphomimetic rescues the morphological defects seen in the dfmr1 null mutants, the data suggest that the excess growth may be due to elevated protein synthesis. In contrast, the dephosphomimetic, S500A-hFMR1, is incapable of providing any functional rescue and closely mimics dfmr1 null phenotypes at molecular, cellular and behavioral levels (Coffee, 2012).

FMRP is an mRNA-binding protein best characterized as a negative regulator of translation, although it may also activate translation in some cases. FMRP is present in stalled polyribosomes and inhibits the translation of mRNA targets. In the absence of FMRP, both single FMRP-target protein (e.g. Chickadee/Profilin) and total protein levels are elevated in the Drosophila brain, particularly acutely during the late developmental stages of synaptogenesis and early-use synaptic refinement. The mouse FMR1 knockout similarly exhibits increased protein synthesis in the brain. Phosphorylation mechanisms regulate activity-dependent protein synthesis. Phosphorylated FMRP preferentially associates with stalled polyribosomes, whereas non-phosphorylated FMRP associates with actively translating polyribosomes. Phosphorylation likely confers a protein-binding site conformational change that modulates ribosomal association. Although the molecular mechanism by which FMRP stalls ribosomes has not been elucidated, it is likely to be dynamic, as it can be acutely reversed by RNA decoys in run-off assays. This reversibility would most likely be modulated by FMRP phosphorylation, but could also involve FMRP degradation. Previously studies have shown that human FMRP is just as effective as the native fly protein in restraining brain protein expression, although neither of the human paralogs (FXR1, FXR2) provides any activity. Using targeted neuronal expression, this study shows that only the phosphomimetic (S500D-hFMR1) can restore the elevated single protein and total brain protein levels back to the wild-type condition in the Drosophila FXS model. Whereas S500D-hFMR1 is both necessary and sufficient for this inhibitory mechanism in neurons, S500A-hFMR1 is unable to provide any molecular function. This provides the first proof that S500 phosphorylation is an essential prerequisite for FMRP's function as a negative translational regulator in the in vivo brain (Coffee, 2012).

The hallmark cellular defect in FXS patients, as well as both murine and Drosophila disease models, is the over-proliferation of synaptic connections, many of which appear to be immature. Although most research has focused on the elevated number of postsynaptic dendritic spines, apposing presynaptic bouton specializations accumulate in parallel. In the Drosophila FXS model, both presynaptic boutons and postsynaptic dendrites are over-grown and over-elaborated in the absence of FMRP, and this has been demonstrated to be a FMRP cell-autonomous requirement within neurons. Previous studies of the well-characterized NMJ synaptic arbor have established a solely presynaptic requirement for FMRP in restraining terminal area, synaptic branching and synaptic bouton differentiation. Null dfmr1 synapses display increased terminal area, synaptic branching and supernumerary synaptic boutons. This work has demonstrated that only the phosphomimetic (S500D-hFMR1) is able to curb growth and restore normal synaptic architecture in the dfmr1 null mutant. In sharp contrast, the dephosphomimetic (S500A-hFMR1) does not possess this ability to any detectable degree. Thus, phosphorylation is required for the FMRP function in regulating synapse architecture (Coffee, 2012).

A defining feature of the overgrown synaptic connections arising in the absence of FMRP is that they appear structurally immature. For example, the dfmr1 null NMJ is characterized by the accumulation of mini/satellite boutons. These immature boutons represent a developmentally arrested state of an otherwise normal stage of bouton maturation. In the absence of FMRP, there is a ~50% increase in the number of structurally mature boutons, but a striking 8-10-fold elevation in the abundance of these immature satellite boutons. Only the transgenic introduction of hFMR1 and S500D-hFMR1 can overcome this developmental arrest, restoring the normal number of mature synaptic boutons and eliminating the accumulation of developmentally arrested satellite boutons. Dephosphorylated S500A-hFMR1, in contrast, exhibits no restorative activity in synaptic bouton differentiation or in alleviating the synaptogenic arrest. Thus, phosphorylation of human FMRP is absolutely required for the protein to regulate synaptogenesis (Coffee, 2012).

It was first shown that FMRP acts to translationally repress Futsch/MAP1B, and that dfmr1 null synaptic structure defects are rescued by restoring normal Futsch expression levels. At the Drosophila NMJ, Futsch binds microtubule loops in a subset of developing synaptic boutons. These Futsch-positive microtubule structures are proposed to regulate synaptic growth and bouton differentiation. In dfmr1 null mutants, there is an increased number of Futsch-positive loops throughout the overgrown synaptic arbor, and these supernumerary structures are eliminated by presynaptic FMRP expression. This current study shows a doubling in the number of Futsch loops in the absence of FMRP, compared with wild-type control. Only the transgenic introduction of hFMR1 and S500D-hFMR1 can overcome this Futsch elevation, restoring the normal number of Futsch-positive loops in mutant synapses. Dephosphorylated S500A-hFMR1, in contrast, exhibits no restorative activity. Thus, phosphorylation of human FMRP is absolutely required for the regulation of Futsch/MAP1B during synaptogenesis (Coffee, 2012).

In the Drosophila central brain, the clock circuit is particularly well characterized. Much attention has focused on the sLNv clock neurons, which secrete the neuropeptide PDF to regulate circadian rhythms. In dfmr1 null mutants, it has long been known that these neurons exhibit over-elaborated and over-extended synaptic arbors in the protocerebrum, a phenotype strikingly similar to the NMJ defect. Introduction of human FMRP can fully rescue this synaptic architecture defect. Moreover, only the phosphomimetic (S500D-hFMR1) is able to rescue the synaptic defect in the central brain. In contrast, the dephosphomimetic (S500A-hFMR1) has absolutely no effect on the null mutant phenotype. Thus, there is the same requirement for human FMRP phosphorylation in very distinctive neural circuits: in a peripheral motor circuit and in a central brain circuit. These results demonstrate for the first time the absolute requirement for FMRP phosphorylation to regulate synaptic connectivity in vivo (Coffee, 2012).

The hallmark of FXS is cognitive dysfunction learning disabilities. Consistently, both the mouse and Drosophila FXS genetic models manifest clear learning impairments. A key brain center of learning in Drosophila is the MB and dfmr1 null mutants have defects in MB organization (β lobe midline crossing) and synaptic connectivity. Consistently, previous work has shown that dfmr1 null mutants have significant defects in MB-dependent learning. Wild-type controls learn to move toward an odor not paired to electrical shock at a T-maze choice point, whereas dfmr1 nulls have strong deficits in this associative learning task. This study shows that MB-targeted expression of human FMRP rescues this defect, and that only the phosphomimetic (S500D-hFMR1) maintains this function. In contrast, the dephosphomimetic (S500A-hFMR1) has absolutely no effect on the null mutant phenotype. These results show that the FMRP functional requirement in learning is conserved from man to fly, that this requirement occurs within the learning circuit in the central brain and that phosphorylation of human FMRP at S500 is an absolute prerequisite for function in behavioral learning output. The current model is that the FMRP-mRNA complex at the synapse exists in a phosphorylated translationally repressed state until a signal, e.g., mGluR activation, triggers FMRP dephosphorylation leading to a burst of local translation. The data show that mRNAs are over-translated in the presence of an unphosphorylated form of FMRP (S500A-hFMRP), but that the phosphomimetic constitutively inhibits translation (Coffee, 2012).

FMRP was first shown to be a translation repressor by in vitro studies using recombinant FMRP in reticulocytes and oocyte. Given the current data showing FMRP phosphorylation is a prerequisite of this function, FMRP must have been introduced in a phosphorylated form or phosphorylated by native kinases present in these systems. It is not clear why S500 phosphorylation confers molecular function on FMRP, but it might modulate a protein-binding site that regulates ribosome association, or the ribosomal 'stalling' proposed to be caused by FMRP. Mouse FMRP is dynamically phosphorylated by ribosomal protein S6 kinase (S6K1) downstream of the mammalian target of rapamycin pathway, and dephosphorylated by the phosphatase PP2A. In murine hippocampal cultures, the non-phosphorylatable murine S499A-mFMR1 fails to associate with S6K1. In Drosophila, FMRP is phosphorylated in vitro by casein kinase II, although S6K1 might similarly be involved. Early work on mouse FMRP phosphomimetic and dephosphomimetic constructs (S499D and S499A, respectively) has strongly suggested that the phosphorylation state regulates translation repressor function. More recently, the loss of hippocampal S6K1 or introduction of S499A-FMRP has been shown to similarly elevate expression of SAPAP3 and PSD-95, both synaptic FMRP targets. The current study supports and expands on this work, showing a similar phosphorylation requirement for human FMRP in the broad context of the Drosophila FXS model. The new conclusion derived from this study is that synaptic mRNAs are similarly over-translated in the absence of FMRP or presence of unphosphorylated FMRP. Constitutively inhibiting translation with a phosphomimic closely resembles the wild-type state of dynamic regulation, suggesting that it is better to have less protein expression in the synapse than more (Coffee, 2012).

It is quite surprising that the constitutive phosphorylation mimicry achieved by human FMRP S500D is quite adequate to recapitulate wild-type FMRP function in all molecular, cellular and behavioral assays pursued in this study. In vivo FMRP is dynamically phosphorylated and dephosphorylated -- shuttling between a functional and non-functional form -- in an activity-dependent mechanism. It was just recently shown that this reversibility is controlled by receptor-stimulated dephosphorylation of FMRP, which removes translational repression. Why then does the S500D transgene not produce gain-of-function phenotypes, or simply fail to function? Perhaps animals expressing the FMRP phosphomimetic develop an adaptive mechanism to manage the constitutive activation induced by the phosphorylation state of the transgenic protein. FMRP is acutely degraded upon synaptic stimulation, and so one possibility is that increased FMRP degradation after synaptic stimulation releases the critical subset of mRNAs from translation repression. Another possibility is that even though there is constitutively mimicked upregulation of the FMRP phosphorylated state, the phosphomimetic may not yield activation comparable with native phosphorylation, but rather more partial phosphorylation mimicry. Experimentally, while this is the best available mimic condition, it is not phosphorylation per se, but rather substitution of a phosphate group with a negatively charged aspartic acid residue. Thus, the phosphomimetic may enable partial function resembling an averaged state between the normal dynamic conformations of phosphorylation and dephosphorylation, thereby rescuing near the wild-type level. Of course, this explanation does not adequately address the need for a dynamic 'switch', which seems dispensable based on all the molecular, cellular and behavioral studies presented in this study. However, it is likely an oversimplification to assume that all FXS phenotypes result from constitutive excess protein synthesis. The loss of stimulus-induced translation in FXS may underlie other phenotypes not studied, such as the ability of synapses to rapidly remodel structure and function in a manner dependent on the synthesis of new proteins (Coffee, 2012).

FMRP and Ataxin-2 function together in long-term olfactory habituation and neuronal translational control

Fragile X mental retardation protein (FMRP) and Ataxin-2 (Atx2) are triplet expansion disease- and stress granule-associated proteins implicated in neuronal translational control and microRNA function. This study shows that Drosophila FMRP (dFMR1) is required for long-term olfactory habituation (LTH), a phenomenon dependent on Atx2-dependent potentiation of inhibitory transmission from local interneurons (LNs) to projection neurons (PNs) in the antennal lobe. dFMR1 is also required for LTH-associated depression of odor-evoked calcium transients in PNs. Strong transdominant genetic interactions among dFMR1, atx2, the deadbox helicase me31B, and argonaute1 (ago1) mutants, as well as coimmunoprecitation of dFMR1 with Atx2, indicate that dFMR1 and Atx2 function together in a microRNA-dependent process necessary for LTH. Consistently, PN or LN knockdown of dFMR1, Atx2, Me31B, or the miRNA-pathway protein GW182 increases expression of a Ca2+/calmodulin-dependent protein kinase II (CaMKII) translational reporter. Moreover, brain immunoprecipitates of dFMR1 and Atx2 proteins include CaMKII mRNA, indicating respective physical interactions with this mRNA. Because CaMKII is necessary for LTH, these data indicate that fragile X mental retardation protein and Atx2 act via at least one common target RNA for memory-associated long-term synaptic plasticity. The observed requirement in LNs and PNs supports an emerging view that both presynaptic and postsynaptic translation are necessary for long-term synaptic plasticity. However, whereas Atx2 is necessary for the integrity of dendritic and somatic Me31B-containing particles, dFmr1 is not. Together, these data indicate that dFmr1 and Atx2 function in long-term but not short-term memory, regulating translation of at least some common presynaptic and postsynaptic target mRNAs in the same cells (Sudhakaran, 2013).

Observations presented in this study lead to three significant insights into the endogenous functions of dFmr1 and Atx2 in the nervous system and their contribution to long-term synaptic plasticity. First, the data strongly indicate that both proteins function in the same pathway, namely translational control, to mediate the form of long-term memory analyzed in this study. Second, the remarkably similar effects of knocking down these proteins in LNs and PNs provide in vivo support for an emerging idea that translational control of mRNAs in both presynaptic and postsynaptic compartments of participating synapses is necessary for long-term synaptic plasticity. Finally, although both dFmr1 and Atx2 have isoforms containing prion-like, Q/N domains, the different effects of loss of Atx2 and dFmr1 on neuronal Me31B aggregates indicate important differences in the mechanisms by which the two proteins function in translational control (Sudhakaran, 2013).

The different molecular and clinical consequence of pathogenic mutations in FMRP and Atx2 encoding genes has led to largely different perspectives on their functions. Fragile X causative mutations cause reduced levels of the encoding mRNA and lower levels of FMRP, leading to increased protein synthesis and a range of pathologies evident in children and young adults. These pathologies importantly do not include the formation of inclusion bodies. In contrast, SCA-2 and amyotrophic laterosclerosis causative mutations in Atx2 result in the dominant formation of inclusion body pathologies and age-dependent degeneration of the affected neuronal types. Observations made in this article indicate that the distinctive pathologies of the two diseases have obscured common molecular functions for the two proteins in vivo (Sudhakaran, 2013).

The genetic, behavioral, and biochemical observations show (1) shared roles of the two proteins in olfactory neurons for long-term but not short-term habituation, and (2) striking transdominant genetic interactions of dfrm1 and atx2 mutations with each other as well as with miRNA pathway proteins, which is not only consistent with prior genetic and behavioral studies of the two respective proteins but also strongly indicative of a common role for the two proteins in translational repression of neuronal mRNAs. This conclusion is supported at a mechanistic level by (3) the finding that both proteins are required for efficient repression mediated by the 3' UTR of CaMKII, a 3' UTR that this study shows to be repressed by the miRNA pathway, and (4) strong evidence for in vivo biochemical interaction among dFmr1 and Atx2 and for binding of these regulatory proteins with the UTR of the CaMKII transcript that they jointly regulate. Thus, dFMR1 and Atx2 function with miRNA pathway proteins for the regulation of a dendritically localized mRNA in identified olfactory neurons (Sudhakaran, 2013).

An unexpected observation was that dFMR1 and Atx2 seemed to be necessary for LTH as well as for CaMKII reporter regulation in both inhibitory LNs and excitatory PNs of the antennal lobe (Sudhakaran, 2013).

Until recently mammalian FMRP was regarded as a postsynaptic protein, consistent with the view that translational control of mRNAs essential for long-term plasticity occurs exclusively in postsynaptic dendrites. In contrast, work in Aplysia indicated that translational control of mRNAs is required in presynaptic terminals for long-term synaptic plasticity. This conflict between vertebrate and invertebrate perspectives is beginning to be resolved by findings that (1) mammalian FMRP is present in axons and presynaptic terminals; and that (2) translational control of both presynaptic and postsynaptic mRNAs is essential for long-term plasticity of cultured Aplysia sensorimotor synapses (Sudhakaran, 2013 and references therein).

Prior studies at the Drosophila neuromuscular junction have strongly indicated presynaptic functions for dFmr1 and translational control but have also pointed to their significant postsynaptic involvement in neuromuscular junction maturation, growth, and plasticity. More direct studies of experience-induced long-term plasticity have been performed in the context of Drosophila olfactory associative memory, wherein a specific dFmr1 isoform in particular and translational control in general are necessary for long-term forms of memory. However, the incomplete understanding of the underlying circuit mechanism has made it difficult to conclude presynaptic, postsynaptic, or dual locations for dFmr1 function in long-term memory. In contrast, recent work showing an essential role for Atx2 and Me31B in PNs for LTH more strongly indicate a postsynaptic requirement for translational control mediated by these proteins; however, this did not address a potential additional presynaptic function (Sudhakaran, 2013).

The finding that dFmr1 and Atx2 are necessary in both LNs and PNs for LTH, a process driven by changes in the strength of LN–PN synapses, provides powerful in vivo support for a consensus model in which translational control on both sides of the synapse is necessary for long-term plasticity. A formal caveat is that the anatomy of LN–PN synapses in Drosophila antennal lobes remains to be clarified at the EM level. If it emerges that these are reciprocal, dendrodendritic synapses, similar to those between granule and mitral cells in the mammalian olfactory bulb, then a clear assignment of the terms 'presynaptic' and 'postsynaptic' to the deduced activities of dFmr1 and Atx2 in this context may require further experiments (Sudhakaran, 2013).

Previous studies in Drosophila have indicated a broader role for Atx2 than dFmr1 in miRNA function in nonneuronal cells. Although Atx2 is necessary for optimal repression of four miRNA sensors examined in wing imaginal disk cells, dFmr1 is not necessary for repression of any of these sensors. The resulting conclusion that dFmr1 is required only for a subset of miRNAs to function in context of specific UTRs is consistent with the observation that only a subset of neuronal miRNAs associate with mammalian FMRP and that the protein shows poor colocalization with miRNA pathway and P-body components in mammalian cells. Parallel studies have shown that Atx2 in cells from yeast to man is required for the formation of mRNP aggregates termed stress granules, which in mammalian cells also contain Me31B/RCK and FMRP. In addition, biochemical interactions between these proteins and their mammalian homologs with each other as well as with other components of the miRNA pathway have been reported. However, neither the mechanisms of Atx2-driven mRNP assembly, nor the potential role for FMRP in such assembly, have been tested in molecular detail (Sudhakaran, 2013).

The demonstration that loss of Atx2 in neurons results in a substantial depletion of Me31B-positive foci in PN cell bodies and in dendrites is consistent with Atx2 being required for the assembly of these two different (somatic and synaptic) in vivo mRNP assemblies. Thus, the mechanisms that govern their assembly, particularly of synaptic mRNPs in vivo, overlap with mechanisms used in P-body and stress granule assembly in nonneuronal cells (Sudhakaran, 2013).

The finding that loss of dFmr1 has no visible effect on these Me31B-positive foci can be explained using either of two models. A simple model is that dFmr1 is not required for mRNP assembly, a function mediated exclusively by Atx2. This would suggest that Atx2 contains one or more functional domains missing in dFmr1 that allow the multivalent interactions necessary for mRNP assembly. This is most consistent with the observation that that although dFMR1 is a component of stress granules in Drosophila nonneuronal cells, it is not required for their assembly. An alternative model would allow both dFmr1 and Atx2 to mediate mRNP assembly but posit that dFmr1 is only present on a small subset of mRNPs, in contrast to Atx2, which is present on the majority. In such a scenario, loss of dFmr1 would only affect a very small number of mRNPs, too low to detect using the microscopic methods used in this study. In the context of these models, it is interesting that both dFmr1 and Atx2 contain prion-like Q/N domains, potentially capable of mediating mRNP assembly. It is to be noted here that the dFmr1 Q/N domain, although lacking prion-forming properties, is capable of serving as a protein interaction domain enabling the assembly of dFmr1 into RNP complexes. This observation would support the view that dFmr1 may be involved in the formation of only a subset of cellular mRNP complexes. Future studies that probe the potential distinctive properties of these assembly domains may help discriminate between these models. In addition, potential interaction of Atx2 with other proteins that are involved in mRNP formation across species, like Staufen, could help to understand the mechanisms behind Atx2-dependent function in mRNP assembly (Sudhakaran, 2013).

However, the observations presented in this study clearly show that despite the remarkable similarities in the roles of dFmr1 and Atx2 for repression of CaMKII expression at synapses and the control of synaptic plasticity that underlies long-term olfactory habituation, both proteins also have distinctive molecular functions in vivo (Sudhakaran, 2013).

Mutations that affect neuronal translational control are frequently associated with neurological disease, particularly with autism and neurodegeneration. Although these clinical conditions differ substantially in their presentation, a broadly common element is the reduced ability to adapt dynamically to changing environments, a process that may require activity-regulated translational control at synapses. Taken together with others, the observations of this study suggest that there may be two routes to defective activity-regulated translation. First, as in dFmr1 mutants, the key mRNAs are no longer sequestered and repressed, leading to a reduced ability to induce a necessary activity-induced increase in their translation. Second, it is suggested that increased aggregation of neuronal mRNPs (indicated by the frequent occurrence of TDP-43 and Atx2-positive mRNP aggregates in neurodegenerative disease) may result in a pathologically hyperrepressed state from which key mRNAs cannot be recruited for activity-induced translation. Thus, altered activity-regulated translation may provide a partial explanation not only for defects in memory consolidation associated with early-stage neurodegenerative disease but also for defects in adaptive ability seen in autism spectrum disorders (Sudhakaran, 2013).

Fragile X mental retardation protein regulates translation by binding directly to the ribosome

Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused by loss of function of the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is involved in the translational regulation of several neuronal mRNAs. However, the precise mechanism of translational inhibition by FMRP is unknown. This study shows that FMRP inhibits translation by binding directly to the L5 protein on the 80S ribosome. Furthermore, cryoelectron microscopic reconstruction of the 80S ribosomeFMRP complex shows that FMRP binds within the intersubunit space of the ribosome such that it would preclude the binding of tRNA and translation elongation factors on the ribosome. These findings suggest that FMRP inhibits translation by blocking the essential components of the translational machinery from binding to the ribosome (Chen, 2014).

FMR1 encodes an RNA binding protein, fragile X mental retardation protein (FMRP) that is highly expressed in the brain and FMRP appears to regulate the expression of many proteins throughout the brain. FMRP has three RNA-binding domains: one RGG domain that is rich in arginines and glycines and two hnRNP K homology domains (KH domains). Consistent with its proposed role in regulating protein synthesis, the majority of FMRP in the cell is associated with polyribosomes. Interestingly, a missense mutation in the KH2 domain (Ile304Asn of human FMRP) abolishes the binding of FMRP to polyribosomes and causes an aggravated form of FXS in humans. This suggests that RNA binding by FMRP plays a key functional role in the brain. In vitro selection experiments identify a G-quadruplex structure and a pseudoknot structure as the potential RNA ligands for the RGG and KH2 domains, respectively. Based on these results it is proposed that FMRP may bind to mRNAs that possess G-quadruplex- or pseudoknot-forming sequences and repress their translation. Additionally, many proteins, microRNAs and noncoding RNAs have been proposed to be important for translational repression by FMRP (Chen, 2014).

To understand the mechanism of translational inhibition by FMRP, this study used the Drosophila FMRP (dFMRP) homolog, in which the RNA-binding domains are nearly 75% identical to human FMRP. Both the full-length and a N-terminally truncated dFMRP (NT-dFMRP) were purified and an in vitro translation system (IVTS) made from Drosophila embryo extract was used to test the activity of dFMRP. Renilla luciferase mRNA was used as the reporter for protein synthesis because it has three G-rich sequences that potentially form G-quadruplex structures, and additionally has 7 ACUK and 6 WGGA sequences. The time course of protein synthesis was monitored by bioluminescence. The addition of dFMRP or NT-dFMRP to the IVTS inhibits the synthesis of luciferase. NT-dFMRP was used in further studies because it is equally active in inhibiting translation as the full-length protein and easier to purify than the full-length dFMRP (Chen, 2014).

Titration experiments show that the inhibition of translation depends on the concentration of NT-dFMRP added to the IVTS. NT-dFMRP also inhibits the translation of luciferase mRNAs that do not have a N7-methyl guanosine cap at the 5′ end or a 3′ poly(A) tail, indicating that translation inhibition is 5′ cap and poly(A) tail independent. To confirm that the inhibition of translation by NT-dFMRP is 5′ cap-independent, uncapped luciferase mRNA with an internal ribosome entry site (IRES) was synthesized from the Reaper mRNA at the 5′ end. IRES-dependent translation of luciferase mRNA is as efficient as the translation with the 5′ capped mRNA. NT-dFMRP inhibits the translation of luciferase mRNA having the IRES element, confirming that the 5′ cap is not essential for inhibition. These results also suggest that FMRP does not affect the initiation step of protein synthesis (Chen, 2014).

Previous studies suggest that FMRP associates directly with the ribosome. However, other reports show that FMRP binds to the ribosome via the mRNA or as an mRNP complex. It is not clear whether mRNA or other components are required for the association of FMRP with the ribosome. By using gel filtration chromatography and SDS-PAGE, it was shown that NT-dFMRP can indeed bind directly to the ribosome in the absence of mRNA. Furthermore, the binding of NT-dFMRP to the ribosome is stoichiometric even though excess amount of NT-dFMRP is present in the binding reaction. Next, the binding of NT-dFMRP with functionally relevant mutations in the KH1 (I244N) or KH2 (I307N) domains was tested. The KH1 mutant shows a 2-fold reduced binding to the 80S ribosome, while the KH2 mutant binds to a similar extent as NT-dFMRP. The binding results are consistent with functional data, which show that the KH1 domain is important for translational inhibition by NT-dFMRP (Chen, 2014).

A cryo-EM map of the Drosophila 80S ribosome•NT-dFMRP complex was obtained to determine the three-dimensional (3D) binding position of NT-dFMRP on the ribosome. Subtraction of the 3D map of the control Drosophila 80S ribosome from that of the 80S ribosome•NT-dFMRP complex shows an elongated mass of density, within the ribosomal inter-subunit space, that spans from central protuberance (CP) to α-sarcin/ricin stem-loop (SRL) region of the 60S subunit. One end of the elongated difference mass interacts with the CP and A-site finger (ASF) of the 60S subunit, while its other end is situated between the protein S12 region of the small (40S) subunit and SRL region of the 60S subunit. Cross-linking data suggests that the N-terminus of the NT-dFMRP construct interacts with the CP protein L5; consequently, that portion of the difference map is assigned to the N-terminus, and the portion between S12 and SRL is tentatively assigned to the C-terminus domain of FMRP. Both the crosslinking and cryo-EM results agree with a previous tandem affinity purification analysis of dFMRP from a cytoplasmic lysate, which showed that FMRP could interact with ribosomal proteins L5 and L18, both located in the CP of the 60S subunit. Indeed, a direct interaction of NT-dFMRP is observed with protein L5. The previous interaction reported with protein L18 could involve the N-terminus of the full-length FMRP that is absent in the construct used in this study. Docking of an I-TASSER homology model of NT-dFMRP into the corresponding cryo-EM map density tentatively places its KH1 and KH2 domains interacting with the CP and ASF, respectively, of the 60S subunit. This region of the 60S subunit would normally be occupied by a tRNA in the peptidyl site (P site) during protein synthesis. Superimposition of the ribosome-bound FMRP and previously known binding position of the tRNA at the ribosomal P site indicates that the KH1 and KH2 domains of FMRP would partially overlap with the anticodon arm of the tRNA. However, future structural studies with a translationally inhibited ribosome•FMRP complex carrying a tRNA in the P site will be essential to understand if and how both the P-site tRNA and FMRP would be accommodated simultaneously on the ribosome (Chen, 2014).

Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation

RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). This study identified Drosophila Fragile X Mental Retardation Protein (dFMRP) as a robust genetic modifier of TDP-43 dependent toxicity in a Drosophila model of ALS. dFMRP overexpression mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with PolyA Binding Protein (PABP) in a variant dependent manner. Furthermore, dFMRP dosage modulates TDP-43 solubility and molecular mobility with overexpression of dFMRP resulting in a significant reduction of TDP-43 in the aggregate fraction. Polysome fractionation experiments indicate that dFMRP overexpression also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP overexpression mitigates Futsch dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. These data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs (Coyne, 2015).

This study used a combination of genetic and molecular approaches to uncover a novel functional interaction between dFMRP and TDP-43. Taken together, the results support a model whereby dFMRP, a well established translational regulator, can modulate the neurotoxicity caused by TDP-43 overexpression. When overexpressed, dFMRP decreases the association of TDP-43 with the aggregate-like fraction. Together with immunoprecipitation and binding experiments, these findings support a model whereby dFMRP promotes the remodeling of the RNP by 'extracting' TDP-43 and freeing the sequestered mRNA from the protein-RNA complex. This in turn may alleviate the negative impact that TDP-43 exerts on its mRNA targets as is the case for futsch mRNA. Indeed, dFMRP OE in the context of TDP-43 restores the expression of futsch, which is a translation target of TDP-43. While the change in Futsch expression is slight in magnitude, it is statistically significant. These findings suggest a scenario whereby the robust synaptic phenotypes observed in ALS may result from the combinatorial effect of decreased expression for multiple TDP-43 targets at the NMJ. In future studies it will be interesting to determine additional synaptic targets of TDP-43 whose expression is restored upon dFMRP OE. While futsch mRNA can be translationally controlled by both dFMRP and TDP-43, in the context of TDP-43 RNA granules, dFMRP appears to favor an association with TDP-43 protein over its translation target, leaving futsch mRNA available for protein synthesis, which explains the translation restoration observed in the context of dFMRP overexpression. Given the wide repertoire of RNA binding protein partners of TDP-43, it will be interesting in the future to determine whether others can also confer neuroprotection to TDP-43 dependent toxicity and whether they do so by a similar molecular mechanism. This would be expected given that Futsch expression is significantly increased but not fully restored by dFMRP OE at the NMJ (Coyne, 2015).

Previous studies have shown that TDPWT and disease linked mutations, although expressed at comparable levels, confer differential toxicity in various phenotypic assays. This study provides evidence that TDPWT and TDPG298S also interact differentially with protein partners. TDPG298S colocalizes with PABP to a lesser extent than TDPWT. Further evidence is provided that TDPWT and TDPG298S exhibit distinct molecular mobilities within neurites, which is consistent with previous reports that although wild-type and disease linked variants both associate with stress granules, their dynamics, persistence and size differ dramatically (Coyne, 2015).

Taken together, these findingssuggest that ALS may be a consequence of chronic translation inhibition. This could result from dysregulation of RNA granule physiology in the context of excess cellular stress as previously suggested. This scenario is consistent with previous findings that inhibition of SG is neuroprotective and provides a plausible mechanism for how TDP-43 mutations lead to disease. Additionally, it can explain the association of wild-type TDP-43 with cytoplasmic aggregates in the majority of ALS cases, regardless of etiology. One possibility is that, in the context of aging related or other cellular stress, wild-type TDP-43 enters the RNA stress granule cycle, contributing to translation inhibition and disease pathophysiology (Coyne, 2015).

The results indicate that FMRP remodels TDP-43 RNP granules and this restores futsch translation and expression at the NMJ. This in turn, can alleviate phenotypes associated with microtubule instability such as the presence of satellite boutons. Altered microtubule stability is emerging as a prominent pathological mechanism underlying the progression of ALS and may provide a useful avenue for the development of therapeutics. In addition, altered ribostasis has emerged as a major hypothesis for explaining the progression from RNA stress granules to aggregates seen in disease. This model suggests altered translational regulation as a molecular mechanism underlying disease progression. The current results support this model and provide evidence that mitigating translational repression can suppress disease phenotypes (Coyne, 2015).

In future studies it will be important to establish whether blanket approaches such as RNA SG inhibition or translation restoration offer more promise than targeted strategies based on specific targets. Two recent studies have shown that TDP-43 suppresses toxicity in CGG repeat expansion models of Fragile X associated tremor/ataxia syndrome (FXTAS). Removing a portion of the C-terminus of TDP- 43 in which interactions with hnRNP A2/B1 typically occur, abolishes the ability of TDP-43 to suppress toxicity. These results suggest that TDP-43 may work to mitigate CGG RNA toxicity via interactions with its protein partners by preventing them from sequestration into toxic RNA foci. Thus, in the case of CGG repeat disorders, TDP-43 may alter RNP complexes similar to how dFMRP overexpression alters RNP complexes in the TDP-43 model of ALS. Together with these studies, the current results provide evidence for common mechanisms underlying neurodegenerative diseases and repeat expansion disorders. In both cases, remodeling of RNP granules and the 'freeing' of RNA binding proteins or mRNA targets mitigates toxicity (Coyne, 2015).

In conclusion, this study identified a novel strategy for mitigating TDP-43 dependent phenotypes in vivo, based on FMRP mediated remodeling of RNA granules, which provides relief to chronic translation inhibition for specific mRNA targets such as futsch. The results suggest that targeting RNP remodeling or translation restoration may prove useful as therapeutic strategies. Future experiments are aimed at identifying additional translational targets of TDP-43 in vivo that will broaden the repertoire of therapeutic strategies for ALS and related neurodegenerative diseases (Coyne, 2015).

Wan, D., Zhang, Z. C., Zhang, X., Li, Q. and Han, J. (2015). X chromosome-linked intellectual disability protein PQBP1 associates with and regulates the translation of specific mRNAs. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26002102

X chromosome-linked intellectual disability protein PQBP1 associates with and regulates the translation of specific mRNAs

X chromosome-linked intellectual disability is a common developmental disorder, and mutations of the polyglutamine-binding protein 1 (PQBP1) gene have been linked to this disease. In addition to existing in the nucleus as a splicing factor, PQBP1 is also found in cytoplasmic RNA granules, where it associates with RNA-binding proteins. However, the roles of cytoplasmic PQBP1 are largely unknown. This study showed that the Drosophila homolog of PQBP1 (dPQBP1) is present in the cytoplasm of photoreceptor cells, and its loss results in defective rhabdomere morphogenesis, which is due to impaired Chaoptin translation. This study also showed that dPQBP1 regulates mRNA translation by interacting with dFMR1, which binds to specific mRNAs and facilitates their assembly into translating ribosomes, a function that is conserved for human PQBP1 and FMRP. These findings reveal the conserved function of PQBP1 in mRNA translation and provide molecular insights into the pathogenic mechanisms underlying Renpenning syndrome (Wan, 2005).

A novel link between FMR gene and the JNK pathway provides clues to possible role in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive form of thoracic cancer with poor prognosis. While some studies have identified the molecular alterations associated with MPM, little is known about their role in MPM. For example, fragile X mental retardation (FMR) gene is up-regulated in MPM but its role in MPM is unknown. Utilizing Drosophila genetics, this study investigated the possible role FMR may be playing in MPM. Evidence is provided that suggests that FMR may contribute to tumorigenesis by up-regulating a matrix metalloprotease (MMP) and by degrading the basement membrane (BM), both important for tumor metastasis. A novel link between FMR and the JNK pathway was demonstrated and it was suggested that the effects of FMR in MPM could in part be mediated by up-regulation of the JNK pathway (Srivastava, 2015).

Zfrp8 forms a complex with fragile-X mental retardation protein and regulates its localization and function

Fragile-X syndrome is the most commonly inherited cause of autism and mental disabilities. The Fmr1 (Fragile-X Mental Retardation 1) gene is essential in humans and Drosophila for the maintenance of neural stem cells, and Fmr1 loss results in neurological and reproductive developmental defects in humans and flies. FMRP (Fragile-X Mental Retardation Protein) is a nucleo-cytoplasmic shuttling protein, involved in mRNA silencing and translational repression. Both Zfrp8 and Fmr1 have essential functions in the Drosophila ovary. This study identifies FMRP, Nufip (Nuclear Fragile-X Mental Retardation Protein-interacting Protein) and Tral (Trailer Hitch) as components of a Zfrp8 protein complex. Zfrp8 is required in the nucleus, and controls localization of FMRP in the cytoplasm. In addition, Zfrp8 genetically interacts with Fmr1 and tral in an antagonistic manner. Zfrp8 and FMRP both control heterochromatin packaging, also in opposite ways. It is proposed that Zfrp8 functions as a chaperone, controlling protein complexes involved in RNA processing in the nucleus (Tan, 2016).

Stem cell maintenance is essential for the generation of cells with high rates of renewal, such as blood and intestinal cells, and for the regeneration of many organs such as the brain and skin. Previous work has shown that Zfrp8 is essential for maintaining hematopoietic, follicle, and germline stem cells (GSCs) in Drosophila melanogaster. Knockdown (KD) of Zfrp8 in GSCs results in the loss of stem cell self-renewal, followed by the eventual loss of all germline cells. Similarly in vertebrates, the Zfrp8 homolog, Pdcd2, is essential for embryonic stem cell maintenance and the growth of mouse embryonic fibroblasts; Pdcd2 mouse embryos die before implantation. PDCD2 is abundantly expressed and essential in highly proliferative cells including cultured cells and clinical isolates obtained from patients with hematologic malignancies. The function of Zfrp8 and PDCD2 is highly conserved, as expression of transgenic PDCD2 is sufficient to rescue Zfrp8 phenotypes. Zfrp8 directly binds to Ribosomal Protein 2 (RpS2), a component of the small ribosomal subunit (40S), controls its stability and localization, and hence RNA processing. Zfrp8 also interacts with the piRNA pathway, which is conserved throughout all metazoans and is also essential for the maintenance of GSCs (Tan, 2016).

The piRNA pathway functions in maintaining heterochromatin stability and regulating the expression levels of retrotransposons. Both processes are thought to occur through piRNA targeting of chromatin modifying factors to the DNA. Guided by piRNAs, the piRNA pathway protein Piwi and associated proteins can set repressive epigenetic modifications to block transcription of nearby genes. Levels of transposon transcripts are also controlled by cytoplasmic PIWI-piRNA complexes, which can bind complementary mRNAs and mark them for translational repression and degradation (Tan, 2016).

Fragile-X Mental Retardation Protein (FMRP) functions as a translational repressor involved in RNA silencing. FMRP is a Piwi interactor and part of the piRNA pathway. FMRP-deficient animals display phenotypes similar to piRNA pathway mutants including genomic instability and de-repression of retrotransposons. While FMRP is predominantly localized within the cytoplasm, FMRP complexes have also been demonstrated within the nucleus. In Xenopus, FMRP has been shown to bind target mRNAs co-transcriptionally in the nucleus. Like Zfrp8, FMRP has been shown to bind ribosomal proteins prior to nuclear export. In the cytoplasm, the FMRP-containing RNP complex controls mRNAs stability, localization, and miRNA-dependent repression. FMRP mRNA targets are not well defined, as different studies show low overlap of putative targets in neuronal tissues (Tan, 2016).

In Drosophila, FMRP is required to maintain GSCs, and loss of Fmr1 is associated with infertility and developmental defects in oogenesis and neural development. Fmr1, the gene encoding FMRP, is essential in both vertebrates and Drosophila for the maintenance of neural stem cells (NSCs). In humans, loss of FMRP is associated with Fragile X-associated disorders, which cover a spectrum of mental, motor, and reproductive disabilities. Fragile X-associated disorders are the most commonly inherited cause of mental disabilities and autism. In vertebrates, FMRP physically interacts in the nucleus with NUFIP1 (Nuclear FMRP-Interacting Protein 1), a nucleo-cytoplasmic shuttling protein involved in ribonucleoprotein (RNP) complex formation. NUFIP1 is found in the nucleus in proximity to nascent RNA, and in the cytoplasm associated with ribosomes. In the cytoplasm, FMRP co-localizes and associates with Trailer Hitch (Tral) to form a translational repressor complex. The Tral complex contains a number of translational repressor proteins, which together control the initiation of translation and the stability of mRNAs, such as gurken (grk). In Drosophila, loss of Tral causes ovary phenotypes similar to piRNA pathway mutants, including oocyte polarity defects and transposon activation (Tan, 2016).

This study has identified Zfrp8 interactors by performing a yeast-two hybrid screen, and also by analyzing the components of the Zfrp8 complex by mass spectrometry. The nature of the proteins in the Zfrp8 complex indicates that it is involved in mRNA metabolism and translational regulation. Zfrp8, Nufip, FMRP, and Tral are all part of the complex, and Zfrp8 interacts antagonistically with Fmr1 and tral, suppressing their oogenesis defects. Furthermore, it was determined that Zfrp8 is required within the nucleus, and controls FMRP localization within the cytoplasm. It was further confirmed that FMRP functions in heterochromatin silencing and that Zfrp8 is required in the same process, but has an opposite function of FMRP. It is proposed that Zfrp8 functions as a chaperone of the FMRP’ containing RNP translational repression complex and controls the temporal and spatial activity of this complex (Tan, 2016).

Zfrp8 is essential for stem cell maintenance, but its molecular functions have not yet been clearly defined. Two distinct approaches were taken to address this question. A yeast-two hybrid screen was performed to identify direct interactors of Zfrp8, and the components of the Zfrp8 complex were characterized by mass spectrometry (Tan, 2016).

Because of the high sequence and functional conservation of Zfrp8 (flies) and PDCD2 (mammals), and because no stem cell-derived cDNA library exists in Drosophila, a mouse embryonic stem cell cDNA library was screened using mammalian PDCD2 as bait. Forty-six initial positives were isolated, and 19 potential interactors were identified after re-testing of the positives (Tan, 2016).

In order to purify the Zfrp8 protein complex a transgenic line was established expressing NTAP-tagged Zfrp8 under the control of the general da-Gal4 (daughterless) driver. Two-step tandem affinity purification was performed on embryonic extracts and the purified proteins were separated by SDS-PAGE electrophoresis. The proteins were eluted and analyzed by mass spectrometry. Thirty proteins were identified as part of the Zfrp8 complex. The threshold for interactors was set to at least 5x peptide enrichment in Zfrp8 over vector control fractions. Eighteen of the proteins are predicted to function in ribosomal assembly or translational regulation, strongly suggestive of a function of Zfrp8 in mRNA processing (i.e. translation, localization, and stability). In the complex six ribosomal subunits were found (five 40S subunits and one 60S subunit); EF2 and eIF-4a, which are required for translation initiation and elongation; and FMRP, Tral and Glorund which function in mRNA transport and translational repression. While Zfrp8 interacts with several ribosomal proteins it does not appear to be part of the ribosome itself (Tan, 2016).

No overlapping interactors were found in the yeast-two hybrid screen and mass spectrometry assay. But interestingly, FMRP was identified as part of the Zfrp8 complex by mass spectrometry and NUFIP1 in a yeast-two hybrid assay. Most likely Nufip (estimated 57 kD) was not identified as part of the Zfrp8 complex in the TAP-purification approach, because proteins with similar size to tagged Zfrp8 (~55 kD) were excluded from the mass spectrometry analysis. To investigate whether these proteins could work together in the same molecular process, the interaction of both Zfrp8 and PDCD2 with Nufip (flies) and NUFIP1 (mammals) was confirmed in tissue culture cells. Immunoprecipitation of human HEK293 cell extracts expressing FLAG-tagged NUFIP1 pulled down endogenous PDCD2. Next whether this protein interaction also exists in Drosophila was examined. It was possible to co-purify endogenous Zfrp8 with NTAP-tagged Nufip from transfected S2 cells. An additional Western blot was performed on the purified NTAP-Nufip isolate and it was shown that FMRP is present in the protein complex, indicating that Nufip physically interacts with both Zfrp8 and FMRP. These results suggest that all three proteins function together in a molecular complex which regulates RNP processing/assembly and translation. Based on these results, and the requirement of both Zfrp8 and Fmr1 in stem cell maintenance, it was decided to characterize the genetic interaction between these genes (Tan, 2016).

To further characterize the connection between the two genes, whether the loss of Zfrp8 can modify oogenesis defects reported for Fmr1 females. Similar to what was previously reported, 100% of Fmr1Δ50M/Df(3 R)Exel6265 and 80% of Fmr1Δ50M/Fmr13 ovaries displayed developmental defects. The ovarioles contained fused egg chambers, aberrant nurse cell numbers. Occasionally, egg chambers with oocyte misspecification/multiple oocytes were also observed. Interestingly, the loss of one copy of Zfrp8 suppressed the majority of Fmr1 ovary defects, restoring cell division in the germline, as well as egg chamber morphology and separation. In Zfrp8/+; Fmr1Δ50M/Df(3R)6265, fusion of the first egg chamber is still observed in most germaria, but despite this, oogenesis appears to proceed normally resulting in normal looking ovarioles. Zfrp8/+; Fmr1Δ50M/Fmr13 ovaries appear almost completely normal even though these ovarioles contain no FMRP (Tan, 2016).

The loss of Fmr1 has also been associated with a strong reduction in egg production. This study found that similar to previous reports, Fmr1Δ50M/Df(3R)Exel6265 and Fmr1Δ50M/Fmr13 mutants display a strong reduction in fertility; females laid on average of 1 and 6 eggs/day, respectively, as compared to 18 eggs/day for wild-type flies. The removal of one copy of Zfrp8 partially suppressed Fmr1 infertility and resulted in 8 eggs/day from Fmr1Δ50M/Df(3R)Exel6265 and 15 eggs/day from Fmr1Δ50M/Fmr13 females. These results demonstrate that Zfrp8 and Fmr1 affect the same process and that even though they are found in the same complex, have opposing functions (Tan, 2016).

To investigate the nature of the Zfrp8 interaction with FMRP, the localization of the proteins within the ovary was examined. Zfrp8 displays ubiquitous distribution in all cells and cell compartments of the wild type ovary. No significant changes in Zfrp8 localization or levels are visible in Fmr1 ovaries. FMRP has a more varied distribution pattern, present in strong, cytoplasmic puncta in the cytoplasm of nurse cells and follicle cells, and also in high levels in the cytoplasm of the maturing oocyte. FMRP is also detectable in low levels in nurse cell nuclei at stage 8 egg chambers at an average of 9.76 puncta per nucleus. As expected, Fmr1 ovaries display no FMRP staining in either the cytoplasm or nucleus (Tan, 2016).

To determine whether Zfrp8 functions in FMRP regulation, Zfrp8 was depleted in the germline by expressing Zfrp8 RNAi under the control of the nos-Gal4 driver, and changes in FMRP expression were assessed. In control nos-Gal4 ovaries, FMRP levels and distribution were similar to that in wild-type ovaries. However, in Zfrp8 KD ovaries, aberrant FMRP localization is observed in the germline; FMRP is more uniformly distributed throughout the cytoplasm and puncta are strongly diminished. Remaining FMRP puncta appear fragmented, reduced in intensity, size and number (~10% of wild-type). These results indicate a Zfrp8 requirement for proper FMRP localization to the cytoplasm. FMRP normally functions by shuttling mRNA cargo from the nucleus to the cytoplasm, where it represses the translation of bound mRNA. The observed change of FMRP localization in Zfrp8 KD ovaries therefore may indicate a regulatory function for Zfrp8 in the nuclear export and localization of FMRP (Tan, 2016).

Zfrp8 protein is present in both the cytoplasm and nucleus and, as demonstrated above, controls the distribution of FMRP in the cytoplasm. It was decided to investigate the cell compartment in which Zfrp8 is required, in order to elucidate how Zfrp8 regulates FMRP. To do so, the capability of Zfrp8 deletion constructs to rescue mutant lethality was examined. Expression of human PDCD2 cDNAs driven by the general driver da-Gal4 is fully capable of rescuing Zfrp8 lethality. Mutated Zfrp8 constructs were created, removing either the two putative NLSs or the putative NES domains. These proteins were expressed under the da-Gal4 driver, and while clearly overexpressed on Western blots, failed to rescue mutant lethality, suggesting that the three domains are essential for the function of the protein (Tan, 2016).

In an alternative approach, the function of Zfrp8 proteins targeted to a distinct cell compartment was examined. Four N-terminal GFP-tagged transgenic proteins were expressed, encoding a wild-type Zfrp8, nuclear-localized NLS-Zfrp8, cytoplasmic-localized NES-Zfrp8, and cell membrane-localized CD8-GFP-Zfrp8. Transgenic Zfrp8 subcellular localization is visible when the proteins are strongly overexpressed. When the transgenes were expressed at lower levels, similar to the endogenous levels, with the hsp70-Gal4 driver at 25 oC, both wild-type and nuclear-localized Zfrp8 were able to rescue mutant lethality at similar rates, whereas the cytoplasmic- and membrane-localized proteins did not show rescue. These results show that Zfrp8 is required in the nucleus and suggest that like FMRP, Zfrp8 may function by shuttling between nuclear and cytoplasmic compartments (Tan, 2016).

This study has shown that FMRP and Zfrp8 are present in the same protein complex. In addition to FMRP, the mass spectrometry results have also identified other translational regulators, such as Tral. Tral has previously been shown to function in conjunction with FMRP to control the translation of mRNAs (Tan, 2016).

To determine whether Zfrp8 functions in Tral/FMRP-associated translational regulation, the genetic interaction between Zfrp8 and tral was investigated. Tral regulates dorsal-ventral (D/V) patterning through the localization and translational control of gurken (grk) mRNA. Eggs laid by tral females display ventralized chorion phenotypes, due to the aberrant Gurken morphogen gradient. If Zfrp8 functions to regulate the translational activity of FMRP/Tral, a suppression of the tral ventralized phenotypes should be apparent when Zfrp8 is reduced. Tral was depleted in the germline by expressing a TRiP RNAi lineunder the control of the nos-Gal4 driver. Tral KD resulted in similar ventralized egg phenotypes as previously observed in eggs laid by tral1 females: 1% of eggs displayed two normal dorsal appendages (Wt), 36% had fused appendages, and 63% had no dorsal appendages. Removing one copy of Zfrp8 in the tral KD background suppressed the tral phenotypes. This genetic interaction suggests that in addition to controlling the localization of FMRP in the cytoplasm, Zfrp8 also influences the translational control by Tral, essential for formation of dorsal-ventral polarity in the egg (Tan, 2016).

Whether Zfrp8 regulates Tral localization as it does FMRP was investigated by examining the distribution of GFP-fusion Tral protein trap line. Tral protein was uniformly present in cytoplasmic compartments of germline and somatic cells, with stronger granules surrounding nuclei, and was highly enriched within the oocyte. Zfrp8 KD results in loss of oocyte identity, and the distribution of Tral was significantly altered in those cells. But in all other germline cells Tral distribution remained unaffected. Tral and its orthologs are cytoplasmic proteins and examination of the Tral protein sequence identifies no NLSs. Zfrp8 may therefore interact only indirectly with Tral and not regulate its localization (Tan, 2016).

Zfrp8 and Fmr1 control position effect variegation piRNA pathway genes have been shown to be essential for heterochromatin packaging in position effect variegation (PEV) experiments. PEV measures expression of endogenous or reporter genes inserted within or adjacent to heterochromatin. Fmr1 is specifically required for chromatin packaging as loss of a single copy of Fmr1 is sufficient to inhibit heterochromatin silencing of a white reporter inserted into the pericentric heterochromatin region 118E10 on the 4th chromosome (Tan, 2016).

PEV of Zfrp8 heterozygotes, Fmr1 heterozygotes and Fmr1, Zfrp8 transheterozygotes were examined using 118E10 (4th chromosome centromeric) and an additional white reporter, inserted into heterochromatin region 118E15 (4th chromosome telomeric). While thewhite+ reporters in Zfrp8null/+ eyes were expressed at levels comparable to those in wild-type controls, expression in Fmr1Δ50M/+ of both white reporters was strongly enhanced. But, the removal of one copy of both Zfrp8 and Fmr1 decreased expression of the reporters back to the Zfrp8/+, near wild-type levels, indicating restored heterochromatin silencing of both 4th chromosomal insertions. These findings suggest that in normal eyes, Zfrp8 functions upstream of Fmr1 and controls Fmr1 effects on heterochromatin packaging (Tan, 2016).

A connection between regulation of heterochromatin silencing and Piwi has clearly been established and the current results show that Zfrp8 and FMRP are part of the mechanism that controls heterochromatin silencing. Heterochromatin is established at the blastoderm stage in Drosophila embryos and is subsequently maintained throughout development. Thus, FMRP and Zfrp8 function together in heterochromatin packaging in the early embryo in the same way as they do during oogenesis (Tan, 2016).

This study has shown that Zfrp8 is part of a complex that is involved in RNA processing, i.e. translation, localization, and stability. It is proposed that Zfrp8 likely forms a ribonucleoprotein complex with Nufip, FMRP and select mRNAs in the nucleus, and is required for localization of this complex in the cytoplasm. After nuclear export, mRNAs within the complex are targeted for translational control and repression by FMRP and Tral. The suppression of the Fmr1 and tral phenotypes in a Zfrp8 heterozygous background, occurs in the absence of Fmr1 and the strong reduction of tral. This suggests that Zfrp8 function is not protein specific, but rather that it controls the FMRP and Tral-associated complex, even in the absence of each of the two proteins. This hypothesis is consistent with Zfrp8 actively controlling the localization of FMRP to cytoplasmic foci, as this localization is affected in Zfrp8 germ cells (Tan, 2016).

Previous studies identified a piRNA pathway protein, Maelstrom (Mael), that is controlled by Zfrp8 in a similar manner as FMRP. Zfrp8 forms a protein complex with Mael, genetically suppresses the loss of mael, and controls Mael localization to the nuage, a perinuclear structure. But the Zfrp8 phenotype is stronger and appears earlier than that of mael, tral, Fmr1, or other piRNA pathway regulatory genes studied so far. Zfrp8 may therefore control a central step in the regulation of specific RNPs. Consistent with this hypothesis, the TAP purification and mass spectrometry analysis identified a number of Zfrp8-associated proteins, the majority of which function in ribosomal assembly or translational regulation, such as the ribosomal protein RpS2. And Zfrp8 KD in the germ line and partial loss of rps2 result in a similar "string of pearls phenotype", caused by developmental arrest in early stages of oogenesis. In addition, a recent study has shown that Zfrp8 and PDCD2 contain a TYPP (TSR4 in yeast, YwqG in E. coli, PDCD2 and PDCD2L in vertebrates and flies) domain, which has been suggested to perform a chaperone-like function in facilitating protein–protein interactions during RNA processing. These observations lead to a hypothesis that Zfrp8 functions as a chaperone essential for the assembly of ribosomes and the early recruitment and localization of ribosomal-associated regulatory proteins, such as FMRP, Tral and Mael (Tan, 2016).

Zfrp8 negatively controls the functions of Fmr1 and tral. In the absence of FMRP and Tral the temporal and spatial control of translation of their associated RNPs is lost. It is proposed that reducing the level of Zfrp8 diminishes the availability of these RNP-complexes in the cytoplasm resulting in suppression of the Fmr1 and tral phenotypes (Tan, 2016).

Zfrp8, Fmr1 and tral have all been shown to genetically and physically interact with components of the piRNA pathway, and to regulate the expression levels of select transposable elements. Transposon de-repression is often associated with the loss of heterochromatin silencing. The molecular mechanisms underlying heterochromatin formation appear to involve maternally contributed piRNAs and piRNA pathway proteins that control the setting of epigenetic marks in the form of histone modifications, maintained throughout development. But transposon expression can also be controlled post-transcriptionally by cytoplasmic PIWI-piRNA complexes, suggesting that transposon deregulation and heterochromatin silencing phenotypes seen in FMRP and Zfrp8 may be linked to translational de-repression. It is proposed that by facilitating the early assembly of ribosomes with specific translational repressors, Zfrp8 regulates several developmental processes during oogenesis and early embryogenesis including dorsal-ventral signaling, transposon de-repression, and position effect variegation (Tan, 2016).



To facilitate characterization of the Fmr1 protein, a monoclonal antibody was produced to it, designated 6A15. By Western blotting, 6A15 recognizes a single major protein of approximately 85 kDa in extracts of Drosophila S2 tissue culture cells. This protein comigrates with the translation product of the Fmr1 cDNA. 6A15 does not cross-react with human FMR1 or FXR proteins, because no bands were detected from HeLa cell extract. The specificity of this monoclonal antibody was further demonstrated by immunoprecipitation of Fmr1 produced by in vitro transcription and translation. hnRNP K, another KH domain-containing protein, was not immunoprecipitated by 6A15. Since 6A15 specifically recognized Fmr1, this antibody was used further to determine the subcellular distribution of the protein in S2 cells. Like its human homologs, Fmr1 is localized predominantly to the cytoplasm at steady state (Wan, 2000).

To determine the timing of expression and transcript complexity of Fmr1, developmental Northern analysis was performed using a probe derived from the Fmr1 cDNA. A prominent 2.8-kb transcript was detected in total RNAs prepared from ovaries and in poly(A)+ RNAs prepared from 0- to 3-h embryos. At later times, from 3 to 6 h and beyond, the major transcript detected was about 4.0 kb, and its level peaked between 9 to 12 h. Although two different-sized Fmr1 transcripts are produced during development, they appear to encode proteins of the same size because a developmental Western blot probed with 6A15 detects a single protein of the expected size of 85 kDa, whose expression level remains unchanged throughout development. This suggests that the difference in the transcript sizes is most likely due to variation in the untranslated regions of the mRNAs. Indeed, fragments of the same size were amplified from RNAs from all different stages of embryogenesis by reverse transcription-PCR using three sets of primers that span the entire Fmr1 coding region (Wan, 2000).

To examine the tissue distribution of Fmr1 during embryogenesis, whole-mount in situ staining was performed using the full-length Fmr1 cDNA as a hybridization probe and whole-mount immunostaining using 6A15. Both approaches reveal the same tissue distribution pattern. From the time that the egg is laid to early gastrulation, Fmr1 protein is uniformly distributed in the embryo. At midgastrulation (stage 11), the protein is expressed everywhere but there is a discernible concentration in the mesoderm. After gastrulation (stage 14), Fmr1 is uniformly distributed, with significantly elevated levels in the mesoderm, ventral nerve cord, and brain. At stage 16, expression in the ventral nerve cord and brain is more pronounced and elevated staining in the muscle is also detected. Overall, Fmr1 expression is widespread, with more pronounced expression in the central nervous system and in muscles. In situ hybridization and immunostaining studies of mammalian FMR1 and FXR in mouse and human embryos reveal expression in all tissues, with FMR1 and FXR2 displaying the highest levels in the central nervous system and testis and FXR1 being more prominent in muscles. Thus, the Fmr1 expression profile resembles the combined expression pattern of its mammalian homologs (Wan, 2000).

A specific monoclonal Fmr1 antibody has been characterized by Western and immunoprecipitation analyses (Wan, 2000). A systematic expression study was performed of the protein throughout the fly life cycle. Fmr1 expression is first widely detected in many tissues in stage 5 embryos. In late stage 16 embryos, strong Fmr1 expression is present in brain lobes, ventral nerve cord (VNC), and muscles (Wan, 2000). In the third instar larva, most (or perhaps all) of the neurons in the VNC and brain express high levels of Fmr1. Double-labeling with Fmr1 antibody and propidium iodide, a dye used to visualize nuclei, has shown that Fmr1 is abundant in soma cytoplasm as well as in neuronal processes within the CNS and peripheral nerves exiting the CNS. In addition to the CNS expression, high levels of Fmr1 are also observed in larval imaginal discs, testis, and ring gland. In adult brain, Fmr1 is also expressed in most (or perhaps all) of the neurons and highly enriched in optic lobes and distinct clusters of cells within the central brain. The conspicuous Fmr1 expression in the central complex is interesting, as this structure regulates coordinated motor control (Zhang, 2001).

Fmr1 was observed in the cytoplasm, rather than the nucleus, of all the cells examined, including all neurons and muscles. Even after Fmr1 overexpression, only cytoplasmic staining was observed. These observations are consistent with the subcellular localization of mammalian FMRP, although occasional nuclear localization has been observed (Devys, 1993; Verheij, 1993). Mammalian FMRP has also been localized at synapses by immunoelectron microscopy (Weiler, 1997) and synaptosomal preparation analyses (Feng, 1997b; Tamanini, 1997). Double-labeling experiments were performed with Fmr1 antibody and synaptic marker antibodies, e.g., Synaptotagmin (Syt) and Discs large (Dlg). Fmr1 is not enriched at central synapses in the VNC neuropil nor in peripheral NMJ synapses. Thus, Fmr1 is highly expressed in neurons, moderately expressed in muscles, is globally cytoplasmic, but it is not enriched in synapses (Zhang, 2001).

Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies

Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. This study shows that staufen- and FMRP-containing RNPs in Drosophila neurons contain proteins also present in somatic 'P bodies,' including the RNA-degradative enzymes Decapping protein 1 (Dcp1p) and Xrn1p/Pacman and crucial components of miRNA (Argonaute), NMD (Upf1p), and general translational repression (Dhh1p/Me31B) pathways. Drosophila Me31B, a DEAD-box helicases, is shown to participate (1) with an FMRP-associated, P body protein (Scd6p/Trailer hitch) in FMRP-driven, Argonaute-dependent translational repression in developing eye imaginal discs; (2) in dendritic elaboration of larval sensory neurons; and (3) in bantam miRNA-mediated translational repression in wing imaginal discs. These results argue for a conserved mechanism of translational control critical to neuronal function and open up new experimental avenues for understanding the regulation of mRNA function within neurons (Barbee, 2006).

Several observations now indicate that P bodies, maternal granules, and a major subclass of neuronal RNP are similar in underlying composition and represent a conserved system for the regulation of cytoplasmic mRNAs. Known RNA transport and translational repressors shared between maternal and neuronal staufen granules now include, Stau, Btz, dFMR1, Pum, Nos, Yps, Me31B, Tral, Cup, eIF4E, Ago-2, and Imp. Strikingly, in human cells, the Me31B homolog RCK/p54, the Tral homolog RAP55, the four human argonaute proteins, eIF4E, and a eIF4E-binding protein analogous to Cup, 4E-T, are all found in P bodies. In yeast, homologs of Me31B (Dhh1p) and Tral (Scd6p) are also known to be in P bodies, and Dhh1p in particular plays a role in recruiting RNA-decapping proteins and exonucleases to these RNPs. Consistent with the above observations in yeast, the enzymes involved in mRNA hydrolysis including the 5′ to 3′ RNA exonuclease Xrn1p/Pcm and the RNA-decapping enzyme DCP1 are present on Drosophila neuronal staufen RNPs and maternal RNA granules. These data unequivocally demonstrate tight spatial proximity of components mediating various RNA regulatory processes in Drosophila neurons (Barbee, 2006).

The large collection of proteins and processes common to P bodies, staufen granules, and likely maternal RNA granules suggests that they share an underlying core biochemical composition and function, which would then be elaborated in different biological contexts. For example, one anticipates that proteins involved in mRNA transport will be more prevalent in maternal and neuronal RNPs, which need to be transported for their biological function (Barbee, 2006).

An interesting aspect of neuronal staufen RNPs described in this study is the diversity of translational repression systems that are present within them. (1) In Me31B, these RNPs contain a protein that works in general translation repression of a wide variety of mRNAs and can also affect miRNA-based repression. (2) In Ago-2, they contain a component specific to miRNA/RNAi-dependent repression. (3) Neuronal staufen granules also contain UPF1, which was originally thought to be solely involved in mRNA degradation. However, because UPF1 can act as a translation repressor and physically interacts with Stau, a reasonable hypothesis is that UPF1 might work in neuronal granules, in conjunction with Stau, to repress the translation of a subset of mRNAs. The presence of multiple mechanisms for translation repression colocalizing in granules in Drosophila neurons may allow for differential translation control of subclasses of mRNA in response to different stimuli (Barbee, 2006).

Evidence accumulating in the literature suggests that there is a potential diversity of RNA granule types in neurons. Observations in Drosophila neurons are most consistent with a model in which a major subclass of neuronal RNP, in which various translational repressor and mRNA turnover proteins colocalize, is related to other compositionally distinct, diverse RNPs. A major subclass of staufen-containing RNP is indicated by data showing substantial colocalization among various proteins analyzed. Diversity is indicated by the lack of 100% colocalization: for instance, 55% of staufen-positive particles in wild-type neurons do not contain detectable dFMR1 (Barbee, 2006).

Two types of observations suggest that the apparent subclasses of particles containing Stau or dFMR1, but not both, are related to the particles in which they colocalize: (1) these two types of RNPs are clearly compositionally related to particles that contain both proteins; (2) this is supported by the observation that colocalization can be substantially increased under some conditions. Overexpression of either dFMR1 or Stau:GFP increases colocalization between Stau and dFMR1 from 45% in wild-type neurons to more than 80%. Concurrent with increased frequency of colocalization, Stau:GFP or dFMR1 induction increases apparent particle size (or brightness) and reduces the total number of particles. The increase in colocalization and brightness, as well as reduction in particle number, is most easily explained by growth and/or fusion of related RNPs. Significantly, similar effects on mammalian neuronal granule size and number have been reported following overexpression of Stau or another granule protein, RNG105. Thus, the underlying regulatory processes appear conserved between Drosophila and mammalian neurons (Barbee, 2006).

While it remains unclear how FMRP, Stau, or RNG105 enhance granule growth or fusion, it is conceivable that individual mRNAs first form small RNPs whose compositions reflect specific requirements for translational repression of the mRNAs they contain. These small RNPs exist in dynamic equilibrium with larger RNPs in which multiple, diverse translational repression complexes are sequestered. Induction of factors that promote granule assembly could push the equilibrium toward mRNP sequestration within large granules. A requirement of this dynamic model, which postulates interactions among different types of RNP, is that the RNPs themselves can change in composition during transport to synaptic domains. This is supported by FRAP analyses showing rapid exchange of Stau:GFP between cytosol and granule (Barbee, 2006).

Additional types of RNPs have also been described in neurons. For example, polysomes apparently arrested in translation have been observed near dendritic spines, and these RNPs show no obvious similarity to large, ribosome-containing particles, termed neuronal RNA granules. In addition, a potentially distinct RNP containing Stau, kinesin, and translationally repressed RNAs, but not ribosomes, has been purified from the mammalian brain. More recently, it has been shown that RNPs containing stress-granule markers TIA-1 and TIA-R as well as pumilio2 are induced by arsenate-treatment of mammalian cultured neurons. Interestingly, as previously shown for somatic cells, these large stress granules appear tightly apposed to domains containing DCP1 and Lsm1, markers of P bodies. Determining the temporal and compositional relatedness of such varied RNPs, their pathways of assembly as well as their functions, is a broad area of future research not only in neuroscience but also in cell biology (Barbee, 2006).

These diverse types of biochemical compartments for individual mRNAs suggest that neural activity or other developmental signaling events would influence translation in two steps: first, by desequestering mRNPs held within large granules and, then, by derepressing quiescent mRNAs in individual mRNPs. Thus, RNPs described in this study could have a complex precursor-product relationship with other RNPs, including polysomes discovered by now-classical studies at dendritic spines (Barbee, 2006).

Despite the complexity revealed by the diversity of neuronal RNPs, the importance and significance of the observed colocalization of Me31B, Tral, argonaute, and dFMR1 in staufen-positive neuronal RNPs is most clearly demonstrated by functional analyses revealing biological pathways in which these proteins function together (Barbee, 2006).

Several independent lines of evidence are consistent with a function for Me31B in neuronal translational repression as part of a biochemical complex that includes dFMR1. (1) Subcellular localization studies indicate that Me31B and Tral localize to dFMR1-containing RNPs especially prominent at neurite branch points in cultured Drosophila neurons. (2) Me31B, Tral, and dFMR1 coimmunoprecipitate from Drosophila head extract, thus confirming the physical association of three proteins. (3) Loss-of-function alleles of either Me31B or Tral suppress the rough eye phenotype seen when dFMR1 is overexpressed in the sev-positive photoreceptors. (4) Overexpression of Me31B in sensory neurons leads to altered branching of terminal dendrites, a phenotype also seen with overexpression analyses of Nos, Pum, and dFMR1. (5) Reduction of Me31B expression in sensory neurons by RNAi results in abnormal dendrite morphogenesis and tiling defects, phenotypes similar to that observed following loss of nanos, pum, or dFmr1 function. Significantly, the effect of Me31B on dendritic growth is correlated with its ability to function in translational repression. These five independent lines of evidence provide considerable support for Me31B (and Tral) function in neuronal translation control processes. While the site of functional interaction between dFMR1, Me31B, and Tral (soma or neuronal processes) is not identified here, the importance of the physical interactions is clearly demonstrated (Barbee, 2006).

Several observations also argue that Me31B acts, at least in part, within neurons to promote translation repression and/or mRNA degradation in response to miRNAs. This possibility was first suggested by the physical and genetic interactions of Me31B with dFMR1, a protein that has been implicated in the miRNA-mediated repression. Using direct assays for miRNA-mediated function in vivo, this study shows that Me31B is required for efficient repression by the bantam miRNA in developing wing imaginal discs. This identifies Me31B as a protein required for efficient miRNA-based repression (Barbee, 2006).

Recently, miRNA-based regulation has been shown to be important for the control of spine growth in hippocampal neurons and to be a target of protein-degradative pathways involved in long-term memory formation in Drosophila. Thus, the data predict that Me31B will be important in modulating miRNA function pertinent to development of functional neuronal plasticity. More generally, because Me31B homologs in yeast and mammals have been shown to function in P body formation in somatic cells, the requirement for Me31B in miRNA function provides evidence to support a model in which formation of P bodies is required for efficient miRNA-based repression in varied cell types and biological contexts (Barbee, 2006).

The conclusion that staufen- and dFMR1-containing neuronal RNPs are similar in organization and function to P bodies has several implications for neuronal translational control. (1) The presence of diverse translational repression systems on these RNPs suggests that, like in P bodies, different classes of mRNAs will be repressed by different mechanisms. This may allow specific RNA classes to be released for new translation in response to different stimuli. Such diversity of control may allow synapses to remodel themselves differently, depending on the frequency and strength of stimulation (e.g., LTD or LTP). (2) FRAP experiments indicate that both P bodies and staufen granules are dynamic structures. This argues that, like P bodies, staufen granules are in a state of dynamic flux, perhaps in activity-regulated equilibrium with the surrounding translational pool. (3) The presence of mRNA-degradative enzymes on staufen granules suggests regulation of mRNA turnover may play an important role in local synaptic events. For example, if synaptic signaling were to induce turnover of specific mRNAs at a synapse, then stimulated synapses could acquire properties different from unstimulated ones that retain a 'naive' pool of stored synaptic mRNAs. Finally, these observations imply that the proteins known to function in translation repression within P bodies will play important roles in modulating translation in neurons. Thus, it is anticipated that proteins of mammalian or yeast P bodies such as Edc3p, Pat1p, the Lsm1-7p complex, GW182, and FAST will be present on and influence assembly and function of neuronal granules (Barbee, 2006).

Characterization of Fragile X Mental Retardation Protein granules formation and dynamics in Drosophila

FMRP is an evolutionarily conserved protein that is highly expressed in neurons and its deficiency causes fragile X mental retardation syndrome. FMRP controls the translation of target mRNAs in part by promoting their dynamic transport in neuronal RNA granules. High expression of mammalian FMRP induces formation of granules termed FMRP granules. These RNA granules are reminiscent of neuronal granules, of stress granules, as well as of the recently described in vitro-assembled granules. In contrast with mammalian FMRP, which has two paralog proteins, Drosophila FMRP (dFMRP) is encoded by a single gene that has no paralog. Using this genetically simple organism, formation and dynamics of FMRP granules were investigated. Increased expression of dFMRP in Drosophila cells induces the formation of dynamic dFMRP RNA granules. Mutagenesis studies identified the N-terminal protein-protein domain of dFMRP as a key determinant for FMRP granules formation. The RGG RNA binding motif of dFMRP is dispensable for dFMRP granules formation since its deletion does not prevent formation of those granules. Deletion of the RGG motif reduced, however, dFMRP trafficking between FMRP granules and the cytosol. Similarly, deletion of a large part of the KH RNA binding motif of dFMRP had no effect on formation of dFMRP-granules, but diminished the shuttling activity of dFMRP. These results thus suggest that the mechanisms controlling formation of RNA granules and those promoting their dynamics are uncoupled. This study opens new avenues to further elucidate the molecular mechanisms controlling FMRP trafficking with its associated mRNAs in and out of RNA granules (Gereau, 2013a).

Characterization of fragile X mental retardation protein recruitment and dynamics in Drosophila stress granules

The RNA-binding protein Fragile X Mental Retardation (FMRP) is an evolutionarily conserved protein that is particularly abundant in the brain due to its high expression in neurons. FMRP deficiency causes fragile X mental retardation syndrome. In neurons, FMRP controls the translation of target mRNAs in part by promoting dynamic transport in and out neuronal RNA granules. Upon stress, mammalian FMRP dissociates from translating polysomes to localize into neuronal-like granules termed stress granules (SG). This localization of FMRP in SG is conserved in Drosophila. Whether FMRP plays a key role in SG formation, how FMRP is recruited into SG, and whether its association with SG is dynamic are currently unknown. In contrast with mammalian FMRP, which has two paralog proteins, Drosophila FMR1 (dFMRP) is encoded by a single gene that has no paralog. Using this genetically simple model, the role of dFMRP in SG formation was assessed, and the determinants required for its recruitment in SG as well as its dynamics in SG were defined. dFMRP is dispensable for SG formation in vitro and ex vivo. FRAP experiments showed that dFMRP shuttles in and out SG. The shuttling activity of dFMRP is mediated by a protein-protein interaction domain located at the N-terminus of the protein. This domain is, however, dispensable for the localization of dFMRP in SG. This localization of dFMRP in SG requires the KH and RGG motifs which are known to mediate RNA binding, as well as the C-terminal glutamine/asparagine rich domain. These studies thus suggest that the mechanisms controlling the recruitment of FMRP into SG and those that promote its shuttling between granules and the cytosol are uncoupled. This is the first demonstration of the regulated shuttling activity of a SG component between RNA granules and the cytosol (Gareau, 2013b).

Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles

Synaptic neuropeptide and neurotrophin stores are maintained by constitutive bidirectional capture of dense-core vesicles (DCVs) as they circulate in and out of the nerve terminal. Activity increases DCV capture to rapidly replenish synaptic neuropeptide stores following release. However, it is not known whether this is due to enhanced bidirectional capture. Experiments at the Drosophila neuromuscular junction, where DCVs contain neuropeptides and a bone morphogenic protein, show that activity-dependent replenishment of synaptic neuropeptides following release is evident after inhibiting the retrograde transport with the dynactin disruptor mycalolide B or photobleaching DCVs entering a synaptic bouton by retrograde transport. In contrast, photobleaching anterograde transport vesicles entering a bouton inhibits neuropeptide replenishment after activity. Furthermore, tracking of individual DCVs moving through boutons shows that activity selectively increases capture of DCVs undergoing anterograde transport. Finally, upregulating fragile X mental retardation 1 protein (Fmr1, also called FMRP) acts independently of futsch/MAP-1B to abolish activity-dependent, but not constitutive, capture. Fmr1 also reduces presynaptic neuropeptide stores without affecting activity-independent delivery and evoked release. Therefore, presynaptic motoneuron neuropeptide storage is increased by a vesicle capture mechanism that is distinguished from constitutive bidirectional capture by activity dependence, anterograde selectivity, and Fmr1 sensitivity. These results show that activity recruits a separate mechanism than used at rest to stimulate additional synaptic capture of DCVs for future release of neuropeptides and neurotrophins (Cavolo, 2016).

Synapses are supplied by anterograde axonal transport from the soma, the site of synthesis of synaptic vesicle proteins and dense-core vesicles (DCVs) that contain neuropeptides and neurotrophins. Delivery to synapses was thought to be based on a one-way anterograde trip until it was discovered that DCVs are subject to sporadic capture while traveling bidirectionally through en passant boutons as part of long-distance vesicle circulation (Wong, 2012). Interestingly, constitutive DCV capture occurs both during fast anterograde and retrograde transport, which are mediated by different motors (i.e., the kinesin 3 family member unc-104/Kif1A and the dynein/dynactin complex, respectively). Balanced capture in both directions is advantageous because DCVs are distributed equally among en passant boutons (Wong, 2012). In principle, bidirectional capture could occur by parallel regulation of anterograde and retrograde motors or by modification of the microtubules that both anterograde and retrograde DCV motors travel on (Cavolo, 2016).

Before the discovery of bidirectional capture of circulating vesicles, activity was shown to replenish the presynaptic neuropeptide pool following release by inducing Ca2+-dependent capture of DCVs being transported through boutons. This result and subsequent experiments (Bulgari, 2014) established that capture, rather than delivery or DCV turnover, limits synaptic neuropeptide stores. Activity-dependent capture was first described with a GFP-tagged neuropeptide in the Drosophila neuromuscular junction (NMJ), but also occurs with neurotrypsin, wnt/wingless, and brain-derived neurotrophic factor. Mechanistically, activity-dependent capture was characterized in terms of the rebound in presynaptic GFP-tagged peptide content following release and correlated with decreased retrograde transport. However, it is now evident that the reduction in retrograde flux could be caused by enhanced bidirectional capture as DCVs travel back and forth through the terminal as part of vesicle circulation (Wong, 2012). Therefore, prior studies support the hypothesis that there is only one synaptic capture mechanism, which is bidirectional and facilitated by activity (Cavolo, 2016).

This study tested the above hypothesis by investigating the directionality of activity-dependent capture. Experiments were performed with multiple approaches, including inhibiting retrograde transport, particle tracking, and simultaneous photobleaching and imaging (SPAIM; Wong, 2012). Furthermore, the effect of fragile X retardation protein (Fmr1, also called FMRP) was examined because it is known to affect bouton size and neuropeptide release. Together, these studies establish that different mechanisms mediate synaptic capture at rest and in response to activity (Cavolo, 2016).

Until recently, it was thought that presynaptic neuropeptide stores were set by controlling synthesis and delivery by fast one-way axonal transport of DCVs. However, studies of the Drosophila NMJ have shown that there is an excess of DCVs delivered to type-I boutons by long-distance vesicle circulation. Therefore, because DCV delivery is not limiting, the presynaptic neuropeptide pool is determined by capture, which was found to be bidirectional (Wong, 2012). However, in addition to constitutive capture, activity induces Ca2+-dependent capture. This is advantageous because tapping into the circulating vesicle pool removes delays associated with synthesis and transport, which can take days in humans, to rapidly replace released peptides. Surprisingly, experiments presented in this study demonstrate that activity-dependent capture is unidirectional and selectively sensitive to a genetic perturbation (i.e., Fmr1 overexpression). Therefore, activity does not simply enhance constitutive bidirectional capture that operates at rest, but instead stimulates an independent synaptic capture mechanism (Cavolo, 2016).

Previously, it was not possible to genetically block activity-dependent capture to determine its contribution to steady-state presynaptic stores. However, this study documented inhibition of activity-dependent capture by Fmr1 overexpression. As this was accompanied by a dramatic decrease in presynaptic DCV number, it is concluded that activity-dependent capture makes a large contribution to steady-state presynaptic peptide stores and hence the capacity for future release. At the Drosophila NMJ, DCVs contain a bone morphogenic protein and neuropeptides. Thus, it is possible that activity-dependent capture affects development and acute synaptic function (Cavolo, 2016).

Capture efficiency measurements revealed that the previously detected decrease in retrograde traffic following activity was an indirect effect of vesicle circulation; activity-induced capture of only anterograde DCVs at each en passant bouton simply leaves fewer DCVs for the retrograde trip back into the axon without changing retrograde capture. Of interest, anterograde selectivity for activity-induced capture rules out mechanisms that would perturb transport in both directions (e.g., microtubule breaks). DCV anterograde transport is mediated by the unc-104/Kif1A motor, which also transports SSV proteins and is required for formation of boutons. Therefore, activity-dependent capture may regulate unc-104/Kif1A to affect synaptic release of both small-molecule transmitters and peptides. However, alternative targets could be involved, including proteins that mediate DCV interaction with this anterograde motor or alter the DCV itself (e.g., its phosphoinositides, which may bind to the unc-104/Kif1A pleckstrin homology domain) (Cavolo, 2016).

Dynamics of glutamatergic signaling in the mushroom body of young adult Drosophila

The mushroom bodies (MBs) are paired brain centers located in the insect protocerebrum involved in olfactory learning and memory and other associative functions. Processes from the Kenyon cells (KCs), their intrinsic neurons, form the bulk of the MB's calyx, pedunculus and lobes (see Mushroom body is a quadruple structure). In young adult Drosophila, the last-born KCs extend their processes in the alpha/beta lobes as a thin core (alpha/beta cores) that is embedded in the surrounding matrix of other mature KC processes. A high level of L-glutamate (Glu) immunoreactivity is present in the alpha/beta cores (alpha/betac) of recently eclosed adult flies. In a Drosophila model of fragile X syndrome, the main cause of inherited mental retardation, treatment with metabotropic Glu receptor (mGluR) antagonists can rescue memory deficits and MB structural defects. To address the role of Glu signaling in the development and maturation of the MB, the time course of Glu immunoreactivity was compared with the expression of various glutamatergic markers at various times, that is, 1 hour, 1 day and 10 days after adult eclosion. It was observed that last-born alpha/betac KCs in young adult as well as developing KCs in late larva and at various pupal stages transiently express high level of Glu immunoreactivity in Drosophila. One day after eclosion, the Glu level was already markedly reduced in the alpha/betac neurons. Glial cell processes expressing glutamine synthetase and the Glu transporter dEAAT1 were found to surround the Glu-expressing KCs in very young adults, subsequently enwrapping the alpha/beta lobes to become distributed equally over the entire MB neuropil. The vesicular Glu transporter DVGluT was detected by immunostaining in processes that project within the MB lobes and pedunculus, but this transporter is apparently never expressed by the KCs themselves. The NMDA receptor subunit dNR1 is widely expressed in the MB neuropil just after eclosion, but was not detected in the alpha/betac neurons. In contrast, evidence is provided that DmGluRA, the only Drosophila mGluR, is specifically expressed in Glu-accumulating cells of the MB alpha/betac immediately and for a short time after eclosion. The distribution and dynamics of glutamatergic markers indicate that newborn KCs transiently accumulate Glu at a high level in late pupal and young eclosed Drosophila, and may locally release this amino acid by a mechanism that would not involve DVGluT. At this stage, Glu can bind to intrinsic mGluRs abundant in the alpha/betac KCs, and to NMDA receptors in the rest of the MB neuropil, before being captured and metabolized in surrounding glial cells. This suggests that Glu acts as an autocrine or paracrine agent that contributes to the structural and functional maturation of the MB during the first hours of Drosophila adult life (Sinakevitch, 2010).

In Drosophila and other arthropods, Glu is well characterized as the excitatory neurotransmitter of the neuromuscular junction. However, this amino acid has important signaling functions in the Drosophila brain as well. The Drosophila genome was predicted to encode 30 iGluR subtypes, including 3 AMPA- and 15 kainate-like, 2 NMDA-like, 4 δ-like and 6 divergent receptors. For now, the best characterized of these are the postsynaptic iGluRs expressed at the neuromuscular junction. Drosophila NMDA-like receptors are expressed in the central nervous system and have been implicated in learning and memory and locomotor control. The Drosophila genome encodes a single functional mGluR, DmGluRA, an ortholog of vertebrate group II mGluRs (Parmentier, 1996). This mGluR is presynaptic and expressed at the periphery of the active zones at the glutamatergic neuromuscular junctions, where it modulates both synapse excitability and fine structure (Bogdanik, 2004). DmGluRA is also expressed in the brain, in particular in lateral clock neurons, where it regulates circadian locomotor behavior (Hamasaka, 2007; Sinakevitch, 2010 and references therein).

The mushroom bodies (MBs) are paired centers located in the protocerebrum of Drosophila and other dicondylic insects that play essential roles in olfactory learning and memory and other brain functions, such as the control of locomotor activity, courtship behavior, courtship conditioning, visual context generalization, and sleep. The intrinsic structure of the MB is provided by the Kenyon cells (KCs), which have their cell bodies in the brain cortex and their dendrites in the MB calyx, where they receive input from the antennal lobe projection neurons. Axon-like processes of KCs project anteriorly and ventrally in the peduncle to form the vertical and medial lobes, which are subdivided into discrete parallel entities, the vertical α, α' and the medial β, β' and γ lobes. In addition to the KCs, there are other MB intrinsic neurons and several classes of MB extrinsic neurons that connect the MB to other areas of the brain neuropil. Emerging evidence suggests that different subtypes of MB KCs may be involved in distinct mechanisms of memory formation due to their connections to different MB extrinsic neurons (Sinakevitch, 2010).

Developmental studies have shown that the KCs are produced in each hemisphere of the brain by the division of four neuroblasts born early during the embryonic stage. The division of these neuroblasts sequentially produces the three morphologically and spatially distinct subtypes of KCs: γ, α'/β' and α/β. The γ neurons are generated up to the mid-third instar larval stage; they form the larval dorsal and medial lobe. The next KC subtype to be generated is the α'/β' neuron, which continues to be produced until puparium formation. Lastly, the α/β neurons are generated from the time of puparium formation until adult eclosion. In the α/β lobes, the KCs are organized in concentric layers. The youngest axon-like processes situated in the inner layer of the lobes are successively displaced outwards as they differentiate and newer α/β processes are added to the structure from the most recently born KCs (Kurusu, 2002). This volume of the α/β lobes into which grow the last-born axons contains densely packed and extremely thin fibers that are rich in actin filaments. This subset of processes has been named the α/β core (α/βc) (Sinakevitch, 2010).

An increased response to mGluR activation may play a prominent role in the fragile X syndrome (FXS), the most common form of inherited mental retardation and the predominant cause of autism. Mutations in dFmr1, the Drosophila homologue of the gene implicated in FXS, lead both to learning deficits and altered development of the MB, of which the most common feature is a failure of β lobes to stop at the brain midline. These behavioral and developmental phenotypes can be successfully rescued in Drosophila by treatment with mGluR antagonists (McBride, 2005), implicating Glu in the pathology, as is the case in mammalian models. Recent studies showed that dFmr1 interacts with DmGluRA in the regulation of synaptic architecture and excitability at glutamatergic synapses (Gatto, 2008; Repicky, 2009). However, until now the precise role of Glu and mGluRs in FXS and MB development has remained obscure (Sinakevitch, 2010).

This study presents evidence that Glu and its receptor DmGluRA are directly involved in construction of the MB neural circuits. Previous studies suggested that the Drosophila last-born α/βc KCs are immunoreactive to anti-Glu antibodies. The present study shows that these neurons express a high level of Glu-like immunoreactivity in newly eclosed adult flies. Interestingly, newborn KCs in late larval and pupal stages also appear to express as a rule a high level of Glu. To understand further the role and fate of Glu during KC maturation, the dynamics of Glu, DmGluRA and other Glu signaling-associated proteins were analyzed in the MB of young adult Drosophila from the time of their eclosion until 10 days post-eclosion. The results indicate that a transient Glu release likely regulates functional maturation of newborn KCs by a paracrine action during Drosophila post-embryonic development and the first hours after adult eclosion (Sinakevitch, 2010).

One intriguing question in neuroscience is how newborn neurons establish a functional network during their period of growth and maturation. This work describes a study of the late maturation of a subset of the α/β intrinsic MB KCs, the α/βc neurons, during a short period after adult eclosion. Glu-like immunoreactivity has been observed in the ingrowth lamina of the cockroach MB, which contains axons of the youngest KCs. Similarly in Drosophila, Glu accumulates in the α/βc, which contains newly generated neurons, whereas taurine-expressing neurons were found in the outer α/βc and aspartate-expressing neurons in the rest of the α/β lobes. It has been shown in vertebrates that Glu can have a strong influence on cone motility and induce rapid filopodia protrusion from hippocampal neurites or cultured astrocytes. In the present study an extensive analysis was performed of the distribution of various glutamatergic markers in the MBs of young adult Drosophila. The results suggest that the α/βc neurons are not simply glutamatergic. Rather, the evidence provided in this study indicates that these newborn KCs may transiently use Glu as a paracrine agent to favor interactions with glial cell processes and become mature neurons forming functional circuits (Sinakevitch, 2010).

Although the last-born α/βc KCs show a high level of Glu immunoreactivity a few hours prior and after adult eclosion, Glu immunostaining is dramatically reduced in these cells 24 hours after eclosion and is entirely absent a few days later. Disappearance of this signal could result from the release or intracellular metabolism of this amino acid. Similarly, it was observed in cockroach MBs that newborn KCs loose Glu immunoreactivity when they become mature and establish contacts with extrinsic neurons. This study also presents the first evidence that Glu transiently accumulates at a high level in developing newborn KCs of Drosophila in late larva and during pupal stages. Therefore, transient Glu expression could correlate with KC growth and maturation not only in the α/βc around eclosion time but also in other lobes during earlier stages of MB development (Sinakevitch, 2010).

Three subtypes of vesicular Glu transporters (VGluTs) have been identified in the mammalian nervous system with similar Glu transport functionality. Two of these (VGluT1 and VGluT2) present complementary distribution in central glutamatergic neurons. The third isoform, VGluT3, appears to be primarily expressed in neurons that release another transmitter (serotonin, dopamine, acetylcholine or GABA), where it may be required for efficient synaptic transmission. In the present study, neither the α/βc neurons nor any other intrinsic MB KCs were found to express the Drosophila vesicular transporter DVGluT. This may indicate that the Glu that is accumulated in the inner α/βc neurons is not stored in synaptic vesicles. However, the possibility cannot be excluded that these cells express another vesicular Glu transporter not yet identified in Drosophila. DVGluT immunoreactivity was observed in the MBs, particularly in the γ lobe and spur region and in the α lobes, but the punctuate labeling and localization suggest that this distribution corresponds to glutamatergic synapses belonging to extrinsic neurons (Sinakevitch, 2010).

Can the Glu transiently stored in the newborn MB neurons be released into the extracellular space? In the absence of DVGluT or another similar transporter, this could involve a non-vesicular release of Glu. Non-conventional release of Glu from immature neurons has been demonstrated in the developing rat hippocampus where Glu release exerts a paracrine action that seems to particularly affect the migration of neighboring maturing neurons. To address this question indirectly, the presence in the MB of other proteins known to be involved in the recycling and degradation of Glu at glutamergic synapses was sought (Sinakevitch, 2010).

An important role of glial cells is to capture Glu released from the synapse with specific transporters and then convert Glu to glutamine with GS. The only Drosophila high-affinity Glu transporter, dEAAT1, is expressed in subtypes of glial cells and is associated with Glu-release sites. GS2 is similarly expressed in glial cells in the Drosophila nervous system. This study shows that glial cells expressing dEAAT1 and GS surround the Drosophila MB lobe neuropiles, closely enwrapping the α/β lobes, thus isolating them from other lobes, and sending a mesh-like system of extensions inside these lobes. Enwrapping and invading of the MB β lobes by glia was also observed to occur in cockroach MBs, where glial cells are implicated in the removal of degenerating transient KC processes that occur during their establishment of mature connections with extrinsic cell dendrites. The high levels of glial dEAAT1 and GS within the Drosophila MB lobes suggest that this neuropil is tightly cordoned off from other parts of the brain and regulates the extracellular Glu level between the axons (Sinakevitch, 2010).

These data show that GS expression is highly dynamic in the MB during the first day of adult life, suggesting that glial cells play a role in establishing the MB's functional network. During the first hour after eclosion, the meshwork of glial processes expressing Glu signaling-associated molecules (GS and dEAAT1) is not present in the inner α/βc region, but within 24 hours this area becomes covered by glial extensions. These glial elements are possibly guided towards the α/βc area by the gradient of Glu released by the last born KCs. Glia could be involved in reducing Glu concentration in this area and play a role in axonal guidance and final maturation of KCs. Evidence that Glu transporters are required for coordinated brain development has been previously reported for mice: the absence of two glial Glu transporters resulted in excess of extracellular Glu and abnormal formation of the neocortex (Sinakevitch, 2010).

Assuming Glu is released by the newborn MB neurons, it has to interact with specific receptors. Therefore, the expression was sought of Glu receptors in MB neuropiles of young adult Drosophila, particularly those receptors that are likely to regulate neuronal growth and maturation through second-messenger pathways. Once activated by simultaneous Glu binding and membrane depolarization, the NMDAR channel allows calcium influx into the postsynaptic cell, where this ion triggers a cascade of biochemical events resulting in synaptic maturation and plasticity. Available antibodies against the constitutive dNR1 subunit of the Drosophila NMDAR were used. Immediately after eclosion, many processes in the MB neuropil were found to be dNR1-positive, with the exception of the α/βc neurons. The Glu released from either these α/βc neurons, or the surrounding glial cells, or extrinsic MB glutamatergic neurons may activate these NMDAR receptors. Thus, a widespread localization of NMDAR characterizes the MB immediately after eclosion, at the beginning of adult life when the MB is expected to receive the least inputs from sensory interneurons. Subsequently, with increasing sensory data being received and relayed to projection neurons, there is a dramatic and concomitant restructuring of NMDAR signaling: the majority of MB neurons no longer express these receptors. It is only those neurons that receive constant glutamatergic signaling that still address the dNR1 subunit in the vicinity of glutamatergic synapses expressing DVGluT. This occurs in particular within the spur region of the MB and the lateral horn. Such developmentally related regulation of NMDAR expression in the MBs of young adult flies may relate to adaptations of synaptic activity in response to sensory experience (Sinakevitch, 2010).

mGluRs are neuromodulatory G-protein-coupled receptors that are involved in many aspects of brain physiology, including neuronal development, synaptic plasticity, and neurological diseases. Whereas eight distinct mGluRs are present in the mammalian genome, a single functional mGluR is expressed in Drosophila, DmGluRA. The fly mGluR is structurally and pharmacologically closer to the mammalian group II mGluRs, which are mainly presynaptic receptors negatively coupled to adenylate cyclase. Attempts to locate DmGluRA with the commercially available monoclonal antibody 7G11 were not successful because the antibody produced by the hybridoma clone recently lost its binding specificity. To monitor DmGluRA distribution, a new GAL4 line was used that carries an enhancer trap insertion close to the mGluR start site of transcription, keeping in mind that expression of this GAL4 reporter may, in part, differ from the mGluR pattern. Strikingly, the DmGluRA-GAL4 line was found to express GFP selectively in the Glu-accumulating α/βc KCs of newly eclosed adult flies. This is in contrast to commonly used MB GAL4 driver lines (17d-, c739- and 201Y-GAL4) that do not express GFP in these neurons immediately after eclosion. Ten days later, the GFP staining in the DmGluRA-GAL4 line appeared strongly reduced in the α/βc; in contrast, the MB drivers now expressed GFP in these neurons (Sinakevitch, 2010).

Because the GAL4 reporter method reveals whole neurons, it could not be determined where the receptor is addressed restrictively in cell bodies, dendrites or axons. A previous study performed with an active lot of 7G11 antibody indicated that DmGluRA is present in nearly all neuropiles of the mature adult fly brain, including the MB calyces, but not in the MB lobes (Devaud, 2008). However, thas study did not report on the localization of DmGluRA in newly eclosed Drosophila. Further work is required to precisely locate the subcellular localization of DmGluRA in the newborn α/βc neurons, either with a new antibody or a DmGluRA-GFP fusion gene. The source of Glu binding to this mGluR receptor may be the neighboring glial cells or newborn KCs themselves, or both. Through activation of these receptors, Glu is likely to have a transient paracrine action on the α/βc neurons during the first day after eclosion that could be required for dendrite growth or synaptic maturation (Sinakevitch, 2010).

Although the α/βc KCs represent a minor part of the α/β lobe neurons, the maturation of these cells appears to be essential for proper MB functioning. Selective expression of the rutabaga (rut)-encoded adenylate cyclase in the α/βc neurons with 17d-GAL4 was shown to partially restore olfactory learning and memory in 2- to 5-day-old rut mutant flies. In contrast, no rescue of the rut defect was observed with c739-GAL4, which expresses in more peripheral α/β neurons at this stage. Therefore, the network involved in olfactory learning and memory apparently requires the α/βc neurons and is already functional in 2- to 5-day-old flies. Furthermore, treatment with mGluR antagonists restored courtship behavior, memory deficits and MB structural defects in DFmr1 mutants, a Drosophila model of FXS. These positive effects are even stronger when the pharmacological treatment is applied both during larval development and after eclosion. This suggests that these behavioral defects relate to an abnormally high level or prolonged duration of DmGluRA expression in the α/βc neurons of DFmr1 mutants. Further study should determine the distribution of Glu and DmGluRA during MB development in Drosophila FXS models (Sinakevitch, 2010).

The ubiquitin-proteasome system is one of the major conserved cellular pathways controlling protein turnover in eukaryotic cells. Substrate protein ubiquitination plays important roles in neuronal differentiation, axonal targeting, synapse formation and plasticity. In addition to strong Glu immunolabeling in the inner α/βc KCs, a high level of anti-ubiquitin immunoreactivity was also observed in these neurons immediately after eclosion. Such a high staining level was no longer detected in 10-day-old flies. In contrast, the spur region of the MB showed a constant high ubiquitin immunoreactivity that did not change with the age of the animal. This could suggest that synaptic plasticity is particularly active in this MB area (Sinakevitch, 2010).

Similarly, labeling of the cockroach MB β lobe with anti-ubiquitin showed, at specific stages in each developmental instar, as well as at an early adult stage, consistent staining of newly generated KC axons. Anti-ubiquitin also labeled the extending transiently Glu-immunoreactive collateral processes from developing KCs in the ingrowth zone, the hemimetabolous homologue of Drosophila's core KCs. This study showed that ubiquitin expression precedes degeneration of these collaterals and their subsequent removal by scavenging glial cells. Glu receptors can be endocytosed by an ubiquitin-dependent mechanism. The down-regulation of Glu and its receptor protein, possibly mediated by ubiquitin, thus appear to be important steps in the maturation and differentiation of the α/βc KCs (Sinakevitch, 2010).

In conclusion the present study suggests that the Glu accumulated in the α/βc KCs of young adult Drosophila is used for cell growth and maturation rather than for neurotransmission. The distribution and dynamics of glutamatergic markers indicates that Glu released from newborn KCs can bind to intrinsic mGluRs in the α/β cores and to NMDARs in the rest of the MB neuropil before being captured and metabolized by surrounding glial cells. As an autocrine or paracrine agent, Glu is likely to play a role in pathway finding within the lobe, namely, interactions between maturing KCs and extrinsic neuron dendrites, guidance of glial cell outgrowth and glial process targets into and around the relevant lobes, and maturation of synaptic networks required for a functional MB. Further study of the paracrine function of Glu in wild-type flies and in the Drosophila FXS model may shed light on similar actions of this neurotransmitter in the developing human brain in normal and pathological conditions (Sinakevitch, 2010).

Sleep and synaptic homeostasis: structural evidence in Drosophila

The functions of sleep remain elusive, but a strong link exists between sleep need and neuronal plasticity. This study tested the hypothesis that plastic processes during wake lead to a net increase in synaptic strength and sleep is necessary for synaptic renormalization. In three Drosophila neuronal circuits it was found that synapse size or number increases after a few hours of wake and decreases only if flies are allowed to sleep. A richer wake experience resulted in both larger synaptic growth and greater sleep need. Finally, it was demonstrated that the gene Fmr1 (fragile X mental retardation 1) plays an important role in sleep-dependent synaptic renormalization (Bushey, 2011).

Sleep is present in every species that has been carefully studied, including Drosophila, but its functions remain elusive. Increasing evidence points to a link between sleep need and neuronal plasticity. A recent hypothesis suggests that a consequence of staying awake is a progressive increase in synaptic strength, as the awake brain learns and adapts to an ever-changing environment mostly through synaptic potentiation. However, such increase would soon become unsustainable, because stronger synapses consume more energy, occupy more space, require more supplies, and cannot be further potentiated, saturating the ability to learn. Thus, according to the synaptic homeostasis hypothesis, sleep may serve an essential function by promoting a homeostatic reduction in synaptic strength down to sustainable levels. Also, the hypothesis predicts that the more one learns and adapts (i.e., the more intense is the wake experience), the more one needs to sleep. Findings in rodents are consistent with this hypothesis. For instance, molecular and electrophysiological markers of synaptic strength are higher after wake and lower after sleep. Moreover, presynaptic terminals of hypocretin neurons in zebrafish larvae undergo both circadian and sleep-wake-dependent structural changes, the latter consistent with sleep-dependent down-regulation. Finally, in the fly brain, overall levels of synaptic proteins increase after wake and decrease after sleep (Gilestro, 2009), and synaptic structural changes have been described after very long sleep deprivation (Donlea, 2009). These results suggest that a role for sleep in synaptic homeostasis may hold in phylogenetically distant species and may thus be of general importance (Bushey, 2011).

The evidence in support of the synaptic homeostasis hypothesis is mainly correlative, and thus it is important to seek direct proof that sleep is necessary for synaptic renormalization and to do so at the level of individual synapses. Moreover, the synaptic homeostasis hypothesis predicts that behavioral paradigms that enhance wake-related plasticity in specific neural circuits should increase synaptic strength in those circuits as well as sleep need, but this prediction has never been tested. Finally, the cellular mechanisms that underlie synaptic and sleep changes remain unexplored. This study exploited the power of Drosophila genetics, combined with confocal microscopy and behavioral analysis, to address these questions (Bushey, 2011).

Changes in synaptic strength are often associated with changes in synaptic structure, including synapse number and size, although the link between structural and functional plasticity is complex. In mammals, the diameter and length of synaptic spines correlate with the size of the postsynaptic density and with the magnitude of electric signals transmitted to the dendritic shaft. Moreover, the induction of synaptic potentiation leads to growth of synapses and spines, whereas synaptic depression causes synapses and spines to retract or shrink. Similarly, in Drosophila, synaptic morphology at the neuromuscular junction changes depending on experience, and these changes correlate with synaptic strength. Previous in vivo experiments in mammals and flies measured overall changes in electrophysiological and molecular markers of synaptic strength, without cellular resolution, and without direct evidence for morphological changes in synaptic terminals. Three specific cell populations in the fly brain were selected, and it was asked whether sleep and wake affect synaptic density and size (Bushey, 2011).

The first cell group studied included the small ventral lateral neurons (LNvs), a subset of circadian oscillator neurons that are part of the wake promoting system and express the neuropeptide pigment dispersing factor (PDF). To visualize changes in presynaptic morphology, a fusion protein between synaptotagmin and enhanced GFP (syt-eGFP) was expressed, whose protein product colocalizes with native synaptic vesicles. PDF expression was also measured, because the latter is another marker of presynaptic boutons in small LNvs. First, adult females (7 days old) collected either during the light period were tested after 7 hours of mainly (>75%) spontaneous wake or during the dark period after 7 hours of mostly sleep (>80%) or sleep deprivation (>90%). Syt-eGFP and PDF staining were both higher in the presynaptic region of sleep-deprived and spontaneously awake flies relative to sleeping flies, whereas no differences were found in the axonal processes extending from the cell bodies to the presynaptic region, suggesting that the changes are independent of circadian time and specific to the presynaptic terminal. Males were then tested because they have less consolidated wake during the day than females. Flies were only tested at night, after sleep or sleep deprivation. Sleep-deprived 3- and 7-day-old males consistently showed higher presynaptic syt-eGFP and PDF staining than sleeping flies. In contrast, 1-day-old flies showed low syt-eGFP and PDF staining after both sleep and sleep deprivation. The lack of PDF staining in very young flies suggests that these neurons are still inactive soon after eclosure. Moreover, because PDF promotes arousal, low PDF staining is consistent with flies being predominantly asleep after eclosure, even if mechanical stimulation was used to try to keep them awake, consistent with high sleep need and elevated arousal threshold in newborn mammals. Syt-eGFP staining did not change in newly eclosed flies, whose PDF levels were very low. Syt-eGFP and PDF expression were also measured in Per01 flies carrying a null mutation of the clock gene Period. Because Per01 mutants have no spontaneous consolidated sleep, flies were collected immediately after 7 hours of sleep deprivation or after 5 additional hours of either recovery sleep or sleep deprivation. Overall, syt-eGFP and PDF staining in presynaptic terminals was reduced in Per01 mutants relative to wild-type (WT) flies but was still high after both 7 and 12 hours of sleep deprivation and low after recovery sleep (Bushey, 2011).

The second cell group analyzed included γ neurons of the mushroom bodies, because they can be targeted by mosaic analysis with a repressible cell marker (MARCM) to visualize single cells, show a relatively simple morphology, and undergo activity-dependent pruning. Moreover, the mushroom bodies are involved in sleep regulation, and mutations altering cyclic adenosine monophosphate-dependent protein kinase signaling or Fmr1 (fragile X mental retardation 1) expression in these brain regions affect both sleep need and experience-dependent structural plasticity . Flies were collected at night after 7 hours of sleep or sleep deprivation, and dissected brains were immunostained for GFP-tagged CD8 to visualize neuronal membranes. It was found that the axonal tips were larger after sleep deprivation than after sleep, consistent with an increase in volume of presynaptic terminals. To confirm this result, fly stocks were generated with γ MARCM clones expressing syt-eGFP, and flies were collected after 7 hours of mostly spontaneous wake, or during the dark period after 7 hours of mostly sleep or sleep deprivation. As expected, syt-eGFP tended to accumulate in puncta along lightly stained processes, in contrast to the diffuse CD8-GFP staining. Syt-eGFP puncta were larger in sleep deprived and spontaneously awake flies relative to sleeping flies (Bushey, 2011).

Next, whether postsynaptic morphological changes also occur as a function of sleep and wake was tested. To do so, focus was placed on the first giant tangential neuron of the lobula plate vertical system (VS). This cell (VS1) is unambiguously recognizable, and its stereotyped dendritic tree shows small actin-enriched protrusions morphologically and functionally similar to mammalian dendritic spines. Flies were compared that were spontaneously awake during the day or that slept or were sleep deprived during the first 7 hours of the night. Single VS1 spines were visualized using an antibody against actin-GFP and counted in one easily identifiable branch. The total number of spines was similar in spontaneously awake and sleeping flies but increased after sleep deprivation relative to both conditions, mainly because of an increase in stubby spines (which were the majority of scored spines). The number of mushroom spines did not change. The increase in spine number after sleep loss was associated with increased branching and lengthening of the dendritic tree, whereas spine density (number of spines divided by branch length) was similar in all conditions. Because sleep-deprived female flies had been mostly awake during the previous light period, this suggests that these postsynaptic changes may need sustained periods of wake. Another possibility, not mutually exclusive, is that changes in VS1 spines require a wake condition richer than that experienced by flies spontaneously awake alone inside small glass tubes. Indeed, sleep-deprived flies were kept awake using vibratory stimuli, resulting in the flies often falling from the top to the bottom of the tubes. Because visually driven responses in VS neurons are stronger during flight than during nonflight, it is possible that these cells were activated by the fall (Bushey, 2011).

To test whether a rich wake experience that engages the VS circuit is sufficient to affect VS1 synaptic morphology, up to 100 flies were housed inside a large lighted chamber ('fly mall') for an entire light period (12 hours). In the mall, flies could fly ad libitum, explore, and interact with each other. Flies were collected immediately after the mall experience and compared with flies that, as usual, had remained awake during the day in single tubes. The enriched experience in the mall had profound morphological effects on the VS1 dendritic tree: Total branch length increased because of the addition of more branches with spines (mainly stubby), resulting in an overall increase in spine number (Bushey, 2011).

Once experience-dependent synaptic changes have occurred, are they stable? If not, is sleep necessary to bring synaptic morphology back to pre-enrichment levels? To answer these questions, two other groups of flies were moved back to single tubes after 12 hours of mall experience; one group was allowed to sleep for 7 hours, whereas the other was kept awake as before using mechanical stimuli. In flies that were sleep-deprived after enrichment, branch length, branch points, and spine number were at levels similar to those seen in flies collected immediately after enrichment. In contrast, in flies that were allowed to sleep after the mall experience, all morphological parameters reverted to the levels observed in awake flies kept in single tubes. Moreover, spine density was negatively correlated with the amount of sleep during the last 7 hours, as well as with the maximal duration of sleep bouts. In another experiment, flies were housed in the mall for 12 hours during the day and then moved back to single tubes to record their sleep. During the 24 hours after the enrichment, flies slept more, both during the day and at. Finally, in the last experiment, flies were housed in the mall for 12 hours during the day, moved back to single tubes and sleep deprived all night (12 hours), and then either collected immediately, allowed to sleep for 6 hours, or kept awake for 6 more hours. Consistent with the previous experiments, decreases in all morphological parameters were seen only in flies that could sleep, and spine density was negatively correlated with the amount of sleep during the last 6 hours, as well as with mean and maximal duration of sleep bouts (Bushey, 2011).

Previous experiments suggest that Fmr1 could mediate at least some of the effects of sleep/wake on synapses. Fmr1 protein product (FMRP) is present in dendritic spines, and loss of FMRP in flies is associated with overgrown dendritic trees, larger synaptic boutons, and defects in developmental and activity-dependent pruning. Notably, Fmr1 overexpression results in the opposite phenotype, with dendritic and axonal underbranching and loss of synapse differentiation. Moreover, Fmr1 expression is reduced by sensory deprivation in flies and increased by sensory stimulation and enrichment in mammals (Bushey, 2011).

It was recently shown that FMRP levels increase in the adult fly brain during wake relative to sleep, independent of time of day or light, suggesting that waking experience is sufficient to affect Fmr1 expression even after the end of development. It has also been shown that Fmr1 overexpression in either the whole brain or in the mushroom bodies is associated with an ~30% decrease in sleep duration, and it is hypothesized that this reduced need for sleep occurs because chronically high Fmr1 levels may allow synaptic pruning to occur at all times, independent of sleep. If so, Fmr1 overexpressing (OE) flies should fail to show increased spine density after prolonged wake. Thus Fmr1 was overexpressed specifically in the vertical and horizontal system of the lobula plate. OE flies were collected at night after 7 hours of either sleep or sleep deprivation and were compared to corresponding sleeping and sleep-deprived WT controls. As expected, Fmr1 expression was concentrated in granules along the VS1 dendritic tree, and overall Fmr1 levels were higher in sleeping and sleep-deprived OE flies than in their corresponding controls, due to larger Fmr1 granules in OE flies. Crucially, in contrast to WT controls, OE flies showed no increase in either spine number, branch length, or branch points after sleep deprivation relative to sleep; all these parameters were similar between the two experimental groups, and their levels were close to those observed in WT flies after sleep. Finally, OE flies slept less than their WT controls during baseline and showed a reduced sleep rebound after 12 hours of sleep deprivation at night. Thus, it seems that Fmr1 overexpression was sufficient to completely abolish the wake-dependent increase in VS1 spine number, whereas the effects on sleep were small. The latter result is not surprising, because sleep need presumably results from the overall amount of synaptic plasticity occurring during wake in many brain areas, whereas Fmr1 overexpression was restricted to a few VS neurons (Bushey, 2011).

Sleep is perhaps the only major behavior still in search of a function. The results of this study support the hypothesis that plastic processes during wake lead to a net increase in synaptic strength in many brain circuits and that sleep is required for synaptic renormalization. A wake-related increase in synapse number and strength, if unopposed, would lead to a progressive increase in energy expenditure and saturation of learning. A sleep-dependent synaptic homeostasis may explain why sleep is required to maintain cognitive performance. How sleep would bring about a net decrease in synaptic strength remains unknown, but in mammals, potential mechanisms favoring synaptic depression during non-rapid eye movement sleep may require the repeated sequences of depolarization/synchronous firing and hyperpolarization/silence at ~1Hz observed in corticothalamic cells, as well as the low levels of neuromodulators such as noradrenaline and of plasticity-related molecules such as brain-derived neurotrophic factor. To what extent such mechanisms may also apply to flies remains to be determined (Bushey, 2011).


Since Fmr1 is an RNA binding protein (Wan, 2000), it might be predicted that Fmr1 overexpression would titrate out its RNA substrates. Therefore, Fmr1 was overexpressed in numerous tissues using the UAS-GAL4 transgenic system. Two Fmr1 UAS constructs, UAS-Fmr1 and UAS-I307N (the same point mutation as the human I304N in the second KH RNA binding domain; Jin, 2000), were transformed into the fly genome (Zhang, 2001).

When either Fmr1 construct was driven by panneuronal elav-GAL4 (dfxrNOE), the progeny exhibited abnormally spread-out wings and could not fly. These animals were uncoordinated and displayed early adult lethality, usually 5-10 days following adult eclosion. When either Fmr1 construct was driven by mhc-GAL4, which drives overexpression in all muscles (dfxrMOE), the progeny showed droopy or held-up wings and could not fly. When either Fmr1 construct was driven by G7-GAL4, which drives higher muscle expression, all progeny died at pupal stages. These results suggest that Fmr1 expression level is critical for normal neuromusculature functions. It is interesting to note that overexpression of human FMRP in the mouse model also produces behavioral abnormalities (Peier, 2000). The overexpression of I307N by GAL4 lines in wild-type background consistently produces similar but weaker phenotypes than does the overexpression of wild-type Fmr1 (Zhang, 2001).

To examine the functional significance of the mutation I307N, phenotypes caused by cooverexpression of wild-type and I307N Fmr1 were compared with that caused by overexpression of each alone. Cooverexpression of both in the nervous system causes lethality with few escapers, whereas overexpression of either wild-type or I307N alone produces viable adults with eye/wing phenotypes. These results suggest that the point mutation I307N is not a dominant negative, but rather acts as a simple hypomorph (Zhang, 2001).

All six Fmr1 mutant alleles, including four deletion nulls, are viable with no discernible morphological phenotypes. A range of behavioral tests including bang sensitivity, temperature sensitivity, and phototaxis assays did not show detectable differences between wild-type and null mutants. However, the mutants showed defective coordinated behavior in a simple flight test. The two EP insertion lines, EP(3)3517 and EP(3)3422, and a Fmr1 null (50M), each over deficiency Df(3R)by62, were flight defective. The flight defect was specific to the Fmr1 mutations, since the phenotype was rescued by precise excision of the EP insertion. Flight tests on single animals confirmed the flight defect of Fmr1 mutants. Flies with neuronal or muscle overexpression of Fmr1 are also flight defective, most likely due to their wing postural defects (Zhang, 2001).

One of the best-established systems to study neuronal structure and function in Drosophila is the adult eye. To assay the effect of Fmr1 on retinal neuronal patterning, the protein was first overexpressed by using UAS-Fmr1 constructs and eye-specific GAL4 drivers. UAS-Fmr1 driven by sev-GAL4 produces significant retinal disorder. The disorder includes misshapen rhabdomeres, abnormal numbers of rhabdomeres per ommatidium, and fused ommatidia. The UAS-I307N lines produce phenotypes similar but milder than wild-type UAS-Fmr1, demonstrating a specific role of the KH2 RNA binding domain in these phenotypes, consistent with Wan (2000). In contrast, Fmr1 null mutants produce no detectable effect on the structure of the eye. Thus, overexpression of Fmr1 can specifically perturb neuronal patterning in the eye, but the protein is not required for the process (Zhang, 2001).

Electroretinogram (ERG) assays were performed on different Fmr1 genotypes to assay phototransduction and synaptic transmission between photoreceptors and laminar interneurons. Both neuronal overexpression of Fmr1 (dfxrNOE) driven by elav-GAL4 and null mutants (Fmr1) display a robust photoreceptor response, as indicated by depolarization of the photoreceptor throughout the 1 second light pulse. The plateau potential is similar to the controls, even when the photoreceptor morphology is disrupted. Thus, neither Fmr1 null mutation nor dfxrNOE perturbs phototransduction (Zhang, 2001).

Depolarization of photoreceptors triggers release of the neurotransmitter histamine, which targets inhibitory Cl- channels in the postsynaptic cell. The synaptic response commonly referred to as the 'off-transient' in ERGs is caused by the closure of the histaminergic Cl- channels and resulting depolarization of the laminar cell. Off-transients were measured as the magnitude of negative potential change at termination of the light pulse. Both dfxrNOE and Fmr1 null mutants show similar significant decreases in the characteristic response of the postsynaptic laminar cell to cessation of photoreceptor depolarization. Compared to control, dfxrNOE shows a 56% decrease and Fmr1 null shows a 51% decrease in off-transient mean amplitude. Thus, changes in the level of Fmr1, both increase and decrease, strongly impair synaptic transmission in the visual system (Zhang, 2001).

The second well-defined system for studying synaptic structure and neurotransmission in Drosophila is the larval neuromuscular junction (NMJ). Importantly, the glutamatergic NMJ is amenable to detailed single-cell assays of pre- and post-synaptic mechanisms. Fmr1 is expressed both in presynaptic motor neurons and in postsynaptic muscles during embryonic development and in larvae. Fmr1 nulls and transgenic lines were analyzed with Fmr1 overexpressed either pre- and postsynaptically. Since the cellular phenotype associated with human FraX patients and FMR1 knockout mice is alterations in synaptic morphology (Hinton, 1991; Comery, 1997; Irwin, 2001; Nimchinsky, 2001), it was first determined if synaptic structural defects are present in Drosophila (Zhang, 2001).

Two significant alterations in NMJ synaptic terminals are observed in Fmr1 mutants. (1) Fmr1 null mutants display pronounce synaptic overgrowth and overelaboration of synaptic terminals. This phenotype is reminiscent of the dendritic spine overgrowth observed in mammalian mutants. Quantification of the number of synaptic boutons on muscle 4 reveal that the Fmr1 nulls have a 51% increase over controls. (2) dfxrNOE causes the opposite phenotype of synaptic undergrowth and displays an average 36% decrease in the number of muscle 4 synaptic boutons. Postsynaptic dfxrMOE causes a similar but a more modest loss of structural elaboration and exhibits a 17% decrease in muscle 4 synaptic boutons. Quantification of the muscle 6/7 NMJ boutons shows a similar trend. In addition to the increased bouton number, Fmr1 null mutants show excessive arboreal branching. The muscle 4 NMJ contained ~50% more synaptic branches than controls. In contrast, Fmr1 overexpression had no significant impact on synaptic branching. Thus, the level of Fmr1 on both sides of the synaptic cleft is an important determinant of synaptic growth (Zhang, 2001).

Fmr1 also plays a role in regulating bouton morphology. Overexpression of Fmr1 presynaptically (dfxrNOE) causes an obvious enlargement of single synaptic boutons. The NMJ bouton diameter in dfxrNOE animals is nearly twice that of wild-type. Increased bouton size is not limited to type 1b boutons; type 1s boutons are also larger. This phenotype is specific to presynaptic overexpression, since null mutants and postsynaptic overexpression show bouton size comparable to controls. In summary, Fmr1 null mutants show synaptic overelaboration with increased synaptic branching and bouton differentiation, whereas overexpression causes the opposite undergrowth phenotype with fewer synaptic boutons which, in the case of dfxrNOE mutants, are structurally enlarged (Zhang, 2001).

Synaptic transmission at the NMJ was assayed. Unlike the eye, the glutamatergic NMJ is amenable to detailed, single-cell recordings of synaptic function using two electrode voltage clamp techniques, which can be used to dissect pre- and post-synaptic transmission mechanisms. It was therefore asked whether the NMJ in Fmr1 mutants displays altered communication, due to changes in either presynaptic glutamate release or postsynaptic glutamate response (Zhang, 2001).

Significant alterations in NMJ neurotransmission are observed in Fmr1 mutants. (1) Evoked synaptic transmission is significantly elevated in Fmr1 null mutants. The mean excitatory junctional current (EJC) amplitude is increased from 35 nA in controls to 66 nA in Fmr1 null mutants. The variance of transmission amplitude (SD/mean current amplitude), a measure of synaptic fidelity, is unaffected in Fmr1 compared to wild-type, demonstrating that the average synaptic efficacy is upregulated in null mutants. Fmr1NOE in the presynaptic terminal does not significantly alter mean EJC amplitude. (2) Quantal analyses of miniature excitatory junctional currents (mEJCs) showed that the frequency of spontaneous glutamate release is increased by 5-fold in dfxrNOE animals but is not changed with postsynaptic dfxrMOE. mEJC frequency in Fmr1 null mutants is mildly increased relative to controls. There is no striking increase in mEJC amplitude in any of the Fmr1 genotypes. It is concluded that Fmr1 modulates synaptic transmission through a primarily presynaptic mechanism. Loss of Fmr1 results in elevated evoked neurotransmission, whereas presynaptic overexpression results in elevated spontaneous vesicle fusion (Zhang, 2001).

To summarize, Fmr1 mutants perturb synaptic neurotransmission at two different synapse types: histaminergic photoreceptor (central) synapses and glutamatergic NMJ (peripheral) synapses. Surprisingly, increase and decrease of Fmr1 levels similarly alters presynaptic function in these two synapse types, suppressing transmission in central synapses and elevating it in peripheral synapses. The fact that the polarity of the regulation differs may be attributable to a multitude of differences between the two synaptic classes (Zhang, 2001).

To study the functions of Fmr1 via a genetic approach, the gene was first mapped via polytene chromosome in situ hybridization to 85F9-12 on the third chromosome. The original insertion EP(3)3517 maps in the 5' UTR of the Fmr1 gene, and another insertion EP(3)3422 maps in the second intron of Fmr1 gene. Subsequent remobilization of EP(3)3517 produced four deletions of Fmr1. All four deletions were characterized by DNA sequencing. Anti-Fmr1 staining of these different alleles shows that the two EP insertion lines are hypomorphs, whereas the four Fmr1 specific deletions appear to be protein nulls, since no Fmr1 staining was detected by immunostaining (Zhang, 2001).

The effect of overexpressing Fmr1 protein was examined. Overexpression of a gene can lead to phenotypes that are often relevant to the function of the expressed gene product. To overexpress Fmr1 in developing tissues, use was made of the GAL4-UAS system or sevenless promoter/enhancer sequences that direct expression of Fmr1 in a subset of cell types in the larval eye-antennal imaginal disc. Transgenic flies containing a copy of UAS-Fmr1 were crossed to a variety of promoters that direct GAL4 expression in easily visualized tissues. Overexpression of Fmr1 under the control of vestigial (vg) or decapentaplegic (dpp) promoters, which direct expression in developing wing tissue, causes transgenic wings to suffer a loss of cells in the region of the wing where the promoters for these genes function. Expression of UAS-Fmr1 through dpp-GAL4, which is strongly expressed at the anterior/posterior margin of the developing wing blade, led to a decrease in cells between longitudinal veins 3 and 4, as well as loss of the anterior crossvein. A loss of cells at the margin of the wings was observed with 100% penetrance when UAS-Fmr1 was expressed under the control of vg-GAL4. Finally, UAS-Fmr1 expression was directed by sevenless-GAL4 (sev-GAL4), which drives expression in a subset of the photoreceptor cells, the mystery cells, and the cone cells behind the morphogenetic furrow during eye development. Overexpression of Fmr1 in the eye leads to a severe rough eye phenotype (Wan, 2000).

The phenotypes caused by overexpression of Fmr1 could result from cells failing to adopt an appropriate fate, defects in cell proliferation, or cell death by apoptosis or necrosis. The staining of cells with the dye acridine orange is a reliable marker for apoptosis and does not mark cells that are dying through necrosis. Eye-antennal imaginal discs from third-instar larvae of control (sev-GAL4/+) flies or those expressing Fmr1 under sev-GAL4 control were dissected and stained with acridine orange to qualitatively assess levels of cell death. In control eye-antennal discs, relatively little apoptosis occurs. In contrast, eye-antennal discs overexpressing Fmr1 show a large number of cells behind the morphogenetic furrow that have taken up the dye. Furthermore, the rough eye phenotype was suppressed when the apoptotic inhibitor DIAP1/THREAD was co-overexpressed in the eye-antennal disc. These results indicate that the overexpression of Fmr1 leads to cell death by apoptosis and that any deficiencies in proliferation or cell fate decisions that may contribute to the observed phenotypes may very well be a secondary consequence of the apoptotic events (Wan, 2000).

One potential problem with an analysis dependent on overexpression is nonspecific effects caused by the overabundance of a protein. To assess whether the overexpression phenotype is specific to a normal function of the Fmr1 protein, Fmr1 alleles containing isoleucine-to-asparagine changes (I244N and I307N) in either or both KH domains were expressed behind UAS. Using sev-GAL4 as a driver, transgenic stocks were selected for lines that exogenously express wild-type and mutant forms of Fmr1 at approximately equal levels to allow for quantitative comparisons of the phenotypic effect (Wan, 2000).

Adult eyes from the transgenic stocks were examined by scanning electron microscopy. Mutations in either KH domain significantly ameliorate the degree of eye roughness compared to flies overexpressing a wild-type copy of Fmr1. These results indicate that the observed overexpression phenotypes are due to increased Fmr1 activity. However, that a milder rough eye phenotype is observed with the overexpression of either of the KH domain mutations indicates that these mutations do not remove all of the activity of Fmr1 as observed by this assay. sev-GAL4-driven expression of a UAS-Fmr1 allele where both KH domains are mutated has little or no discernible phenotype, suggesting that most or all activity of the sev-GAL4-expressed Fmr1 requires functional KH domains. Examination of ommatidial cross sections taken through the photoreceptor cells from eyes of transgenic flies show several phenotypes. Ommatidia overexpressing wild-type Fmr1 have occasional missing photoreceptor cells, and pigment cells are often missing. These phenotypes can be explained from the apoptotic events occurring in the developing eye imaginal disc. Ommatidial assembly is an ordered process of cell recruitment with photoreceptor cells forming a cluster, subsequently joined by cells destined to function as cone cells, followed by pigment cells and bristle cells. The apoptotic events likely reduce the number of cells available to form an ommatidial cluster. In addition, rhabdomere structure is altered in photoreceptor cells overexpressing Fmr1. An interesting observation in this regard is that overexpression of the wild type or I307N mutant, both of which contain an intact first KH domain, alters the shape of the rhabdomeres within photoreceptor cells, whereas overexpressing the I244N mutant or the double mutant does not. Whether the two KH domains function cooperatively in binding all RNA substrates or whether they can have independent functions cannot be determined at this time (Wan, 2000).

A severe case of fragile X has been described in which there is a single missense mutation in the second KH domain of the FMR1 gene that substitutes an asparagine residue for the normal isoleucine residue (I304N). The extreme phenotype of this patient, as well as observations that the I304N mutation causes abnormally sized RNP particles to form and that mutations mapping to the KH domains of some genes elicit stronger phenotypes than null alleles of the same gene, has suggested that the effects of the I304N mutation in hFMR1 may be due to a dominant-negative rather than a loss-of-function effect. To determine whether the phenotype elicited by the Fmr1 I307N substitution was due to a dominant-negative or a loss-of-function effect, Drosophila stocks were generated that in addition to overexpressing Fmr1 I307N had a deficiency in a wild-type copy of Fmr1. The deficiency Df(3R)by62 (85D11-85F16) removes one copy of Fmr1 as assessed by quantitative Southern hybridization. If the I307N mutation of Fmr1 acts as a dominant negative, removal of one wild-type copy of the gene would be predicted to increase the severity of the rough eye phenotype. In contrast, if the I307N mutation represented a partial loss of function, removal of one wild-type copy of Fmr1 would be expected to have no effect on the eye or perhaps even lessen the rough phenotype. The comparison of UASdfmr1I307N/+; sev-GAL4/+, and UASdfmr1I307N/+; sev-GAL4/Df(3R)by62 flies indicates no obvious enhancement of eye roughness and perhaps shows a lessening of the defect in flies with the Fmr1 deficiency. Identical results were observed for the mutation in the first KH domain of Fmr1 (I244N). These results indicate that the isoleucine-to-asparagine changes in the KH domains of Fmr1 act as loss-of-function mutations in the genetic background used for these experiments (Wan, 2000).

Drosophila Fragile X protein, DFXR, regulates neuronal morphology and function in the brain

Mental retardation is a pervasive societal problem, 25 times more common than blindness for example. Fragile X syndrome, the most common form of inherited mental retardation, is caused by mutations in the FMR1 gene. Fragile X patients display neurite morphology defects in the brain, suggesting that this may be the basis of their mental retardation. The role of Drosophila Fmr1 in neurite development was examined in three distinct neuronal classes. Fmr1 is required for normal neurite extension, guidance, and branching. Fmr1 mutants also display strong eclosion failure and circadian rhythm defects. Interestingly, distinct neuronal cell types show different phenotypes, suggesting that Fmr1 differentially regulates diverse targets in the brain (Morales, 2002).

Four different Fmr1 alleles, one hypomorphic P element insertion allele and three null deletion alleles, as well as Fmr1 transgenic flies, were used to study the effects of Fmr1 loss and gain of function. The expression and distribution of Fmr1 in the fly brain was examined. The mammalian FMR1 protein is enriched in the brain, but its expression is specific to neurons and excluded from glia. To determine the developmental regulation of Fmr1 brain expression, pupal brains were examined immunohistochemically, at two stages of pupal development, and adult brains were examined using three methods, anti-Fmr1 antibodies, a neuronal specific marker (elav [c155] driven GFP), and glial specific (REPO) marker. Fmr1 was constitutively expressed in most, if not all, neuronal cell bodies and excluded from glia. This pattern persists in adults. Importantly, no Fmr1 protein was detected in the brains of any Fmr1 deletion mutant analyzed (Morales, 2002).

Considering that the most significant phenotype of fragile X patients is mental retardation and that the mammalian gene has been implicated in the regulation of neurite morphology, it was of significant interest to show that this model could be used to study neuronal phenotypes in the Drosophila brain. DC neurons were analyzed because these cells exhibit a simple stereotypical pattern of axon branching in the distal medulla, allowing any abnormalities to be readily observed. Also, the morphology of the LNv, the major circadian rhythm centers in the fly brain were examined Analysis of DC and LNv cells in Fmr1 mutants shows that the loss of Fmr1 causes axon extension defects. In DC neurons, all mutant brains examined showed failure of axon extension. Importantly, this extension defect was less severe in hypomorphic mutants expressing low levels of the protein; i.e., the level of Fmr1 activity is proportional to the observed phenotype. In contrast, overextended axons were observed for LNv neurons, indicating that Fmr1 acts to inhibit axon extension in these cells. Interestingly, not all Fmr1 mutant brains exhibited LNv neuronal defects, suggesting that the role of Fmr1 may be redundant in these cells. Finally, photoreceptor neurons appear morphologically normal in mutants, demonstrating that Fmr1 activity is differentially required within the nervous system. Interestingly, overexpression of Fmr1 in DC neurons, both in wild-type and mutant backgrounds, results in a complete failure of axon extension. Therefore, loss and gain of function led to similar phenotypic defects, potentially suggesting that the dosage of Fmr1 is critical for its function. In vertebrates, it has been shown that the dosage of the protein is correlated with behavioral abnormalities. In addition to neurite extension defects, neurite branching abnormalities are observed for the DC neurons in Fmr1 mutants. It is important to note that even hypomorphic allelic combinations showed strong branching defects. This suggests that Fmr1 regulates extension and branching independently, and this can be most easily explained by assuming that each process requires a different set of Fmr1 targets (Morales, 2002).

In addition to neuronal morphology, the role of Fmr1 in neuronal function was examined. Several defects were observed. Large numbers of Fmr1 mutants fail to eclose and die as fully developed pharate adults with no visible morphological defects. This phenotype may be the consequence of an underlying neural defect since Fmr1 is expressed within the DC neurons, and a similar eclosion phenotype is observed with lesions affecting the DC neurons. However, since Fmr1 is expressed in many different neurons, defective eclosion may arise from a perturbation of another cell type (Morales, 2002).

Fmr1 mutants exhibit abnormal circadian behavior, although the characterization of eclosion rhythms and Period cycling in mutant populations indicates that the molecular clock is intact. Defects affecting the DC neuronal population are known to cause eclosion failure. Indeed, for several different Fmr1 alleles, homozygous mutants develop until the pharate adult stage and then fail to eclose. Did Fmr1 mutations affect the circadian timing of adult eclosion, an independent rhythm controlled by the fly circadian clock system? Interestingly, eclosion exhibits normal circadian periodicity in constant daylight (DD); i.e., the medium of eclosion peaks was at ~CT12 on all 3 days of DD, indicating a normal 24 hr circadian period. However, peaks of eclosion were phase delayed each day in the mutant by as much as 6-8 hr, relative to heterozygous (control) siblings, and such a phenotype is consistent with an effect on rhythmicity that occurs downstream of the clock mechanism. The vast majority of homozygous mutant adult 'escapers' exhibited weak and erratic rhythmicity or were statistically arrhythmic (Morales, 2002).

The alteration of circadian behavior may arise as a consequence of defects in the LNV neuronal projections or because of functional changes within the LNV population. While the LNV morphological phenotypes were variable in severity and observed in fewer than half the mutants, circadian locomotor activity defects were seen in most mutant flies. One possibility is that some individuals have subtle synaptic morphology defects that are nonetheless severe enough to impair LNv function. Alternatively, it may be that loss of Fmr1 impairs a different aspect of LNv function such as the rhythmic release of the PDF neuropeptide, which is known to be critical for the circadian regulation of behavior. Interestingly, it has been reported that fragile X patients exhibit increased variability in total sleep time and problems with sleep maintenance, phenotypes that might be due to abnormal circadian regulation (Morales, 2002).

Observations on Fmr1 function in the central brain, NMJ, and the retina/optic lobe complex indicate that the protein not only regulates multiple processes within a class of neurons, but also regulates these processes differentially between different classes of neurons. Mechanistically, this can be best explained by assuming that the phenotypically relevant targets of Fmr1 regulation are (1) diverse and (2) vary from one cell type to another. Thus, while the rescue of Fmr1 phenotypes by MAP1B mutants suggests that upregulation of MAP1B in the absence of Fmr1 may be sufficient to explain the peripheral phenotypes, it is very difficult to imagine how it would suffice to explain the diverse, variable, and contrasting central defects. That is, a perturbation of other targets must be the cause of the central defects. The description of neuronal phenotypes in Drosophila Fmr1 mutants will permit genetic studies to identify potential targets and other components of the signaling pathways that are relevant for an understanding of fragile X pathogenesis (Morales, 2002).

Drosophila lacking Fmr1 activity show defects in circadian output and fail to maintain courtship interest

Adult Fmr1 mutant flies display arrhythmic circadian activity and have erratic patterns of locomotor activity, whereas overexpression of Fmr1 leads to a lengthened period. Fmr1 mutant males also display reduced courtship activity which appears to result from their inability to maintain courtship interest. Molecular analysis fails to reveal any defects in the expression of clock components; however, the CREB output is affected. Morphological analysis of neurons required for normal circadian behavior reveals subtle abnormalities, suggesting that defects in axonal pathfinding or synapse formation may cause the observed behavioral defects (Dockendorff, 2002).

One known clock-controlled gene in Drosophila is the cAMP response element binding protein (CREB). To determine if the circadian oscillation of this protein is affected in the Fmr1 mutant flies, Fmr1 mutant flies carrying the CRE-luciferase (CRE-luc) reporter gene were examined in a luminometer continuously in constant daylight (DD) for up to 4 days. Although cycling of the CRE-luc reporter is detected in the Fmr1 mutant background, the amplitude of the oscillations is clearly reduced compared to the oscillations in the control background. This result indicates that dfmr1 affects a known molecular output of the clock. Normal cycling of PDF levels was seen in the termini of the small lateral neurons in the Fmr1 mutant brains. Thus this output of the clock is not affected at the normal site of its release, providing further evidence for normal central clock functioning in the Fmr1 mutant flies (Dockendorff, 2002).

Fmr1 mutants failed to activate advanced stages of courtship in response to the courtship-stimulating cues of virgin females. To ask whether the reduced courtship seen in Fmr1 mutant males represents a defect in central courtship-activation systems or a specific sensory deficit, courtship assays were performed in the presence of immature males, which possess pheromonal profiles different from virgin females, but nonetheless they stimulate older males to court. Similar to results obtained with virgin females, Fmr1 mutant males display significantly less courtship activity than control males toward immature males, and largely fail to proceed to the more advanced stages of courtship exhibited by controls. The results indicate that Fmr1 mutants display the same lack of interest in courting two anatomically and pheromonally distinct objects, suggesting that this behavioral phenotype is not likely a result of a specific sensory deficit. This interpretation is supported by results of analyzing the fine structure of courtship behavior. The duration of individual bouts of courtship varied greatly in control males, but was consistently very short in the mutants. Indeed, only 7% of courtship bouts with immature males lasted longer than a single sampling interval in Fmr1 mutants. During pairings with immature males, Fmr1 mutant males initiated courtship just as often as controls, averaging 4-5 bouts of courtship in 10 min. These results indicate that the reduced courtship phenotype of Fmr1 mutant males is largely the result of a failure to engage in sustained bouts of courtship and not in the initiation of courtship (Dockendorff, 2002).

It is interesting to note that the defects observed in the Fmr1 mutant flies for circadian and courtship behavior have similarities to behavioral phenotypes observed in patients with fragile X syndrome. Fragile X patients commonly have abnormal sleep patterns with shortened periods of sleep and longer wake episodes, suggesting defects in their circadian systems. Consistent with this observation, many fragile X patients have been found to have an altered melatonin profile, a well-known output of the circadian clock. Cognitive analysis of fragile X patients has identified mild to severe mental retardation, associated with specific weaknesses in short-term memory, sequential information processing, and other more complex abilities. Psychiatric diagnoses are also common among fragile X patients, with a high rate of attention deficit hyperactivity disorder. The similarities between the human and fly mutant phenotypes and the shared biochemical properties of the two proteins suggest that studies of Fmr1 will be a useful model to identify physiological pathways and substrates affected by the mammalian FMR1 gene (Dockendorff, 2002).

A role for the Drosophila Fragile X-related gene in circadian output

Fly strains bearing deletions in Drosophila Fmr1 have been produced. Since human fragile X patients show a number of abnormal behaviors, including sleep problems, this study investigated whether a loss-of-function mutation of Drosophila Fmr1 affects circadian behavior. Under constant darkness (DD), a lack of Fmr1 expression causes arrhythmic locomotor activity, but in light:dark cycles, behavioral rhythms appear normal. In addition, the clock-controlled eclosion rhythm is normal in Fmr1-deficient flies. These results suggest that Fmr1 plays a critical role in the circadian output pathway regulating locomotor activity in Drosophila (Inoue, 2002).

To determine the pattern of expression of Fmr1, the levels of Fmr1 protein accumulating during a circadian cycle were examined by Western blot analysis. No significant difference in Fmr1 protein levels was detected in head extracts from each of the time points examined in both LD and DD conditions, showing that levels of Fmr1 protein are not under circadian control (Inoue, 2002).

The signaling mechanism that mediates output from central clock proteins to behavior is poorly understood. Several output genes have been identified so far in Drosophila. The circadian phenotype displayed by dfmr1B55 Fmr1 deficiency flies is reminiscent of what happen in flies deficient in either protein kinase A (PKA) or NF1, the protein product of the neurofibromatosis-1 gene. However, whether mutations in Fmr1 and PKA or NF1 lead to arrhythmic activity by similar or different pathways is currently not clear. An intriguing finding is that Fmr1-deficient flies manifest normal eclosion rhythms, suggesting that the daily timing of developmental rhythm might not require Fmr1. These results suggest that eclosion and locomotor rhythms are mediated by different neurons that use the same pacemaker molecules (Inoue, 2002).

How, then, might Fmr1 participate in an output pathway associated with the manifestation of overt locomotor activity rhythms? Fmr1 is a cytoplasmic RNA binding protein associated with ribosomes, as is the case for mammalian FMR1. Therefore, Fmr1 could regulate posttranscriptionally the expression of specific target mRNAs that control output functions. Given the recent findings showing that a secreted neuropeptide, pigment-dispersing factor (PDF) is a critical circadian mediator that couples a molecular clock to circadian rhythms in locomotor activity and can in turn influence function of the clock, it will be important to follow the fate of this peptide in dfmr1B55 flies. Alternatively, since in both the human and Drosophila, the fragile X protein (FMR1 and Fmr1 respectively) has been found to have a role in synaptic growth, Fmr1 might regulate expression of mRNAs required for synaptic function and structure such as mRNA for microtubule-associated protein MAP1B. Proteins that affect neuronal development and/or function are expected to affect circadian rhythms that are driven by neuronal pacemakers. For example, similar results to those found in dfmr1B55 flies were obtained when synaptic transmission was blocked using the tetanus-toxin light chain in per/tim-expressing cells, i.e., less effect on locomotor activity during LD but largely arrhythmic during constant dark conditions (Inoue, 2002).

It is tempting to speculate that sleep problems observed in fragile X patients are attributable to alterations of circadian rhythmicity because sleep propensity is modulated by a circadian clock. Since the molecular mechanisms involved in the generation of circadian rhythms are remarkably similar between Drosophila and mammals, the Drosophila model of fragile X syndrome provides insight into the sleep-wake cycles of animals. The discovery of modifiers involved in Fmr1-mediated regulation of circadian rhythms reveals additional molecular mechanisms in the fragile X syndrome (Inoue, 2002).

Control of dendritic development by the Drosophila fragile X-related gene involves the small GTPase Rac1

Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 gene. How these mutations affect neuronal development and function remains largely elusive. Specific point mutations or small deletions have been generated in the Drosophila fragile X-related (Fmr1) gene, and the roles of Fmr1 in dendritic development of dendritic arborization (DA) neurons have been examined in Drosophila larvae. Fmr1 can be detected in the cell bodies and proximal dendrites of DA neurons and Fmr1 loss-of-function mutations increase the number of higher-order dendritic branches. Conversely, overexpression of Fmr1 in DA neurons dramatically decreases dendritic branching. In dissecting the mechanisms underlying Fmr1 function in dendrite development, it was found that the mRNA encoding small GTPase Rac1 is present in the Fmr1-messenger ribonucleoprotein complexes in vivo. Mosaic analysis with a repressor cell marker (MARCM) and overexpression studies reveals that Rac1 has a cell-autonomous function in promoting dendritic branching of DA neurons. Furthermore, Fmr1 and Rac1 genetically interact with each other in controlling the formation of fine dendritic branches. These findings demonstrate that Fmr1 affects dendritic development and that Rac1 is partially responsible for mediating this effect (Lee, 2003).

Each abdominal hemisegment of Drosophila contains 44 sensory neurons that can be grouped into dorsal, lateral and ventral clusters. To test whether Fmr1 affects the dendritic growth of DA neurons, the expression of Fmr1 in these neurons was confirmed. Fmr1 mRNA is expressed at high levels in the embryonic nervous system and in body wall muscles. To examine the subcellular localization of Fmr1 in DA neurons of live larvae, a UAS-Fmr1-GFP transgenic fly line was generated. When Fmr1-GFP was expressed in DA neurons driven by Gal4 109(2)80, Fmr1 expression was observed in the cytoplasm of DA neurons and in particle-like structures in dendrites. To further confirm that the endogenous Fmr1 is expressed in DA neurons, immunostaining analysis was performed on dissected larvae using a monoclonal antibody raised against Fmr1. Fmr1 is present in DA neurons and is expressed predominantly in the cytoplasm. The expression of Fmr1 in the proximal dendrites of DA neurons and in body wall muscle fibers was also detectable. Owing to the high level of Fmr1 expression in muscles, the localization of endogenous Fmr1 in distal dendrites was barely visible with confocal microscopy. The subcellular localization of Fmr1 in DA neurons is consistent with the subcellular localization of FMR1 in mammalian neurons (Lee, 2003).

Because Fmr1 mutants are viable, it is possible to directly examine the effects of Fmr1 mutations on dendritic development of specific neurons in a large number of live flies. To label all dendritic processes, UAS-mCD8::GFP, which targets to the cell membrane, was expressed in all DA neurons. Third instar larvae were selected 4-5 days after egg laying (AEL) and the images of dendrites of ventral DA neurons were recorded from segments 5 and 6 in live animals. The Fmr1 mutant larvae exhibit more dendritic processes than wild-type larvae. To quantify the difference, the number of ends of all dendritic terminal processes were counted. On average, Fmr1 mutations increase the number of terminal dendritic processes of ventral DA neurons by 25%. To demonstrate that the increased number of terminal dendritic processes in Fmr1 mutants are indeed due to the absence of Fmr1 activity, one copy of the wild-type Fmr1 gene was introduced into the Fmr14 mutant background; the transgene rescues the dendritic defects in Fmr1 mutants. A large number of segments in wild-type and Fmr1 mutant larvae exhibit a similar number of terminal dendritic processes, indicating that there is a large variation among individual larvae of a given genotype and that Fmr1 mutations cause subtle changes in neuronal morphology (Lee, 2003)

To further understand the function of Fmr1 in regulating dendritic growth, Fmr1 was overexpressed in all DA neurons of wild-type wandering larvae. To do so, UAS-Fmr1 flies were crossed with Gal4 109(2)80 flies and the third-instar larvae were examined 4 days AEL. The numbers of terminal dendritic processes were dramatically reduced in both ventral and dorsal DA neurons when Fmr1 was overexpressed. The length of remaining terminal processes was also greatly reduced. This phenotype caused by Fmr1 overexpression is 100% penetrant (Lee, 2003)

Drosophila larvae increase their body surface over 50-fold from the first to the third instar larval stages. Correspondingly, the dendritic fields of DA neurons increase substantially during this period of development. In larvae overexpressing Fmr1, the major dendritic branches are still capable of extending more than fivefold during larval development. However, most terminal processes fail to form or fully extend even at the first instar stage. This demonstrates that overexpression of Fmr1 blocks the formation of higher-order dendritic branches and reduces the complexity of DA neuron dendrites during development (Lee, 2003)

The KH domains of Fmr1 share more than 70% identity with the mammalian FMR1 proteins. Indeed, Fmr1 and human FMR1 have similar RNA-binding properties in vitro. A number of studies have identified a large number of mRNAs that are associated with FMR1 in mammalian systems. However, systematic identification of Fmr1-binding targets in flies has not been carried out. To gain mechanistic insights into Fmr1 function in controlling dendritic growth in flies, co-immunoprecipitation experiments were carried out to identify mRNAs that are associated with the Fmr1-mRNP complex in vivo. In this study, using primers specific for genes encoding small GTPase Rac1, alpha-tubulin, and the voltage-gated K+ channel molecule Hyperkinetic, RT-PCR analyses was performed on either total RNAs or the RNAs that were immunoprecipitated by an anti-Fmr1 monoclonal antibody, from lysates derived from third instar larvae. All three mRNAs could be readily detected from total RNAs, while only the Rac1 mRNA was associated with Fmr1 in lysates derived from wild-type larvae as shown by coimmunoprecipitation experiments. These studies demonstrate that Rac1 mRNA is associated with Fmr1-mRNP complexes in vivo (Lee, 2003)

Based on the finding that Rac1 mRNA is present in Fmr1-mRNP complexes in vivo, it was hypothesized that the effect of Fmr1 on dendritic development in DA neurons may be partially mediated by Rac1. This hypothesis was tested genetically. First, the function of Rac1 in dendritic growth and branching of DA neurons was examined in Drosophila embryos. A null allele, Rac1J11, was tested. Gal4 109(2)80 was used to drive the expression of GFP in DA neurons in Rac1J11 mutant embryos and no gross defects were observed in dendritic branching patterns in later embryogenesis stages. DA neuron dendrites develop in discrete phases from the embryonic to larval stages. In embryos, dorsal dendrites of DA neurons extend from cell bodies first, and stop elongation 16-17 hours AEL, falling short of the dorsal midline. The lateral dendrites start to extend toward adjacent segment boundaries and cover the hemisegment before hatching (22-23 hours AEL). These findings in Rac1J11 mutant embryos suggest that Rac1 is not required for the initial growth of dorsal dendrites during embryogenesis (Lee, 2003)

During larval stages, the dendritic fields of DA neurons expand many-fold in accordance with the increase of larval body size. Higher-order dendritic branches further develop to cover the whole epidermal surface of each hemisegment. The MARCM technique was used to examine the role of endogenous Rac1 in dendritic growth in the third instar larval stage. Single GFP-labeled wild-type or Rac1 mutant DA neurons were generated in abdominal segments and the number of terminal dendritic branches was counted. Rac1 mutant ddaC neurons fewer dendritic branches than wild-type neurons, a phenotype similar to that caused by Fmr1 overexpression. Different Rac1J11 mutant ddaC neurons exhibit varying severities of dendritic defects. On average, there was a 23% reduction in the number of dendritic branches due to the Rac1 mutation. Similar dendritic defects were also found in other DA neurons. These findings demonstrate that Rac1 is required for normal dendritic branching of DA neurons in vivo, consistent studies that rely on the ectopic expression of dominant mutant forms of Rac1 (Lee, 2003)

To support the notion further that Rac1 is partially responsible for the effect of Fmr1 on dendritic development, Rac1 was overexpressed in DA neurons in third instar larvae with the UAS-Gal4 system. Consistent with the finding that Rac1 loss-of-function results in a decreased number of terminal dendritic branches, overexpression of Rac1 promotes dendritic branching of DA neurons with 100% penetrance. This result is also in line with previous studies that ectopic expression of the constitutively active form of Rac1 promotes dendritic branching. The enhanced dendritic branching caused by Rac1 overexpression is much more dramatic than that caused by Fmr1 loss-of-function, and this is presumably due to the high level of ectopic expression of Rac1 (Lee, 2003)

Because Fmr1 (or its mammalian homolog FMR1) can function as a translation inhibitor, it was of interest to enquire whether the elevated Rac1 expression obtained by using the UAS-Gal4 system would partially rescue the dendritic phenotype caused by Fmr1 overexpression. To test this hypothesis, Fmr1 and Rac1 were expressed simultaneously in DA neurons driven by Gal4 109(2)80. Overexpression of Fmr1 decreases the number of higher-order dendritic branches, but could be partially rescued by co-expression of Rac1. In addition, the number of terminal dendritic branches in Fmr14 mutants with a reduced rac1 dosage was significantly lower that that in Fmr14 mutants. These findings support the notion that Rac1 is one of the downstream components of Fmr1 function in controlling dendritic development (Lee, 2003)

RNA-mediated neurodegeneration caused by the Fragile X premutation rCGG repeats in Drosophila

Fragile X syndrome, a common form of inherited mental retardation, is caused by a massive CGG trinucleotide repeat expansion in the 5' untranslated region (UTR) of the FMR1 gene that leads to transcriptional silencing and the absence of the encoded Fragile X Mental Retardation Protein (FMRP). Fragile X syndrome carriers have FMR1 alleles, called premutations, with an intermediate number of 5' untranslated CGG repeats between patients (>200 repeats) and normal individuals (<60 repeats). A novel neurodegenerative disease has recently been appreciated in some premutation carriers. Since no neurodegeneration is seen in fragile X patients, who do not express FMR1, it is hypothesized that lengthened rCGG repeats of the premutation transcript may lead to neurodegeneration. Here, using Drosophila, it is shown that 90 rCGG repeats alone are sufficient to cause neurodegeneration. This phenotype is neuron specific and rCGG repeat dosage sensitive. Although devoid of mutant protein, this neurodegeneration exhibits neuronal inclusion bodies that are Hsp70 and ubiquitin positive. Overexpression of Hsp70 suppresses the neurodegeneration. These results demonstrate that neurodegenerative phenotype associated with fragile X premutation is indeed caused by the lengthened rCGG repeats and provide the first in vivo experimental demonstration of RNA-mediated neurodegeneration (Jin, 2003).

Neurodegenerative diseases are a heterogeneous group of disorders that usually strike in mid-life and include the polyglutamine diseases, the tauopathies, and Parkinson's disease. Most of these mutations are found within the coding region of the relevant loci and share a common feature of misfolding of the mutant proteins. However, several neurodegenerative disorders, including Spinocerebellar ataxia type 8, 10, 12, and Huntington's disease-like type 2, have been linked to noncoding repeat expansions. While the underlying mechanisms for these disorders remains obscure, a toxic RNA-mediated gain-of-function has been suggested along with other possibilities. Apparently similar to these disorders is the recently described neurodegenerative syndrome characterized by progressive intension tremor and ataxia in fragile X premutation carriers. The common feature, besides neurodegeneration, is the hypothetical link of a noncoding RNA-mediated neurodegeneration. Since full mutation patients with fragile X syndrome, who do not express FMR1 message, do not show evidence of neurodegeneration, the causal focus has fallen upon the premutation message. Using Drosophila as a model system, this study demonstrates that indeed a portion of the human FMR1 5' UTR of a premutation allele containing 90 rCGG repeats is alone sufficient to cause neurodegeneration. A normal CGG repeat of 60 triplets, when moderately expressed, has little phenotype, and this same allele, when overexpressed, does lead to neurodegeneration, supporting the notion that overall rCGG abundance is critical. Therefore, it is likely for the human disorder that a combination of CGG repeat length and FMR1 message abundance together may define a threshold for the clinical phenotype (Jin, 2003).

The intriguing observation from the neuroanatomical studies on fragile X premutation carrier males with neurodegenerative phenotype is the presence of ubiquitin-positive intranuclear neuronal inclusions. The origin of the intranuclear inclusions is unknown; however, some features are also observed with the polyglutamine disorders. In this study, it has been shown that fragile X premutation rCGG repeats not only cause neurodegeneration but also induce the formation of inclusions. The presence of ubiquitin and proteasome complex within the inclusions suggests a role of the protein degradation pathway in the pathogenesis of this tremor/ataxia syndrome associated with fragile X premutation carriers. Interestingly, recent neurohistological studies on expanded-CGG repeat mouse also show the presence of ubiquitin-positive inclusions. These results suggest that high level of rCGG repeat can lead to the formation of inclusions. One discrepancy between this fly model and human pathological study is the presence of inclusion in both nuclei and cytoplasm in the fly model. However, in the expanded CGG repeat mouse model, both nuclear and cytoplasmic inclusions are also observed. This difference may be human specific; however, in some polyglutamine diseases, such as Huntington disease, inclusions were also found present in both nuclei and cytoplasm. Alternatively, it might be an age-related phenomenon or disease state-dependent, since human pathological study was done using postmortem brains (Jin, 2003).

Molecular chaperone, Hsp70, is a constituent of the inclusions, and variable expression of Hsp70 can modify the degenerative phenotype in the eye. Studies in both flies and mice show that overexpression of chaperones or HSPs, particularly Hsp70, which help fold proteins or target them for degradation, increase resistance to polyglutamine-induced toxicity. However, in the fly model, the CGG repeats are only transcribed but not translated, so there is no mutant protein to misfold and be a chaperone target. (It is noted that CGG, in any reading frame, cannot code for polyglutamine nor is any polyglutamine detected immunohistochemically in the inclusions, thereby ruling out the trivial explanation of these results by upstream promiscuous translation.) It has been well known that long triplet repeats can form stable hairpin structure, and it is possible that the protein(s) interacting with long rCGG repeats (possible double-stranded RNA) may fold into a stable alternative conformation, which results in aggregation, and become the target for protein degradation. In addition, Hsp70 may also confer the protection by inhibiting signal transduction pathways leading to cell death, by preventing activation of stress kinases, or by blocking pro-caspase processing or caspase activation. Finally, this data implicating the role of protein degradation in RNA-mediated neurodegeneration links this form of neurodegeneration to the larger class of neurodegenerative diseases exhibiting features of protein misfolding. By inference of this Drosophila model, it might now be speculated that the human disorders linked to noncoding repeat loci are likely to involve RNA-mediated neurodegeneration and to share this overall feature of protein misfolding, thus linking all human neurodegenerative diseases together (Jin, 2003).

Pathogenic RNAs that alter cellular functions have been associated with several human diseases. In myotonic dystrophy type 1 (DM1), a CTG expansion in the 3' UTR sequesters CUG binding proteins from their normal cellular functions, leading to abnormal RNA splicing of several genes. It is likely that fragile X premutation rCGG may behave similarly. In the fragile X premutation carriers with elevated FMR1 mRNA, the long rCGG tract may attract and sequester rCGG binding protein(s) from its normal functions, affect RNA metabolism, increase cellular toxicity, and lead to progressive cell death, particularly in the brain since it has highest expression of FMR1 gene. Indeed, it has been shown that rCGG repeats can be bound by the proteins from mouse brain. Identification of these rCGG binding proteins will be important to test this hypothesis and understand the pathogenesis of this novel disorder (Jin, 2003).

In conclusion, it has been demonstrated that RNA alone is sufficient to cause neurodegeneration and that this form of neurodegeneration shares the feature of protein misfolding involvement common to most other forms of genetic neurodegeneration. These data also strongly support the emerging clinical picture of a specific neurodegenerative disease associated with fragile X premutation carriers and suggest this disorder may exhibit a clinical threshold based upon total rCGG abundance. Finally, based upon the data presented here, the power of Drosophila genetics can now be used to dissect the molecular basis of RNA-mediated neurodegeneration through enhancer and suppressor screens and to test novel therapeutic approaches (Jin, 2003).

The Drosophila Fragile X protein dFMR1 is required during early embryogenesis for pole cell formation and rapid nuclear division cycles

The FMR family of KH domain RNA binding proteins are conserved from invertebrates to humans. In humans inactivation of the X-linked FMR gene, Fragile X, is the most common cause of mental retardation and leads to defects in neuronal architecture. While there are three FMR family members in humans, there is only a single gene, dfmr1, in flies. As in humans, inactivation of dfmr1 causes defects in neuronal architecture and in behavior. dfmr1 has other functions in the fly besides neurogenesis. This study analyzed its role of during early embryonic development. dfmr1 embryos were found to display defects in the rapid nuclear division cycles that precede gastrulation, in nuclear migration and in pole cell formation. While the aberrations in nuclear division are correlated with a defect in the assembly of centromeric/centric heterochromatin, the defects in pole cell formation are associated with alterations in the actin-myosin cytoskeleton (Despande, 2006; full text of article).

Recent studies have shown that dFMR1 is physically associated with Ago-2 and other components of the RNAi machinery and have suggested that this association may be important for the regulatory functions of the dFMR1 protein. Analysis of early embryogenesis in dfmr1 mutant embryos provides additional evidence for a connection between dfmr1 and the RNAi machinery. Ago-2 activity is required for the proper execution of the rapid nuclear division cycles that precede the formation of the cellular blastoderm and a variety of defects are evident in ago-2 mutant embryos. These include asynchronous nuclear division, incomplete chromosome condensation, defects in chromosome segregation, and chromosome fragmentation. Significantly, the same defects in the nuclear division cycles are evident in dfmr1 embryos: nuclear division is asynchronous, mitotic figures have incompletely condensed and lagging chromosomes, and there is evidence of chromosome fragmentation. In addition, the assembly/functioning of the spindle apparatus is abnormal, and like ago-2, many dfmr1 embryos have 'orphaned' centrosomes that have duplicated and migrated to 'opposite poles' even though no nucleus is apparent. In the case of ago-2, around half of the embryos exhibit at least one of these nuclear division phenotypes, while for dfmr1 25%-30% of the embryos show at least one of these phenotypes (Despande, 2006).

Many of the defects in nuclear division seen in ago-2 embryos can be attributed to a failure in the assembly of centromeric and centric heterochromatin. The centromeric-specific histone H3 variant CID is often present in greatly reduced amounts or completely absent from the centromeric regions of ago-2 mitotic chromosomes. There are also abnormalities in the localization of the centric heterochromatin protein HP1. Although CID was not examined in dfmr1 embryos, it was found that the centric heterochromatin protein HP1 is mislocalized in dfmr1 embryos much like that observed in ago-2 mutants. Tests were performed for the establishment/maintenance of functional heterochromatin by examining the effects of dfrm1 mutations on the silencing of a white transgene inserted on the fourth chromosome. A reduction in dfmr1 activity was quite effective in suppressing the silencing of a white transgene inserted into a heterochromatic region of the fourth chromosome. Although the strongest suppression was observed when the mothers were homozygous for dfmr13, suppression was also evident in the reciprocal cross in which fathers were homozygous for dfmr13. These findings indicate that dfmr1 is required to maintain the silenced state as the fly develops. In addition, these results point to a role for dfmr1 in the initial establishment of the silenced state in the embryo. First, suppression is stronger when the mother is homozygous mutant than when the father is. Second, suppression is also seen in all progeny of heterozygous dfrm13 females. In this case, two equal classes were observed. In the first class, suppression is relatively strong, and these flies are presumed to be heterozygous for the dfmr13 mutation. Weak suppression is observed in the second class and these flies are presumed to be wild type for dfmr1. These findings indicate that homochromatic silencing is dependent upon maternally contributed dFMR1 and would be consistent with the idea that dFMR1 functions in the establishment of the silenced state at a point early in embryogenesis (Despande, 2006).

Taken together with the physical association between dFMR1 and the components of the RNAi machinery, these results would support the idea that dFMR1 functions as a cofactor in an RNAi pathway required in early embryos for the proper execution of the nuclear division cycles and for the assembly of functional centric/centromeric heterochromatin. In contrast, there remains the question of why the defects in the nuclear division cycles in dfmr1 and also in ago-2 embryos are not fully penetrant. In the case of ago-2, there are other Argonaute-related genes in the fly that could potentially perform partially redundant or overlapping functions that compensate for the loss of Ago-2. Consistent with this idea, a similar spectrum of nuclear division defects is evident in embryos from mothers deficient in piwi activity. Moreover, like ago-2, the nuclear division phenotypes in piwi embryos are not fully penetrant. However, this explanation would not account for the incomplete penetrance of dfmr1, since it is the only fragile X family member in the fly. One plausible idea is that dfmr1 functions as a facilitator in the nuclear division cycle RNAi pathway, but is not absolutely essential for the operation of this pathway. Alternatively, there may be other RNA-binding proteins that can substitute for dFMR1 (Despande, 2006).

While the various nuclear division cycle abnormalities in dfmr1 embryos closely resemble those observed in ago-2 (or in piwi), this is only partially true for the pole cell formation phenotypes. Like ago-2, there is a small but significant reduction in the number of pole cells in dfrmr1 embryos. Like ago-2, this could be due at least in part to defects in the migration of nuclei into the posterior pole, in which case it probably arises from the disruptions in the nuclear division cycles during the presyncytial blastoderm stages. However, this is not the most striking phenotype in dfmr1 embryos. In contrast to either ago-2 or wild-type embryos, the pole cells in dfmr1 embryos fail to properly segregate from the surrounding somatic nuclei/cells and instead remain intermingled with somatic nuclei in syncytial blastoderm embryos and somatic cells in cellular blastoderm embryos. Unlike the various nuclear division cycle phenotypes, the pole cell segregation phenotype is fully penetrant and is seen in virtually every appropriately staged dfmr1 embryo. dfmr1 also differs from ago-2 in that transcriptional quiescence is not properly established in all pole cells (Despande, 2006).

The pole cell segregation phenotype in dfmr1 embryos can be traced back to the budding stage. In wild-type embryos, nuclei migrating into the pole plasm induce the formation of buds. The outside surface of the buds is marked by actin and Anillin, while there is a Chickadee ring surrounding the pole bud nucleus. Once the buds, including the pole cell nuclei, have been fully extruded from the surface of the embryo, the actin/Anillin ring at the base of the bud contracts, completing the cellularization process. With the completion of cellularization, the exterior surface of the embryo is redefined with the pole lying outside of the embryo. In dfmr1 embryos, the pole buds fail to emerge from the surface of the embryo or, if they do, they do not enlarge properly. The process of pole bud emergence and/or enlargement appears to be short circuited by 'precocious' cellularization. In these experiments, this is marked by the formation of partially closed and closed Anillin rings around unbudded or incompletely budded pole cells. Once the pole cells form, many remain embedded in the somatic layer instead of locating on the exterior of the embryo and, when the blastoderm cellularizes, the pole cells are often intermingled with somatic cells (Despande, 2006).

The dfmr1 pole cell segregation phenotype resembles that reported for rhogef2 mutants. In rhogef2 mutant embryos, the pole buds fail to form cortical actin networks that are separated from the somatic layer of nuclei and, when the buds cellularize, they remain embedded in the soma. Because of the apparent similarities in phenotypes, it was of interest to find out whether the expression of Rhogef2 was impaired in dfmr13 embryos. Although there was no reduction in expression, it was found that the localization of Rhogef2 in the pole buds/cells and in the soma of dfmr13 embryos was abnormal much like that observed for Anillin. Taken together with the effects of dfmr1 on Profilin and Pnut, these findings would suggest that dfrm1 activity is required for the proper assembly and/or functioning of the actin–myosin cytoskeleton during pole cell formation (and later during the cellularization of the somatic nuclei) (Despande, 2006).

Two different but not mutually exclusive models could potentially account for the disruptions in the actin–myosin cytoskeleton in dfmr1 embryos. In the first, dFMR1 would play a more or less direct role in organizing the cytoskeleton through protein–protein interactions with effector molecules. In this view, the organization of the cytoskeleton in dfmr1mutants would be perturbed because dFMR1 is not available to influence the activity of these effector molecules. This possibility is supported by a number of findings. First, dFMR1 interacts with the Rac1-GTP-binding protein Sra-1/CYFIP. Sra-1/CYFIP is part of a multi-component complex (SCAR/WAVE) that regulates actin nucleation through the activation of Arp2/3. The complex is activated when Sra-1/CYFIP is displaced from the complex by the GTP-bound form of Rac1. Second, dFMR1 has also been shown to interact with the lethal-(2)-giant-larvae (LGL) protein. LGL is a component of the PAR complex, which is involved in establishing cellular asymmetries. Additional support for this idea comes from the association of mammalian FMR proteins with the actin cytoskeleton. The second model is that dfmr1 influences the organization of the cytoskeleton indirectly through its ability to downregulate the translation of target mRNAs. In this case, the cytoskeleton would be perturbed in dfmr1 embryos because the stoichiometric balance between components of the cytoskeletal that are dfmr1 targets and those that are not would be altered. Consistent with this idea, several of the known mRNA targets for dfmr1 regulation in the nervous system encode components of both the actin–myosin and the microtubule cytoskeleton (rac1, chic, and futsch). Since antibody-staining experiments suggest that the profilin Chic may be overexpressed in dfmr1 mutant embryos, it would be reasonable to think that the translation of chic mRNA may also be regulated by dFMR1 during early embryogenesis. Chic is probably not the only cytoskeletal target for dfmr1 that is misregulated in mutant embryos. While neither Rac1 or Futsch expression was examined in this study, antibody-staining experiments indicate that Anillin is present at higher levels in dfmr1 embryos than in similarly staged wild-type embryos. Like many of the other known targets for dFMR1, Drosophila Anillin mRNA has several potential dFMR1 recognition motifs. In this respect, it is interesting to note that Orb protein does not seem to be overexpressed in dfmr1 pole cells, while it is overexpressed in dfmr1 ovaries (in both nurse cells and the oocyte). This finding suggests that the mRNA targets for dfmr1 regulation may vary with different tissues or stages of development. Presumably, this depends upon what other regulatory cofactors are present or absent (Despande, 2006).

The role of PIWI and the miRNA machinery in Drosophila germline determination

The germ plasm has long been demonstrated to be necessary and sufficient for germline determination, with translational regulation playing a key role in the process. Beyond this, little is known about molecular activities underlying germline determination. This study reports the function of Drosophila Piwi, Dicer-1, and dFMRP (Fragile X Mental Retardation Protein) in germline determination. Piwi is a maternal component of the polar granule, a germ-plasm-specific organelle essential for germline specification. Depleting maternal PIWI does not affect Osk or Vasa expression or abdominal patterning but leads to failure in pole-plasm maintenance and primordial-germ-cell (PGC) formation, whereas doubling and tripling the maternal piwi dose increases Osk and Vasa levels correspondingly and doubles and triples the number of PGCs, respectively. Moreover, Piwi forms a complex with dFMRP and Dicer-1, but not with Dicer-2, in polar-granule-enriched fractions. Depleting Dicer-1, but not Dicer-2, also leads to a severe pole-plasm defect and a reduced PGC number. These effects are also seen, albeit to a lesser extent, for dFMRP, another component of the miRISC complex. Because Dicer-1 is required for the miRNA pathway and Dicer-2 is required for the siRNA pathway yet neither is required for the rasiRNA pathway, the data implicate a crucial role of the Piwi-mediated miRNA pathway in regulating the levels of Osk, Vasa, and possibly other genes involved in germline determination in Drosophila (Megosh, 2006).

It has been nearly a century since the discovery of germ plasm and its function in germline fate determination in diverse organisms. In recent decades, the components and assembly of the polar granule in Drosophila and its equivalent in C. elegans have been effectively explored. Translational regulation has also been implicated in pole plasm for abdominal patterning and germline determination. In addition, germ cell-less (gcl) and mitochondrial large-subunit ribosomal RNAs (mtlr RNAs) have been shown to be required for germline determination. However, the biochemical activities of these molecules remain largely unknown. This study identified Piwi and likely the miRNA machinery as a germ-plasm regulatory activity that is involved in germline fate determination (Megosh, 2006).

Germ-plasm assembly occurs in a stepwise fashion. Step 1 involves the transport of polar granule materials to the posterior end of the oocyte during oogenesis, a process that involves a microtubule-based transport system as well as genes such as cappuccino and staufen. Step 2 is the assembly of polar-granule components at the posterior end, a process that is almost concurrent with the transport and that is completed by stage 12 of oogenesis. A critical component for the assembly is Osk, which determines the pole-cell number in a dose-dependent manner and has the ability to recruit Vasa and Tud as well as to induce pole-cell formation at ectopic sites within the embryo. Three lines of data suggest that Piwi is downstream of Osk, Tud, and Vasa in the assembly process: (1) Osk, Tud, and Vasa appear to assemble normally into the pole plasm in Piwi-depleted developing oocytes; (2) Piwi cannot recruit Osk or Vasa ectopically to the anterior pole, yet Osk can recruit Piwi to the anterior pole; (3) Osk, Tud, and Vasa all have both germline determination and posterior-patterning functions, but Piwi does not appear to have a detectable function in patterning (Megosh, 2006).

Although the assembly of polar-granule components occurs in a hierarchical fashion, there is growing evidence for interactions between polar-granule components beyond what is required for assembly. For example, a regulatory relationship between nanos and tudor has been reported. In nanos mutant embryos, both Tudor levels and the number of pole cells increase. Other experiments suggest that the presence of mtlrRNA in the polar granules is required for stabilization of the polar-granule components Vasa, Gcl, nos mRNA, and pgc mRNA. The regulatory function reported in this study for Piwi toward Osk, Vasa, and Nos further supports the interplay and interdependency among pole-plasm components. A previous study implicates osk as a rate-limiting factor for all aspects of pole-plasm function. The results suggest that Piwi, likely working through the miRNA pathway, is also a limiting factor for germ-cell formation. This function of Piwi is likely achieved via regulation of the levels of Osk, Tud, and Vasa, and possibly that of other polar-granule components, in a dose-dependent fashion (Megosh, 2006).

The regulation of Piwi toward the expression of Osk, Tud, Vasa, and Nos appears to be dispensable; Piwi-deficient oocytes and early embryos do not display detectable defects in their expression of Osk, Tud, Vasa, and Nos. This redundancy is likely due to an overlapping function of Piwi with other proteins involved in the RNAi pathway and/or colocalized in nuage during oogenesis; such proteins might include Maelstrom, Armitage, and Aubergine. Among these proteins, Aubergine, a close homolog of Piwi, is a known polar-granule component in early embryos. It regulates the translation of Osk during oogenesis and is required for both pole-cell formation and posterior patterning during embryogenesis (Megosh, 2006).

It is intriguing that Piwi regulates Osk and Vasa expression yet does not display a posterior-patterning phenotype. This function is different from that of Aubergine, so it is possible that Piwi and Aubergine each have their own regulatory targets in addition to Osk and Vasa. The Piwi targets may be specifically involved in maintaining polar-granule localization and may not be subject to Aubergine regulation, whereas Aubergine targets might be involved in both germline determination and posterior patterning. In support of this possibility, it has recently been shown that the generation of certain rasiRNAs shows varying dependencies on Piwi and Aubergine. The regulation of Piwi toward its specific target genes may be activated during oocyte maturation, similar to the oocyte maturation-dependent activation of RNAi as observed for aubergine and spindle-E. Thus, Piwi is not required for Osk and Vasa localization during oogenesis but is required for maintaining their localization during embryogenesis. An alternative hypothesis is that Piwi, like Aubergine, also regulates patterning genes but that this function is redundant. This hypothesis, however, does not explain the fact that neither ectopic expression nor overexpression of Piwi causes a detectable defect in posterior patterning (Megosh, 2006).

Given the association of Piwi with Dcr-1 and dFMRP, the Piwi-mediated regulation is likely via the miRNA but not the siRNA mechanism, which is Dcr-2-dependent, or the rasiRNA mechanism, which does not depend on either Dcr-1 or Dcr-2. This hypothesis is further supported by the similar phenotypes observed in embryos depleted of Piwi, Dcr-1, and dFMRP but not Dcr-2. It is possible that Piwi might bind to novel small RNAs to achieve this function, given recent findings that mammalian Piwi subfamily proteins bind to Piwi-interacting RNAs (piRNAs). If so, these novel RNAs must function in a Dcr-1-dependent pathway in the cytoplasm given Piwi's localization to the cytoplasm in early pole cells. The function of the Piwi/DCR-1-mediated miRNA or novel small-RNA pathway in germline specification is very similar to that of other germ-cell regulators, such as gcl and mtlr RNAs, in that these genes are required for pole-cell formation but not for abdominal segmentation. However, unlike embryos from the gcl-bcd females, embryos from the piwi-bcd females exhibit no cell-cycle delays in the anterior nuclei and no significant changes in the morphology of anterior nuclei. Furthermore, GCL mediates a transcriptional repression mechanism [72]. Thus, the effect of the Piwi-miRNA mechanism on pole-cell formation may be distinct from the gcl-mediated mechanism (Megosh, 2006).

It is important to note that the Piwi-mediated miRNA pathway positively regulates the expression of Osk and Vasa, in contrast to the known translational repression role of the miRNA pathway. In support of this observation, the Piwi ortholog in the mouse, MIWI, also appears to positively regulate gene expression, likely by enhancing mRNA stability and translation. Alternatively, it is possible that Piwi regulates an unidentified intermediate protein whose function is to repress the expression of Osk and Vasa (Megosh, 2006).

piwi is essential for the self-renewal of adult germline stem cells in Drosophila. Recent studies have demonstrated that the miRNA pathway is involved in division and self-renewal of adult germline stem cells in the Drosophila ovary. This study further connects Piwi and the miRNA pathway and reveals their crucial role in germline fate determination during embryogenesis. These observations suggest that the germline and stem cells may share a common miRNA-mediated mechanism in defining their fates. Given the high degree of conservation of the miRNA machinery during evolution, this pathway may function in diverse organisms in determining the germline and stem cell fates (Megosh, 2006).

Drosophila fragile X mental retardation protein and metabotropic glutamate receptor A convergently regulate the synaptic ratio of ionotropic glutamate receptor subclasses

A current hypothesis proposes that fragile X mental retardation protein (FMRP), an RNA-binding translational regulator, acts downstream of glutamatergic transmission, via metabotropic glutamate receptor (mGluR) Gq-dependent signaling, to modulate protein synthesis critical for trafficking ionotropic glutamate receptors (iGluRs) at synapses. However, direct evidence linking FMRP and mGluR function with iGluR synaptic expression is limited. This study used the Drosophila fragile X model to test this hypothesis at the well characterized glutamatergic neuromuscular junction (NMJ). Two iGluR classes reside at this synapse, each containing common GluRIIC (III), IID and IIE subunits, and variable GluRIIA (A-class) or GluRIIB (B-class) subunits. In Drosophila fragile X mental retardation 1 (dfmr1) null mutants, A-class GluRs accumulate and B-class GluRs are lost, whereas total GluR levels do not change, resulting in a striking change in GluR subclass ratio at individual synapses. The sole Drosophila metabotropic glutamate receptor, DmGluRA, is also expressed at the NMJ. In dmGluRA null mutants, both iGluR classes increase, resulting in an increase in total synaptic GluR content at individual synapses. Targeted postsynaptic dmGluRA overexpression causes the exact opposite GluR phenotype to the dfmr1 null, confirming postsynaptic GluR subtype-specific regulation. In dfmr1; dmGluRA double null mutants, there is an additive increase in A-class GluRs, and a similar additive impact on B-class GluRs, toward normal levels in the double mutants. These results show that both dFMRP and DmGluRA differentially regulate the abundance of different GluR subclasses in a convergent mechanism within individual postsynaptic domains (Pan, 2007).

The finding of elevated group I mGluR5-dependent hippocampal LTD in the fmr1 knock-out mouse has elicited a great deal of attention and excitement. This type of LTD is caused by loss of surface expression of AMPA GluRs, in a mechanism requiring protein synthesis. Because synaptic protein translation is regulated by FMR, these observations suggest a mechanistic connection between mGluR signaling and FMRP translation regulation in the control of GluR expression at the synapse and indicate that this pathway may be a critical regulator of functional synaptic plasticity. This idea has been formally expressed as the mGluR theory of FXS (Bear, 2004). This study directly investigates this hypothesized connection between mGluR signaling, FMRP regulatory function, and the synaptic expression of GluRs using the Drosophila FXS model. In Drosophila, there is a single FMR1 protein (dFMRP) and a single mGluR (DmGluRA). dFMRP structure, expression, and regulative functions closely resemble mammalian FMRP. In contrast, DmGluRA is more homologous to mammalian group II/III mGluRs, not the group I mGluRs implicated in the FMRP mechanism. However, these mGluR class distinctions may mean little in Drosophila, with its single mGluR. The mammalian group I mGluR antagonist MPEP rescues morphological and behavioral phenotypes in dfmr1 null mutants (McBride, 2005), and DmGluRA modulates synaptic architecture (Bogdanik, 2004), which is a known function of mammalian group I mGluRs. These findings suggest that DmGluRA likely also occupies the group I mGluR niche in Drosophila. In any case, DmGluRA is the only mGluR capable of mediating glutamatergic signaling in the Drosophila system (Pan, 2007).

Both dfmr1 and dmGluRA mutants have been shown to have strong defects in glutamatergic synaptic function at the Drosophila NMJ. Neurotransmission at this synapse is mediated by A- and B-class AMPA-type GluRs, which have distinctive functional properties and subsynaptic distributions and are regulated by distinct mechanisms. This study shows in dfmr1 null mutants that A-class GluRs accumulate and B-class GluRs are lost. The total GluR content does not change, but rather there is a striking shift in the GluR class ratio within single postsynaptic domains. This subclass-specific regulation of GluRs is a novel finding. In dmGluRA null mutants, it was shown that both GluR classes, and therefore the total GluR population, are significantly increased. This is a novel finding for DmGluRA but consistent with findings in mammals showing that GluR1 AMPA receptors are decreased in synaptic terminals when mGluR activity is induced. Moreover, this study showed that postsynaptic overexpression of DmGluRA induces exactly opposite changes of A- and B-class GluRs compared with dfmr1 null mutants. By testing active zone density and targeted presynaptic rescue of dFMRP in the dfmr1 null, we show that the regulatory function of dFMRP on the GluR classes is a postsynaptic mechanism. Finally, it was show in dfmr1; dmGluRA double null mutants that both GluR class phenotypes are additive; A-class GluRs increase further with the additive increases of dfmr1 and dmGluRA single mutants, and B-class GluRs tend toward normal levels, with the additive downregulation in the dfmr1 single mutant and upregulation in the dmGluRA single mutant. These results suggest that DmGluRA signaling and dFMRP function converge to regulate the synaptic expression of these two GluR classes but that independent pathways of DmGluRA signaling and dFMRP function also exist (Pan, 2007).

This study suggests that dFMRP and DmGluRA perform in both overlapping and independent pathways in the regulation of postsynaptic GluR classes. Targeted presynaptic expression of dFMRP in the dfmr1 null fails to provide any rescue of class-specific GluR misregulation, showing that the dFMRP requirement is in the postsynaptic compartment. Consistently, targeted postsynaptic overexpression of DmGluRA causes the opposite class-specific GluR misregulation of the dfmr1 null, suggesting an intersection of DmGluRA signaling and dFMRP function in the postsynaptic compartment. In the dfmr1 null, quantal size is increased, a hallmark postsynaptic defect. A mechanistic cause suggested by this study is the elevated A-class GluR level, consistent with former reports that GluRIIA overexpression increases quantal size. Moreover, GluRIIA overexpression increases active zone number per bouton, based on the NC82/bruchpilot probe, but does not alter active zone density, which is identical to the phenotype reported in this study for dfmr1 mutants. These results support the conclusion that both dFMRP and DmGluRA function in the postsynaptic domain in class-specific GluR regulation and that this mechanism may feedback to alter presynaptic properties (Pan, 2007).

In addition to the postsynaptic mechanism, there appears to also be presynaptic roles of both dFMRP and DmGluRA that can impact the postsynaptic GluR domains. It was shown previously that both proteins are expressed in the presynaptic neuron of the Drosophila NMJ. Single null mutants show differential misregulation, with the dfmr1 null displaying the class-specific change reflecting its postsynaptic function but the dmGluRA null increasing both GluR classes in common. This must reflect a presynaptic function for DmGluRA. Consistently, presynaptic overexpression of DmGluRA depresses the level of both A- and B-class GluRs, the opposite phenotype as the dmGluRA null. Likewise, presynaptic overexpression of dFMRP also reduces B-class GluR expression, although it does not change the abundance of A-class receptors. Presumably, these presynaptic roles reflect the know functions of dFMRP and DmGluRA in regulating presynaptic glutamate release properties, and therefore the GluR changes reflect transynaptic signaling in a homeostatic mechanism (Pan, 2007).

By strict genetic criteria, the prediction for the interaction of two proteins within a common regulatory pathway is that the mutant phenotype for the gene product downstream in the pathway should be epistatic to that of the gene product upstream in the pathway. Clearly, the FMRP translation regulatory activity should be downstream of mGluR surface glutamate reception. Such a strict epistatic relationship is not observed for DmGluRA and dFMRP in the control of GluR expression. Rather, the null mutant phenotypes are obviously additive in double mutants. The A-class GluR goes up in both single mutants and goes up further in the double mutant. The B-class GluR goes down in dfmr1 and up in dmGluRA and shows an intermediate, additive level in the double mutant. Such additive phenotypes show that dFMRP and DmGluRA have overlapping functions but can be operating in the independent pathways. Together, these results suggest that dFMRP and DmGluRA pathways converge on the regulation of GluR synaptic expression and that this involves both presynaptic and postsynaptic interactions (Pan, 2007).

Regulating GluR class composition in the postsynaptic domain is an important mechanism controlling neurotransmission strength and synaptic plasticity properties. The subunit composition of mammalian NMDA and AMPA receptors are both known to be regulated in this manner. Similarly at the Drosophila NMJ, the independent regulation of GluR classes is critical, because each receptor class has distinct functional properties, e.g., the A-class specifically is negatively regulated by protein kinase A phosphorylation, is modulated by atypical protein kinase C, is important in retrograde signaling, and mediates larger, slower-decaying transmission events with a smaller single channel conductance. The molecular mechanisms for controlling each GluR therefore must be distinct and, indeed, distinct mechanisms have been identified. For example, the PDZ [postsynaptic density 95 (PSD-95)/Discs Large (DLG)/zona occludens-1]-domain scaffold DLG, a PSD-95 homolog, is involved in the localization of many synaptic proteins but plays a specific role in B-class GluR regulation: GluRIIB abundance correlates with DLG level, but GluRIIA localization is unaffected in dlg mutants. Similarly, the Rho-type guanine nucleotide exchange factor dPix (the Drosophila homolog of the Pak interacting exchange factor), its interacting Drosophila p-21 activated kinase (dPak), a serine threonine kinase activated by GTPases Rac and cell division cycle 42, and a dPak binding partner, the adaptor Dreadlocks (Dock; Nck homolog), are all required to facilitate synaptic expression of A-class GluRs, but GluRIIB is reportedly not affected in mutants of this pathway. Trafficking mechanisms likely involve GluR tethering to the cytoskeleton. The actin-interacting Coracle (mammalian brain 4.1 protein) binds only GluRIIA to specifically regulate its abundance, with no role in B-class GluR tethering. Thus, separable mechanisms for A- and B-class GluR regulation clearly exist (Pan, 2007).

FMRP/dFMRP is an RNA-binding protein and a regulator of protein translation at the synapse. Although FMRP/dFMRP is best defined as a negative regulator of translation, it may also positively regulate the translation of a distinct set of synaptic mRNAs. Presumably, these translation regulation mechanisms underlie the differential, and opposing, regulation of A- and B-class GluRs by dFMRP. Some newly synthesized proteins may promote synaptic expression of A-class GluRs, whereas others promote the diminution of B-classes GluRs. One protein whose synaptic translation is regulated by both mGluR signaling and FMRP function is PSD-95, implicated in both NMDA and AMPA GluR synaptic expression. Thus, altered regulation of the Drosophila PSD-95 homolog DLG is an attractive candidate mechanism for GluR phenotypes discovered in this study. In addition, the synaptic cytoskeleton affects synaptic expression of both NMDA and AMPA GluRs. Notably, a key function of FMRP/dFMRP is regulating microtubule and actin filament dynamics via regulating expression of key cytoskeleton-binding proteins, such as Futsch/microtubule-associated protein 1b. The future goal of these studies will be to test these candidate downstream factors as regulators of GluR synaptic expression in Drosophila, downstream of both DmGluRA signaling and dFMRP translational control (Pan, 2007).

Metabotropic glutamate receptor-mediated use-dependent down-regulation of synaptic excitability involves the fragile X mental retardation protein

Loss of the mRNA-binding protein FMRP results in the most common inherited form of both mental retardation and autism spectrum disorders: fragile X syndrome (FXS). The leading FXS hypothesis proposes that metabotropic glutamate receptor (mGluR) signaling at the synapse controls FMRP function in the regulation of local protein translation to modulate synaptic transmission strength. In this study, the Drosophila FXS disease model was used to test the relationship between Drosophila FMRP (dFMRP) and the sole Drosophila mGluR (dmGluRA) in regulation of synaptic function, using two-electrode voltage-clamp recording at the glutamatergic neuromuscular junction (NMJ). Null dmGluRA mutants show minimal changes in basal synapse properties but pronounced defects during sustained high-frequency stimulation(HFS). The double null dfmr1;dmGluRA mutant shows repression of enhanced augmentation and delayed onset of premature long-term facilitation (LTF) and strongly reduces grossly elevated post-tetanic potentiation (PTP) phenotypes present in dmGluRA-null animals. Null dfmr1 mutants show features of synaptic hyperexcitability, including multiple transmission events in response to a single stimulus and cyclic modulation of transmission amplitude during prolonged HFS. The double null dfmr1;dmGluRA mutant shows amelioration of these defects but does not fully restore wildtype properties in dfmr1-null animals. These data suggest that dmGluRA functions in a negative feedback loop in which excess glutamate released during high-frequency transmission binds the glutamate receptor to dampen synaptic excitability, and dFMRP functions to suppress the translation of proteins regulating this synaptic excitability. Removal of the translational regulator partially compensates for loss of the receptor and, similarly, loss of the receptor weakly compensates for loss of the translational regulator (Repicky, 2008).

Drosophila is an excellent, simplified genetic system for comprehensively testing interactions between mGluR signaling and FMRP function in the nervous system. There is a single Drosophila homolog of the three member mammalian FMR gene family (dFMRP) and a single Drosophila homolog of the eight member mammalian mGluR family (dmGluRA). Antagonists of different mammalian mGluR classes have been shown to rescue several dfmr1 null phenotypes (McBride, 2005; Pan, 2008), suggesting that dmGluRA signaling does indeed have a mechanistic connection with dFMRP function. More importantly, the double mutant combination of the two Drosophila null alleles provides an excellent opportunity to test genetic relationships between all FMR family function and all mGluR signaling, particularly in the regulation of synapse development, function, and plasticity. Indeed, it has been shown, using double mutants, that dFMRP and dmGluRA interact in the regulation of ionotropic glutamate receptor trafficking at the NMJ synapse (Pan, 2007), as well as in the modulation of movement behavior and the control of NMJ gross architecture and synaptic ultrastructure (Pan, 2008) (Repicky, 2008).

The goal of this study was to examine the key question of the role of dFMRP in synaptic transmission properties and to determine whether a genetic block in dmGluRA signaling would modulate functional defects caused by loss of dFMRP. All work was done in low external Ca2+ concentrations, as required to permit amplitude facilitation driven by high-frequency stimuli. Previous research has shown that dmGluRA is not required to maintain basal neurotransmission, but is critical for the regulation of activity-dependent synaptic plasticity processes, particularly in establishing the threshold for LTF and limiting the expression of PTP (Bogdanik, 2004). Null dmGluRA phenotypes resemble the consequences of applying K+ channel blockers to wildtype synapses, as well as mutants that either increase Na+ currents (pumilio) or decrease K+ currents (hyperkinetic, frequenin). Interestingly, the pumilio gene encodes an RNA-binding translational suppressor, like dFMRP, whose activity down-regulates the paralytic RNA encoding voltage-gated Na+ channels. The hyperkinetic gene encodes a K+ channel β subunit, and frequenin regulates the function of K+ channels. Thus loss of dmGluRA generates synaptic phenotypes caused by increased neuronal membrane excitability. These comparisons suggest that the dmGluRA receptor monitors glutamate release, particularly during periods of high activity, to feedback and down-regulate the membrane excitability controlling Ca2+ influx and glutamate release, thus dictating neurotransmission strength. There are clear indications that dmGluRA interacts with dFMRP in synapse regulation (Pan, 2007; Pan, 2008), but dFMRP has not previously been shown to control synaptic excitability. This study aimed at discovering whether or not dFMRP and dmGluRA interact in the regulation of activity-dependent synaptic modulation, particularly through mechanisms of altered synaptic excitability (Repicky, 2008).

No striking difference was found in basal transmission properties at low external Ca2+ concentrations in either dfmr1 or dmGluRA single mutants or the dfmr1;dmGluRA double mutant. There is a clear tendency of increased basal transmission strength, particularly in dfmr1, as well as shifts in the power relationship of Ca2+-dependent synaptic vesicle release. Together these changes explain the elevated dfmr1 synaptic strength and more rapid presynaptic vesicle cycle at higher Ca2+ concentrations. Nevertheless, under the low [Ca2+] conditions used in this study, it can be infered that altered neurotransmission in response to HFS must be caused by activity-dependent changes in transmission probability in these genotypes. During short HFS trains, the single null mutants behave similar to control, but there is strongly elevated STF in the dfmr1;dmGluRA double null mutant. Similarly, previous work on synaptic structure also has shown that some phenotypes interact in a synergistic fashion (Pan, 2008). The STF defect shows a clear interaction between dmGluRA-dependent glutamatergic signaling and the requirement for dFMRP function at the synapse, although it does not necessarily demonstrate that the two proteins work in the same pathway(s) in the manifestation of short-term changes in synapse function. It is possible that mutation of the two genes concurrently shows an overlapping function that is not evident otherwise (Repicky, 2008).

The interaction between dmGluRA and dFMRP in long-term, activity-dependent changes in neurotransmission strength is more extensive and informative. During prolonged HFS, dmGluRA null mutants display a profound increase in augmentation, suggesting loss of a glutamate negative feedback loop to reign in glutamate release. Interestingly, removal of dFMRP in the dfmr1;dmGluRA double mutant modifies this defect during the early stages of HFS, extending the time it takes to elevate response amplitudes to the fully augmented level. However, as HFS continues, the full augmentation defect is expressed in the dfmr1;dmGluRA double mutant, indicating that other, dFMRP-independent pathways play a large role in the dmGluRA augmentation phenotype. Following prolonged HFS, wild-type synapses exhibit low-level, persistent PTP, but dmGluRA mutants show grossly elevated PTP (>5-fold normalized amplitude). Importantly, removal of dFMRP in dfmr1;dmGluRA double mutants produces nearly complete loss of this potentiation defect. This restoration toward wild-type seems to indicate converging pathways for dFMRP and dmGluRA in the regulation of activity-dependent synaptic potentiation. The third plasticity defect apparent in dmGluRA nulls is the premature, sudden step-wise appearance of long-term facilitation (LTF). The dfmr1;dmGluRA animals retain this lowered LTF threshold phenotype, but aspects of the defect are reduced by co-removal of dFMRP. First, the time of LTF onset is longer in double mutants compared with dmGluRA (11.8 vs. 9.7 s). Second, the dmGluRA null always shows an abrupt, single step increase in response amplitude between two consecutive stimulations in the HFS train, whereas dfmr1;dmGluRA characteristically oscillates between augmented and LTF amplitudes multiple times before finally succumbing to LTF. These data suggest that removal of dFMRP alleviates the consequences of lost mGluR signaling, albeit insufficiently to block presentation of enhanced transmission phenotypes (Repicky, 2008).

Taken together, the above results show clearly that removal of dFMRP can modulate defects caused by loss of dmGluRA. Removal of dFMRP by itself fails to present any defects in assayed forms of activity-dependent plasticity. However, two new phenotypes were identified in the dfmr1 null synapse. First, during prolonged HFS, dfmr1 mutants fail to maintain consistent transmission amplitudes, but rather manifest striking and characteristic cycling of amplitudes between a low and high transmission state. This cycling presents with sudden, drastic changes in amplitude size in bursts of quite regular periodicity. This is a novel phenotype without clear comparisons in the literature. Second, dfmr1 mutants display multiple excitatory junction current (EJC) events in response to single nerve stimuli during and after HFS. Similar hyperactivity is characteristic of Shaker K+ channel mutants, and double mutation combinations with ether-a-go-go, acting synergistically to increase membrane excitability. Interestingly, synaptic hyperexcitability in Shaker, and also bang-senseless mutants, is rescued with loss of no action potential (nap), to reduce Na+ channels. The similar phenotypes of these mutants compared with dfmr1 suggests dFMRP regulates synaptic excitability, perhaps via regulating membrane excitability, providing a clear mechanistic relationship with mGluR-mediated negative feedback control (Repicky, 2008).

Consistent with this feedback loop, co-removal of mGluR signaling appreciably diminishes these dfmr1 defects. The dfmr1;dmGluRA double null still displays the EJC response amplitude cycling, and requires a similar duration of HFS prior to the onset of cycling. However, the cycling defect is present in far fewer dfmr1;dmGluRA animals compared with the dfmr1 single mutant, and the limited cycling manifest in the double mutants has a slower cycling period, showing partial alleviation of the phenotype in the double mutant. The threshold for manifestation of this intriguing synaptic modulation is clearly lowered by removal of dFMRP but raised again by co-removal of mGluR signaling, albeit not to wild-type levels. In dfmr1 mutants, multiple separate and distinct EJCs occur in response to a single stimulus during HFS. This hyperexcitability defect is effectively lowered in dfmr1;dmGluRA double mutants. Indeed, the hyperactive response was recorded only in two isolated incidents in two separate dfmr1;dmGluRA animals. In dfmr1 mutants, the hyperexcitable responsiveness persists following the HFS train during basal stimulation, but post-HFS hyperexcitability was never observed in dfmr1;dmGluRA animals. Thus co-removal of dmGluRA does indeed diminish the consequences of loss of dFMRP, only partially in the case of the cyclic transmission defect, but quite strongly to block dfmr1 hyperexcitability. Together, these data support the conclusion of a partial co-dependency of dmGluRA receptor signaling on dFMRP regulative function, and vice versa in a feedback loop, to modulate synapse properties critical for the maintenance of transmission fidelity and activity-dependent plasticity (Repicky, 2008).

Both rescue and synergistic interactions of dfmr1 and dmGluRA null mutations in a range of synaptic mechanisms has been shown (Pan, 2007; Pan, 2008). However, a major, persistent limitation has been the lack of any functional data on synaptic transmission, a primary focus of FXS dysfunction. This crucial question has similarly not as yet been addressed in the mouse fmr1 KO model, despite evidence of rescue in other fmr1 defects. This study has shown that activity-dependent synaptic plasticity defects in dmGluRA nulls, including elevated augmentation, potentiation, and premature LTF, are each reduced by the co-removal of dFMRP. Similarly, the synaptic defects in dfmr1 nulls, including transmission amplitude cycling during HFS and multiple EJCs in response to a single stimulus, are decreased by the co-removal of dmGluRA, and hence loss of all mGluR signaling at the synapse. The striking exception to this trend is STF, which is somehow enhanced in the dfmr1;dmGluRA double null compared with both single mutants. These interactions clearly support the conclusion of a relationship between dFMRP function and dmGluRA signaling, but argue against a simple direct signaling cascade. Rather, dFMRP function is likely controlled by several converging signaling pathways, of which dmGluRA-mediated glutamatergic synaptic signaling is only one (Repicky, 2008).

Mechanistic relationships between Drosophila fragile X mental retardation protein and metabotropic glutamate receptor A signaling

Fragile X syndrome is caused by loss of the FMRP translational regulator. A current hypothesis proposes that FMRP functions downstream of mGluR signaling to regulate synaptic connections. Using the Drosophila disease model, relationships between dFMRP and the sole Drosophila mGluR (DmGluRA) were tested by assaying protein expression, behavior and neuron structure in brain and NMJ; in single mutants, double mutants and with an mGluR antagonist. At the protein level, dFMRP is upregulated in dmGluRA mutants, and DmGluRA is upregulated in dfmr1 mutants, demonstrating mutual negative feedback. Null dmGluRA mutants display defects in coordinated movement behavior, which are rescued by removing dFMRP expression. Null dfmr1 mutants display increased NMJ presynaptic structural complexity and elevated presynaptic vesicle pools, which are rescued by blocking mGluR signaling. Null dfmr1 brain neurons similarly display increased presynaptic architectural complexity, which is rescued by blocking mGluR signaling. These data show that DmGluRA and dFMRP convergently regulate presynaptic properties (Pan, 2008).

Drosophila fragile X mental retardation protein developmentally regulates activity-dependent axon pruning

Fragile X Syndrome (FraX) is a broad-spectrum neurological disorder with symptoms ranging from hyperexcitability to mental retardation and autism. Loss of the fragile X mental retardation 1 (fmr1) gene product, the mRNA-binding translational regulator FMRP, causes structural over-elaboration of dendritic and axonal processes, as well as functional alterations in synaptic plasticity at maturity. It is unclear, however, whether FraX is primarily a disease of development, a disease of plasticity or both: a distinction that is vital for engineering intervention strategies. To address this crucial issue, the Drosophila FraX model was used to investigate the developmental function of Drosophila FMRP (dFMRP). dFMRP expression and regulation of chickadee/profilin coincides with a transient window of late brain development. During this time, dFMRP is positively regulated by sensory input activity, and is required to limit axon growth and for efficient activity-dependent pruning of axon branches in the Mushroom Body learning/memory center. These results demonstrate that dFMRP has a primary role in activity-dependent neural circuit refinement during late brain development (Tessier, 2008).

In the absence of dFMRP, elevated levels of total RNA/protein are evident during a restricted period of late pupal brain development, with the protein increase persisting into an early-use refinement period. These increases are transient and disappear in the mature brain thereby defining a limited developmental window of dFMRP function. The increase in protein is predicted as FMRP/dFMRP negatively regulates translation. The elevated RNA is more surprising. dFMRP/FMRP can both negatively and positively regulate mRNA stability, and, therefore, dFMRP may have a developmentally-restricted role primarily as a negative regulator of mRNA stability. Alternatively, the RNA increase may be caused by elevated transcription, via an uncharacterized direct or indirect transcriptional inhibition function of dFMRP. Because the increase in total protein/RNA is not biased towards selected dFMRP targets, these results suggest globally upregulated transcription/translation in the dfmr1 mutant brain during a restricted window of late maturation and early-use refinement (Tessier, 2008).

During brain development, dfmr1 mRNA and dFMRP protein levels tightly correlate with the above changes, but, surprisingly, dfmr1 mRNA levels inversely correlate with dFMRP protein levels in the mature brain. By 4 days after eclosion (AE), dfmr1 mRNA levels rise to levels nearly as high as those present during development, but dFMRP protein is maintained at a basal level in the mature brain. This change strongly suggests a distinct switch in dFMRP regulation, in which transcription and translation become uncoupled. Because dFMRP/FMRP represses the translation of its own mRNA, it is tempting to speculate that this negative-feedback mechanism specifically regulates dFMRP in the mature brain. FMRP modulates synaptic plasticity at maturity, as evidenced by decreased LTP and enhanced LTD in fmr1 knock-out (KO) mice. Consistent with such a mature function, elevated total protein levels are once again evident in the fully mature dfmr1-null brain. A similar increase in cerebral protein synthesis occurs in adult fmr1-KO mice. Together, these data suggest that a switch in dFMRP/FMRP regulation defines separate windows of function in development versus maturity (Tessier, 2008).

It was crucial to determine whether dFMRP function correlates with its developmental expression profile. A known dFMRP target is chickadee/profilin; dFMRP binds chickadee mRNA and negatively regulates its translation. Importantly, the dynamics of chickadee misregulation in the dfmr1-null brain indicate that the dFMRP functional requirement mirrors its developmental expression profile. Chickadee expression normally peaks during late-stage brain development (P4), and it is during this development window, and shortly following, that overexpression is manifested in the dfmr1-null brain. Generally, the increase in chickadee transcripts parallels the increase in protein, suggesting that dFMRP regulation may be at the level of the mRNA, for example, by affecting mRNA stability. dFMRP reportedly interacts with miRNA machinery to control mRNA levels of the sodium channel Pickpocket1. A similar mechanism for chickadee regulation would be consistent with the current results. Interestingly, the increase in Chickadee protein levels coincides with the period of use-dependent neural circuit refinement at eclosion. At least one dFMRP/FMRP target mRNA, futsch (MAP1B), is regulated specifically at postnatal day 10 in fmr1-KO mice. These new insights suggest it will be vital to ascertain the developmental expression of all putative FMRP targets in the context of these distinct windows of regulation in order to validate in vivo functions (Tessier, 2008).

During the peak period of dFMRP expression, there are two phases of dFMRP-dependent axon maturation. During late pupal development, dFMRP inhibits axon elongation, with dfmr1-null neurons exhibiting branches 25% longer than controls. This function is restricted to very late stages (P4), with no differences in branch length or number being observed earlier (P3). Immediately upon eclosion, dFMRP is required for use-dependent pruning, causing a decrease in both axon branch length and number. Pruning is most evident in the smallest presynaptic branches (<5 µm) and occurs quickly (hours) following the onset of adult activity. Targeted overexpression of dFMRP causes inverse defects in both phases of dFMRP requirement. Axon undergrowth is apparent early (P3) and axons fail to grow later (P4). Axon branches present in these neurons are short, filipodial-like structures, and, at eclosion, there is excessive pruning to result in ~30% fewer branches than in P4 and ~3 times fewer branches than in controls. Thus, both axonogenesis and axon branch pruning are bidirectionally modified by inverse changes in dFMRP expression (Tessier, 2008).

Blocking sensory input activity maintains dFMRP in its early development regulative state, with a correlative reduction in both dfmr1 mRNA and dFMRP protein. Both olfactory (Or83b) and phototransduction (ninaE) mutants similarly suppress dFMRP levels, indicating that these two primary modes of brain sensory input positively drive dFMRP expression. Similarly, mammalian FMRP expression is elevated following activity stimulation by both environmental enrichment and mGluR signaling activation. Blocking mGluR activity in Drosophila and mice can rescue some dfmr1 defects, including impaired learning and memory. From these similar findings, it is tempting to suggest that dFMRP/FMRP may function downstream of mGluR signaling activity, perhaps differentially in development versus maturity. Importantly, both Or83b and ninaE sensory mutants cause elevation of chickadee/profilin at the same time dFMRP is suppressed. This finding is consistent with activity-dependent regulation of dFMRP to regulate chickadee/profilin expression (Tessier, 2008).

This study shows that Drosophila neurons undergo activity-dependent pruning coincident with the onset of use. In the absence of dFMRP, pruning does not occur during the normal developmental window. Indeed, blocking sensory input activity leads to further increases in the axon branch number and length in dfmr1-null neurons. Moreover, at maturity, sensory stimulation following sensory deprivation does not induce pruning, probably because the dFMRP level has fallen too low. It is hypothesized that there is a threshold of dFMRP required for efficient activity-dependent pruning during the early-use period, which is normally defined by the window of high dFMRP expression. Reinstated sensory stimulation following sensory deprivation does cause a significant dFMRP-dependent increase in the number of long axon branches. These data are consistent with the need for high dFMRP expression to both limit axonal growth and mediate the early-use refinement of circuits. Importantly, it was confirmed, by using targeted expression of the exogenous light-gated channelrhodopsin-2 channel, that neuronal activation bidirectionally drives the pruning process. Light-driven activation of CHR2 channels induces pruning of the same small (<5 µm) axonal processes that aberrantly persist in the dfmr1-null brain. As predicted, the induced pruning process fails to occur in the absence of dFMRP (Tessier, 2008).

Delayed pruning eventually occurs in dfmr1-null neurons to ultimately rescue the overbranching defect present in younger animals. A similar transient elongation of dendritic spines occurs in young postnatal Fmr1-KO mice, although a secondary overgrowth phenotype may appear months later in adult animals. In Drosophila, the delayed axon pruning in dfmr1-null neurons actually goes too far, resulting in reduced neuronal complexity in mature adult animals. The small presynaptic branches (<5 µm) are reduced 35% in dfmr1-null neurons compared with controls at 4 days. Because pruning normally occurs very rapidly (<3 hours after eclosion), coincident with initial use, it is likely that the pruning process is strictly controlled for that developmental time. By delaying pruning in the absence of dFMRP, it appears that other factors that buffer the extent of process elimination fail to provide adequate regulation of the mechanism. Indeed, this mitigation may be a function of dFMRP itself, as dFMRP levels drop drastically immediately following the normal pruning window. FMRP potentially regulates many proteins involved in a diverse set of functions. Understanding the developmental regulation of proteins that associate with FMRP and FMRP target mRNAs will be crucial to unraveling the underlying pruning mechanisms of activity-dependent neural circuit refinement (Tessier, 2008).

Mutational analysis establishes a critical role for the N terminus of Fragile X Mental Retardation Protein FMRP

Fragile X syndrome is the most common form of heritable mental retardation caused by the loss of function of the fragile X mental retardation protein FMRP. FMRP is a multidomain, RNA-binding protein involved in RNA transport and/or translational regulation. However, the binding specificity between FMRP and its various partners including interacting proteins and mRNA targets is essentially unknown. Previous work demonstrated that dFMRP, the Drosophila homolog of human FMRP, is structurally and functionally conserved with its mammalian counterparts. This study performed a forward genetic screen and 26 missense mutations were isolate at 13 amino acid residues in the dFMRP coding dfmr1. Interestingly, all missense mutations identified affect highly conserved residues in the N terminal of dFMRP. Loss- and gain-of-function analyses reveal altered axonal and synaptic elaborations in mutants. Yeast two-hybrid assays and in vivo analyses of interaction with CYFIP (cytoplasmic FMR1 interacting protein) in the nervous system demonstrate that some of the mutations disrupt specific protein-protein interactions. Thus, mutational analyses establish that the N terminus of FMRP is critical for its neuronal function (Reeve, 2008).

The genetic screen, in which the EP3517 line was mutagenized to screen for mutations in the endogenous dfmr1 downstream of the EP element, is efficient and productive. Because overexpression or ectopic expression of many genes can produce a simple recognizable phenotype whereas a mutation compromising the function(s) of the endogenous gene would rescue the phenotype, the screen strategy is of general utility and can be readily extended to other genes of interest, particularly those with no obvious LOF phenotypes or whose functions are unknown. Another attractive feature of the screen is that the mutations can be readily used for GOF analyses without the need of making transgenic flies carrying engineered mutations (Reeve, 2008).

All the mutations isolated cluster to the N-terminal 411 residues, with no mutations found at the C-terminal 270 amino acids. Specifically, 18 of the 26 missense mutations are located at the N-terminal 220 amino acid region, mainly involved in protein-protein interaction. There are a few nonexclusive possibilities for the clustered distribution of mutations. First, the function of the C-terminal 270 amino acids may be subtle such that one residue change in the region does not sufficiently compromise dFMRP function. Indeed, this region is the least conserved in the FXR family through evolution. Second, the C-terminal 270 amino acids may not contribute to the lethality on which the genetic screen is based. Third, the functions of the four RNA binding domains NDF (N-terminal domain of FMRP), KH1, KH2, and RGG box of dFMRP may be somewhat redundant, so that disruption of one is compensated by others. Therefore, more sensitive or specific screening paradigms have to be devised to isolate mutations in the RNA binding domains. Importantly, however, most of the mutations isolated affect the neuronal functions of dFMRP in both LOF and GOF assays, demonstrating the relevance of the screen paradigm (Reeve, 2008).

The FMRP NT218 interacts directly with several proteins, such as CYFIP1 (Schenck, 2001), FXR1/2 (Zhang, 1995), and 82-FIP (Bardoni, 2003). This work identified critical amino acids for the specific interactions. For example, E66, R113, and S172 are vital for the interaction with CYFIP1, whereas L184, located in the coiled-coil domain, is critical for the FMRP-FXR1 heterodimerization. Conversely, mutations at E66, C78, and R113 abolished the interaction with 82-FIP, which is in full agreement with the early biochemical and structural studies. Importantly, the interaction with 82-FIP and CYFIP1 shares similar residues but distinct from that with FXR1. One of the alleles isolated, E68K, appears to interfere with the function of wild-type dFMRP. This allele also fails to bind to or interact with CYFIP, suggesting that this is not the mechanism of its function as a dominant suppressor of wild-type dFMRP. One tempting speculation is that E68K may interfere with dFMRP homomeric or heteromeric complex formation, leading to interference with the function of the wild-type allele. At any rate, it is clear that mutational analyses identified critical residues for specific protein interactions in the N terminus of FMRP (Reeve, 2008).

Finally, dFMRP has different functions in the NMJ, in which it regulates synaptogenesis, and in the central LNv, in which it regulates axonal outgrowth. However, the residues identified are critical for the interaction between CYFIP and dFMRP in both the LNv and NMJ, suggesting that the dFMRP-CYFIP interaction may be crucial to dFMRP function in different neuronal types and at different developmental stages. Based on the high conservation and functional importance of these and other residues in the FMRP protein, it is speculated that the strong or null mutations in the critical residues of FMRP identified here might cause rare cases of FraX patients without diagnostic CGG expansion (Reeve, 2008).

Genetic modifiers of dFMR1 encode RNA granule components in Drosophila

Mechanisms of neuronal mRNA localization and translation are of considerable biological interest. Spatially regulated mRNA translation contributes to cell-fate decisions and axon guidance during development, as well as to long-term synaptic plasticity in adulthood. The Fragile-X Mental Retardation protein (FMRP/dFMR1) is one of the best-studied neuronal translational control molecules and this study describes the identification and early characterization of proteins likely to function in the dFMR1 pathway. Induction of the dFMR1 in sevenless-expressing cells of the Drosophila eye causes a disorganized (rough) eye through a mechanism that requires residues necessary for dFMR1/FMRP's translational repressor function. Several mutations in dco, orb2, pAbp, rm62, and smD3 genes dominantly suppress the sev-dfmr1 rough-eye phenotype, suggesting that they are required for dFMR1-mediated processes. The encoded proteins localize to dFMR1-containing neuronal mRNPs in neurites of cultured neurons, and/or have an effect on dendritic branching predicted for bona fide neuronal translational repressors. Genetic mosaic analyses indicate that dco, orb2, rm62, smD3, and dfmr1 are dispensable for translational repression of hid, a microRNA target gene, known to be repressed in wing discs by the bantam miRNA. Thus, the encoded proteins may function as miRNA- and/or mRNA-specific translational regulators in vivo (Cziko, 2009).

It is suggested, that as for previously identified sev-dfmr1 suppressors Ago1, Lgl, and Me31b, analysis of PABP, Smd3, Rm62, Orb2, and Dco proteins, encoded by the sev-dfmr1 suppressor genes identified in this study, will help elucidate how dFMR1 works in translational regulation, RNA targeting and localization, and ncRNA pathway function (Cziko, 2009).

Three lines of evidence indicate that the genes identified encode proteins with translational repressor activity. First, with the exception of Dco, all of these proteins have been previously implicated in some aspect of RNA metabolism and are present on dFMR1-containing neuritic granules in which RNA is repressed and transported. Second, the rough-eye phenotype observed in sev-dfmr1 has been linked to the ability of FMRP to repress mRNA translation. Thus, it would be expected that the phenotype would be alleviated by mutations that reduce the efficiency of translational repression. Third, overexpression of Dco, Pabp, Orb2, or Rm62 inhibits the dendritic growth of neurons, a phenotype predicted for neuronal translational repressors. These observations are consistent with the idea that translation of RNAs in neurites, which promotes dendritic branching, is inhibited by overexpression of Dco, Pabp, Orb2, or Rm62. Thus, genetic interaction data, molecular localization, and one functional test in dendrites indicate that Dco/Dbt, PABP, Rm62, or SmD3 function as neuronal translational repressors (Cziko, 2009).

The identification of several canonical translational-factor encoding genes as suppressors of sev-dfmr1 highlights the point that individual translational control molecules work in multicomponent complexes and therefore have several functional interactions. PABP is one example of a protein that is currently believed to perform two opposing functions of translational control. In addition to its well-studied role as a translational activator, PABP can mediate translational repression, e.g., of Vasopressin mRNA although the exact mechanism remains unclear. Dual roles in activation and repression are also suggested by the observation that reduced or elevated levels of PABP have similar effects at the Drosophila neuromuscular junction (NMJ). Additionally, PABP associates with particles containing BC1, a neuron-specific noncoding RNA with translational repressor function, as well as a CYFIP-FMRP complex that may function as a repressor in some contexts but as an activator in others. Similarly, Orb2 homologs (CPEBs) though required for translational activation of CPE-containing mRNAs via poly-A polymerase, also allow translational repression in combination with Maskin or Cup proteins (Cziko, 2009).

It was somewhat surprising that SmD3, a splicing factor, was identified in a screen for translational repressors. However, SmD3 has additional nonsplicing functions: in Caenorhabditis elegans, the Sm proteins are required for germ cell mRNP assembly and RNA localization. Such a role in translational regulation and mRNP assembly is more consistent with functions predicted by the genetic experiments (Cziko, 2009).

Rm62/Dmp68 is a member of the DEAD-box helicase family that has been shown to be associated with a dFMR1-containing RNAi silencing complex. It also has additional roles during transcription and mRNA processing as well as potentially in miRNA processing as part of the Drosha complex. Based on the biochemical evidence for Rm62's presence in FMRP-containing complexes, it is not surprising that rm62 mutations show strong genetic interactions with dfmr1. However, the mechanism of suppression remains unknown (Cziko, 2009).

Finally Dco/Dbt, is by far the most elusive protein in regard to its potential function in the translational regulatory pathway. Dco/Dbt, a casein kinase I (CKI) is best known from circadian biology where it phosphorylates Per and expedites its degradation. dFMR1 protein has several phosphorylation sites, one of which in S2 cells has been demonstrated to be phosphorylated by a CKII protein. While the functional requirement for CKI-dependent dFMR1 phosphorylation is as of yet not understood, there is considerable evidence that the phosphorylation state of FMRP may actually determine its role in translation. Biochemical data demonstrate that most FMRP in granules is in the phosphorylated state while FMRP in the polysome fraction is dephosphorylated, suggesting a mechanism to switch state from an activator to a repressor, and an important regulatory role for kinases that phosphorylate FMRP (Cziko, 2009).

Another interesting potential link between the two proteins is the behavioral observation that patients with Fragile-X Mental Retardation often display circadian disturbances. This altered circadian rhythm is also present in the Drosophila dfmr1 mutants that usefully model fragile-X syndrome (Cziko, 2009).

The identification of these proteins as sev-dfmr1 modifiers illustrates the many possibly regulatory roles of RNA-associated proteins. In addition, the data associating Dco/Dbt with RNA regulation indicates unexplored and novel mechanisms of RNA regulation in neurons (Cziko, 2009).

Given that dFMR1/FMRP is thought to function in miRNA-dependent translational repression, it was of particular interest to asking whether these dFMR1 interactors had any role in this pathway. To address this issue, a sensitive in vivo assay that uses a fluorescent reporter was employed to reveal the strength of translational repression via an endogenous (bantam) miRNA. When combined with genetic mosaic analysis, this assay can be used to study null mutations in candidate genes, as long as the mutations do not cause cell lethality. The assay appears more sensitive than typically used cell-based assays on the evidence of prior analysis of Me31B, whose requirement for miRNA function, clearly seen in the in vivo assay, is only evident in double-knockdown experiments in the more commonly used cell-culture assays (Cziko, 2009).

In vivo experiments revealed no requirement for the sev-dfmr1 interacting proteins Dco, Orb2, Rm62, and SmD3 in miRNA repression. For reasons explained above, it is unlikely that this reflects a weakness in the experimental assay for miRNA function. A bigger surprise was the finding that the dFMR1 itself appeared dispensable for miRNA function in vivo. Because the allele used is a well-characterized null allele, and the absence of dFMR1 in the mutant clones is confirmed by antibody staining, the conclusion that dFMR1 is not a core, essential component of the RISC/miRNA pathway is strong. This conclusion is not inconsistent with any of the existing data showing biochemical association between RISC and FMRP and genetic interactions between Ago1 and FMRP. However, it is also consistent with recent observations indicating the dispensability of FMRP for RISC function in cultured cells. It is suggested that the function of dFMR1 and, by extension, FMRP may be restricted to a subset of transcripts, for instance those with UTRs containing both FMRP binding motifs and miRNA target elements. Indeed similar models that account for the mRNA specificity of FMRP have been previously proposed (Cziko, 2009).

These data provide a foundation on which to design further experiments to understand the specific roles of FMR1 and its interacting proteins in translational control (Cziko, 2009).

Fragile X mental retardation protein is required for programmed cell death and clearance of developmentally-transient peptidergic neurons

Fragile X syndrome (FXS), caused by loss of fragile X mental retardation 1 (FMR1) gene function, is the most common heritable cause of intellectual disability and autism spectrum disorders. The FMR1 product (FMRP) is an RNA-binding protein best established to function in activity-dependent modulation of synaptic connections. In the Drosophila FXS disease model, loss of functionally-conserved dFMRP causes synaptic overgrowth and overelaboration in pigment dispersing factor (PDF) peptidergic neurons in the adult brain. This study identified a very different component of PDF neuron misregulation in dfmr1 mutants: the aberrant retention of normally developmentally-transient PDF tritocerebral (PDF-TRI) neurons. In wild-type animals, PDF-TRI neurons in the central brain undergo programmed cell death and complete, processive clearance within days of eclosion. In the absence of dFMRP, a defective apoptotic program leads to constitutive maintenance of these peptidergic neurons. Tests were performed to see whether this apoptotic defect is circuit-specific by examining crustacean cardioactive peptide (CCAP) and bursicon circuits, which are similarly developmentally-transient and normally eliminated immediately post-eclosion. In dfmr1 null mutants, CCAP/bursicon neurons also exhibit significantly delayed clearance dynamics, but are subsequently eliminated from the nervous system, in contrast to the fully persistent PDF-TRI neurons. Thus, the requirement of dFMRP for the retention of transitory peptidergic neurons shows evident circuit specificity. The novel defect of impaired apoptosis and aberrant neuron persistence in the Drosophila FXS model suggests an entirely new level of 'pruning' dysfunction may contribute to the FXS disease state (Gatto, 2011).

It has long been known that loss of dFMRP compromises the architecture of the small and large ventrolateral PDF neuron circuitry in Drosophila brain. This study identified an additional, novel defect harbored within the PDF neuron population; the dfmr1 null mutation enables inappropriate life-long survival of PDF-TRI neurons normally destined for death via an apoptosis program immediately after eclosion. Although PDF-TRI neurons are not circadian pacemakers, the retention of these cells into adulthood introduces functional neurons with elaborate synaptic contacts that may alter information flow and processing by contributing an aberrant source of PDF. The role of dFMRP in regulating the initiation of apoptotic events appears to be somewhat neuron-type specific and selective to PDF-TRI neurons. Nevertheless, the dfmr1 null mutants do present a generalized delay in peptidergic neuronal process removal upon cell death, including the delayed elimination of CCAP and bursicon cells. As these studies were conducted in ubiquitously dfmr1 null animals, determination of the cell autonomy assigned to these novel dFMRP functions, as compared to potentially coordinate and impinging function(s) of dFMRP in neighboring cells, will require future experimentation (Gatto, 2011).

The key finding of this study is a novel role for dFMRP in mediating neuronal survival by influencing apoptotic initiation. In contrast, the recent focus of cell population regulation has centered on the role of dFMRP in controlling proliferation and differentiation. For example, dFMRP has been implicated in germline stem cell maintenance and proliferative capacity in the Drosophila ovary and the E3 ubiquitin ligase cbl (Casitas B-lineage lymphoma). Earlier mammalian embryonic studies demonstrated altered cellular differentiation from both Fmr1 deficient mouse and human stem cells in vitro. Fmr1-deficient neurospheres has been shown to yield 3-5 fold increases in cells of the TuJ1-positive neuronal lineage and marked decreases in their GFAP-positive glial counterparts, likely due to increased apoptosis. In the mouse Fmr1 knockout, there is also over-proliferation in the subventricular zone. However, human neural progenitor cells isolated from FMR1-deficient fetal cortex were later shown to have proliferative and specification indices comparable to control, albeit with altered gene expression profiles. It has also been demonstrated that FMRP plays a role in regulating the differentiation and proliferative capacity of adult neural stem cells. In the mouse Fmr1 knockout, this was examined in relation to hippocampal neurogenesis, thought to be involved in the plasticity required for learning and memory. FMRP insufficiency yielded increased proliferation, with a bias toward decreased neuronal and increased glial differentiation, both in vitro and in vivo. In studies of the Drosophila FXS model, dfmr1 deficiency resulted in increased mitotic activity in neuroblasts during larval development, from which increased numbers of adult neurons were derived. The dfmr1 null neuroblasts exit quiescence prematurely to execute excess proliferative activities, demonstrating cell cycle misregulation (Gatto, 2011).

Despite this evidence that FMRP plays a role in neural stem cell proliferation, it was found that normal peptidergic neuron numbers were present in the PDF, CCAP and bursicon populations in the dfmr1 null mutant. Thus, there is no evidence for a proliferative defect in these neuronal types. In contrast, adult retention of the PDF-TRI neuron subpopulation was observed that is usually present only transiently during development, owing to a requirement for dFMRP in apoptotic initiation following eclosion. The novel role of dFMRP in this mechanism is presently unknown. As an RNA-binding protein known to either repress or activate the translation of particular target mRNAs, does dFMRP serve as a negative regulator of a survival or anti-apoptotic factor, like Drosophila inhibitor of apoptosis 1 (DIAP1)? Does dFMRP itself promote the expression of a pro-apoptotic factor, such as Reaper, Head involution defective, or Grim, which could antagonize DIAP1 function? While no obvious candidates in these RNA target classifications have yet been uncovered for dFMRP in extensive screening efforts, it is of potential interest that down-regulation of the ENA/VASP actin regulatory proteins has been recently implicated in neuronal degeneration via an apoptosis program (Gatto, 2011).

The ENA/VASP family is crucial for appropriate filopodia formation, serving to block F-actin filament capping and encourage elongation. The sole representative of this family in Drosophila, enabled (ena), has been shown to promote dendritic branching and actin-rich spine-like protrusions in dendritic arborization (DA) sensory neurons. In dfmr1 null mutants, DA neurons in the peripheral nervous system display an increased number of higher-order branches, whereas dFMRP over-expression reciprocally yields dramatically decreased branching. Mechanistically, these defects have been linked to the small GTPase Rac1; however, the structural phenocopy suggests that ena could likewise be upregulated in the Drosophila FXS model. Precedent for this type of misregulation has been demonstrated by the observations that dFMRP negatively regulates other cytoskeleton organizing proteins, including Futsch, the mictrobule-binding MAP1B homolog, and Chickadee, the actin-binding Profilin homolog. Thus, if the PDF-TRI neurons are sensitive to ena-mediated cell death, the dfmr1 mutation may be neuroprotective via this mechanism (Gatto, 2011).

An outstanding question centers on the relative specificity of dFMRP-mediated influence on the commencement of programmed cell death. While serving to largely prevent developmental eradication of the PDF-TRI neurons, the dfmr1 mutation conferred no such long-term maintenance to neurons expressing the neuropeptides CCAP and/or bursicon. What segregating factors might differentiate these classes of peptidergic neurons and the relative requirement of dFMRP for their survival? Mechanistically, apoptotic initiating factors may differ between these neuron classes. The apoptosis of CCAP neurons has been linked to declining levels of the steroid hormone 20-hydroxyecdysone, which allow the pro-apoptotic accumulation of reaper and grim mRNA. No such requirement has yet been defined for the PDF-TRI neurons. Perhaps within the subset of CCAP neurons expressing bursicon, the temporal paracrine role of these neurons requires more stringent ablational regulation, and their effective removal is therefore less susceptible to reprogramming. Moreover, it is plausible that these other neuron classes may be more reliant upon other RNA-binding proteins, rather than dFMRP. For example, LARK is an RNA-binding protein that interacts with and stabilizes dFMRP and also notably influences the development of PDF LNv neurons. Although reportedly pan-neuronal, LARK is known to be enriched within the cytoplasm of CCAP neurons where it oscillates in a circadian manner. If key opportunistic translational regulation of an apoptotic factor underlies initiation of programmed cell death, and if LARK is the predominant translational regulator in these cells, it would be of interest to examine the cell death profiles of the CCAP/bursicon neurons in LARK-deficient animals to assess this mechanistic possibility (Gatto, 2011).

Although dfmr1 mutation did not result in long-term survival of CCAP/bursicon neurons, the dfmr1 nulls did display a significant delay in the clearance of these cells, suggesting a delay in the initiation or progression of the apoptotic process. This is similar to observations in the bursicon receptor mutant rickets (rk4). In rk4 mutants, one-third of the abdominal bursicon neurons were still evident at 24 h post-eclosion, which was then reduced to approximately 8% in 2-day old mutants. Clearly then, normal bursicon signaling is required for the appropriate initiation and regulation of death in these neurons. This suggests that the dfmr1 mutation may alter bursicon signaling or its downstream consequences. As immunocytochemistry failed to reveal any obvious differences in the observable levels of bursicon in dfmr1 nulls as compared to controls, dFMRP may more likely influence bursicon release, the stability or function of the bursicon receptor, or the downstream signaling cascade of this neurotransmission. Importantly, post-ecdysial wing epidermis cell death mediated via bursicon requires activation of the cAMP/PKA pathway, and it is therefore interesting to note that significant impairment in cAMP production occurs both in dfmr1 mutants and human FXS patients. Thus, defective cAMP pathway signaling may underlie the neuronal clearance defect in the FXS condition (Gatto, 2011).

Another factor that could contribute to the delayed or defective removal of these peptidergic neurons and their processes is aberrant glial activation. Drosophila glia have been implicated as active participants in the phagocytosis of apoptotic neurons, developmental pruning of excessive axonal tracts, and clearance of degenerating axonal processes. In addition, neuronal programmed cell death induces glial cell division during a critical period in the first week post-eclosion in adult Drosophila, perhaps facilitating cell corpse removal. Though dFMRP has not been readily detected in CNS repo-expressing glial cells, hFMRP has been detected in the early development of both astrocytic and oligodendrocyte lineages. If in this specialized role glial responsiveness is altered in the dfmr1 null, this could effect the impaired elimination of the transient peptidergic neurons (Gatto, 2011).

Future studies of PDF-TRI neuron retention in the dfmr1 null brain will focus on the potential role of synaptic activity in the critical survival versus apoptosis decision. Other PDF-expressing neurons, including the s- and lLNvs, appear to function downstream of GABAergic synaptic inputs and express the Rdl (resistance to dieldrin) GABAA receptor subunit. In addition, the LNvs also express the DmGluRA metabotropic glutamate receptor although, confoundingly, glutamate may serve an inhibitory role on the LNvs, as its dosage dependent application decreases sLNv intracellular calcium levels. By determining how the PDF-TRI neurons may integrate such signals, it is hoped to gain further insight as to their contribution to neural dysfunction in the Drosophila FXS model. Such investigations will also allow assessment of the circuit-specific applicability of the 'mGluR Theory of FXS', implicating FMRP function downstream of mGluR activation, compared to the 'GABAAR Theory of FXS', which suggests disruption of inhibitory GABAergic signaling is a key to the progression of the disease state. Although these theories are by no means mutually exclusive, each disruption may have a differential relative contribution to neuronal defects in the disease state, including the new discovery here of altered developmental pruning of neurons from specific modulatory neural circuits (Gatto, 2011).

Elevated levels of the vesicular monoamine transporter and a novel repetitive behavior in the Drosophila model of fragile X syndrome

Fragile X Syndrome (FXS) is characterized by mental impairment and autism in humans, and it often features hyperactivity and repetitive behaviors. The mechanisms for the disease, however, remain poorly understood. The dfmr1 mutant in the Drosophila model of FXS grooms excessively, which may be regulated differentially by two signaling pathways. Blocking metabotropic glutamate receptor signaling enhances grooming in dfmr1 mutant flies, whereas blocking the vesicular monoamine transporter (VMAT) suppresses excessive grooming. dfmr1 mutant flies also exhibit elevated levels of VMAT mRNA and protein. These results suggest that enhanced monoamine signaling correlates with repetitive behaviors and hyperactivity associated with FXS (Tauber, 2011).

There are four major findings from this study: 1) age-dependent abnormal climbing in dfmr1 mutant flies can be genetically rescued, 2) excessive grooming is identified as a new behavioral defect in dfmr1 mutant flies, 3) excessive grooming can be suppressed by reserpine, and 4) dVMAT mRNA and protein levels are increased in the absence of dFMRP (Tauber, 2011).

A previous study abnormal climbing activity was observed in dfmr1 mutant flies that progresses with age. These results in this study confirm this finding, and additionally show that introducing a wild-type dfmr1 transgene into the dfmr1 mutant background restores normal climbing behavior. Further, a frameshift mutation in the open reading frame of the transgene abolishes the rescue of climbing behavior. These results demonstrate that the abnormal climbing in dfmr1 mutant flies is directly caused by the loss of dFMRP (Tauber, 2011).

In this study, excessive grooming was identified as an important and novel behavioral defect in the fly model of FXS. The results show that dfmr1 mutant flies groom significantly more than control flies, and that mutant flies also have significantly longer grooming bouts. Further, this excessive grooming intensifies with age in dfmr1 mutant flies, whereas control flies show essentially no change in grooming activity over time. A wild-type copy of the dfmr1 gene can rescue the excessive grooming defect in dfmr1 mutant flies. It is worth noting that FS mutants show more dramatic climbing and grooming defects compared to dfmr1 mutants. The exact underlying cause is not known, but it may be due to the presence of either mRNA or a truncated peptide produced from the FS rescue fragment, having gain of function effects (Tauber, 2011).

In video recordings, control flies mostly walk around the observation chamber and groom occasionally, but rarely stand motionless. In contrast, the mutant fly spends more time grooming. It is possible that grooming is a default activity that occurs whenever a fly is not walking. If this is the case, excessive grooming in dfmr1 mutant flies could result indirectly from problems in walking. This explanation would be consistent with the observation that dfmr1 mutant flies exhibit postural problems and uncoordinated movement. However, the dfmr1 flies are capable of climbing after a brief period of mechanical disturbance (i.e., knocking them down in the graduated cylinder), albeit at a slower speed compared to control flies. Further, reserpine suppresses grooming in dfmr1 mutant flies without improving walking. These observations suggest that grooming is not simply a default behavior in the absence of walking and that dfmr1 mutations specifically cause excessive grooming. Notably, Fmr1 KO mice have also been reported to exhibit excessive grooming when presented with social stimuli. Hence, heightened repetitive activity such as grooming is a common behavioral defect in FXS (Tauber, 2011).

Although reducing mGluR signaling has been shown to rescue learning and memory defects in both mouse and fly FXS models, this study found that the mGluR antagonist MPEP enhances excessive grooming in dfmr1 mutant flies. This is not completely surprising, as the absence of dFMRP likely alters numerous signaling pathways and developmental processes of the nervous system. MPEP also fails to rescue abnormal sleep and circadian rhythm in dfmr1 mutant flies, which may impact locomotor activity like grooming. It is worth noting that dfmr1 mutant flies did not groom more when treated with LiCl, suggesting that mGluR antagonists and LiCl may have different neuronal targets. An interesting question that arises from these results is whether an mGluR agonist might suppress grooming in dfmr1 mutant flies. Previous results have shown that glutamate at concentrations as low as 5 µM is toxic to dfmr1 mutant flies and significantly affects various behaviors in the fly. This makes it difficult to assess the potential benefit of mGluR agonists on grooming (Tauber, 2011).

Previous work shows that dopamine plays a role in FXS in both mice and Drosophila, and that biogenic monoamines stimulate fly grooming. In the current studies, blocking dVMAT with reserpine suppresses excessive grooming in dfmr1 mutant flies, but only significantly at 50 µM. Control flies groom significantly less when treated with just 10 µM. These results indicate that dfmr1 mutant flies are less sensitive to reserpine's effect on grooming. However, the possibility cannot be excluded that reserpine has additional targets and therefore generally sedates the fly. Both suppression of dVMAT as well as a non-specific target could slow down most motor activities including grooming. Alternatively, it is possible that basal monoamine activity is required for grooming, and therefore shutting down monoamine signaling may block the behavior (Tauber, 2011).

Elevated levels of dVMAT transcript and protein were found in dfmr1 mutant flies. Although these increases are not statistically significant in some instances, they are consistent in both mutant lines. However, it is not clear from the results how the loss of dFMRP leads to increased dVMAT expression. The transcription of dVMAT may be directly increased. Alternatively, degradation of dVMAT mRNA may decrease in the absence of dFMRP, a distinct possibility as FMRP has been previously indicated to regulate mRNA stability. How dFMRP regulates dVMAT protein levels is also unclear. Elevated dVMAT protein levels may occur exclusively because of increased transcript levels, but could also result from increased translation or reduced degradation of the protein. Nonetheless, these observations are in agreement with the known function of FMRP as a regulator of transcription and translation (Tauber, 2011).

Many factors may contribute to the excessive grooming in dfmr1 mutant flies, and the data do not resolve whether upregulation of dVMAT directly influences this behavior. Overexpression of dVMAT stimulates grooming in flies, and dopamine levels are increased in dfmr1 mutant brains. Monoamines could directly or indirectly modulate multiple downstream signaling pathways involved in grooming. The hyposensitivity to reserpine seems to suggest that a greater number of dVMATs are present on mutant synaptic vesicles, as a higher concentration of the drug is required to reduce grooming. It is noted that overexpression of dVMAT in serotonergic and dopaminergic neurons leads to hypersensitivity to reserpine on grooming. One likely explanation of these differences is that dfmr1 mutations affect not only monoamine cells but also other cells such as neurons in the mushroom bodies and neurons postsynaptic to monoamine cells. Dopamine signaling is also reduced in the forebrain of Fmr1 KO mice. Thus, while plausible given the effect of reserpine, a clear causal relationship cannot be established between excessive grooming and dVMAT expression levels (Tauber, 2011).

Understanding how FMRP functions in development and aging will be crucial for effective treatment of FXS. Studies in mouse and Drosophila indicate that FMRP is temporally regulated and that treatment requires proper timing. The current results add to the growing evidence of the importance of FMRP in age-related processes, and also demonstrate that hyperactivity and repetitive behavior increase with age in the Drosophila model. Interestingly, the severity of autistic behavior and anxiety has been found to increase with age in studies of FXS patients. The results indicating that reserpine is effective in adult dfmr1 mutant flies could help develop or improve treatment, as they suggest that hyperactive and repetitive behavior in older patients is potentially reversible (Tauber, 2011).

Although it is thought that excessive grooming in dfmr1 mutant flies is a model of an impulsive and repetitive behavior, animal models can never completely recapitulate human disorders. The mechanisms underlying repetitive behaviors in FXS patients are likely much more complex. Nonetheless, this study demonstrate a correlation between monoamine signaling and the excessive grooming phenotype in dfmr1 mutant flies and that VMAT is a protein that merits further study in FXS. Importantly, this study provides potentially useful information for improving the pharmaceutical treatment of FXS symptoms in human patients (Tauber, 2011).

Neural circuit architecture defects in a Drosophila model of Fragile X syndrome are alleviated by minocycline treatment and genetic removal of matrix metalloproteinas

Fragile X syndrome (FXS), caused by loss of the fragile X mental retardation 1 (FMR1) product (FMRP), is the most common cause of inherited intellectual disability and autism spectrum disorders. FXS patients suffer multiple behavioral symptoms, including hyperactivity, disrupted circadian cycles, and learning and memory deficits. Recently, a study in the mouse FXS model showed that the tetracycline derivative minocycline effectively remediates the disease state via a proposed matrix metalloproteinase (MMP) inhibition mechanism. This study used the well-characterized Drosophila FXS model to assess the effects of minocycline treatment on multiple neural circuit morphological defects and to investigate the MMP hypothesis. Drosophila Fmr1 (dfmr1) null animals were treated with minocycline to assay the effects on mutant synaptic architecture in three disparate locations: the neuromuscular junction (NMJ), clock neurons in the circadian activity circuit and Kenyon cells in the mushroom body learning and memory center. Minocycline was found to effectively restore normal synaptic structure in all three circuits, promising therapeutic potential for FXS treatment. Next the MMP hypothesis was tested by assaying the effects of overexpressing the sole Drosophila tissue inhibitor of MMP (TIMP) in dfmr1 null mutants. It was found that TIMP overexpression effectively prevents defects in the NMJ synaptic architecture in dfmr1 mutants. Moreover, co-removal of dfmr1 similarly rescues TIMP overexpression phenotypes, including cellular tracheal defects and lethality. To further test the MMP hypothesis, dfmr1;mmp1 double null mutants were generated. Null mmp1 mutants are 100% lethal and display cellular tracheal defects, but co-removal of dfmr1 allows adult viability and prevents tracheal defects. Conversely, co-removal of mmp1 ameliorates the NMJ synaptic architecture defects in dfmr1 null mutants, despite the lack of detectable difference in MMP1 expression or gelatinase activity between the single dfmr1 mutants and controls. These results support minocycline as a promising potential FXS treatment and suggest that it might act via MMP inhibition. It is concluded that FMRP and TIMP pathways interact in a reciprocal, bidirectional manner (Siller, 2011).

Drosophila FMRP participates in the DNA damage response by regulating G2/M cell cycle checkpoint and apoptosis

Fragile X syndrome, the most common form of inherited mental retardation, is caused by the loss of the fragile X mental retardation protein (FMRP). FMRP is a ubiquitously expressed, multi-domain RNA-binding protein, but its in vivo function remains poorly understood. Earlier studies show that FMRP participates in cell cycle control during development. This study used Drosophila mutants to test if FMRP plays a role in DNA damage response under genotoxic stress. It was found that significantly fewer dfmr1 mutants survive to adulthood than wild-types following irradiation or exposure to chemical mutagens, demonstrating that the loss of Drosophila FMRP (dFMRP) results in hypersensitivity to genotoxic stress. Genotoxic stress significantly reduces mitotic cells in wild-type brains, indicating the activation of a DNA damage-induced G2/M checkpoint, while mitosis is only moderately suppressed in dfmr1 mutants. Elevated expression of cyclin B, a protein critical for the G2 to M transition, is observed in the larval brains of dfmr1 mutants. CycB mRNA transcripts are enriched in the dFMRP-containing complex, suggesting that dFMRP regulates DNA damage-induced G2/M checkpoint by repressing CycB mRNA translation. Reducing CycB dose by half in dfmr1 mutants rescues the defective G2/M checkpoint and reverses hypersensitivity to genotoxic stress. In addition, dfmr1 mutants exhibit more DNA breaks and elevate p53-dependent apoptosis following irradiation. Moreover, a loss-of-heterozygosity assay shows decreased irradiation-induced genome stability in dfmr1 mutants. Thus, dFMRP maintains genome stability under genotoxic stress and regulates the G2/M DNA (Liu, 2012).

Phenotypic analysis of animal models of FXS continues to reveal novel functions for FMRP. This study presents multiple lines of experimental evidence demonstrating for the first time that dFMRP is involved in DNA damage response. DNA damage responses are executed through coordinated interplays and cross-talks of multiple players from sensors to transducers, and finally to effectors. There are four distinct pathways involved in the DNA damage response: cell cycle arrest (also known as DNA damage checkpoint), transcriptional induction, DNA repair and apoptosis; the four pathways act independently under certain conditions, but frequently, they interact to repair the damaged DNA or commit apoptosis. The hypersensitivity to irradiation, G2/M checkpoint defects, excessive apoptosis and increased number of DNA breaks in dfmr1 mutants after irradiation all support that dfmr1 plays a critical role in DNA damage response. In addition, dfmr1 mutants show an elevated rate of loss-of-heterozygosity (LOH) upon DNA damage, indicating reduced genome stability in dfmr1 mutants. It is well established that proteins involved in checkpoint control and DNA repair play a critical role in maintaining genome integrity. Thus, the decreased genome stability in dfmr1 mutants also supports the conclusion that dfmr1 participates in DNA damage response (Liu, 2012).

Loss of dfmr1 affects cell cycle progression in different developmental processes. This study speculated that the hypersensitivity to DNA damage in dfmr1 mutants might be due to a defective cell cycle control. However, in the absence of genotoxic stress, normal expression of the G1/S checkpoint regulator CycE, normal DNA synthesis activity and normal G2/M checkpoint were detected in dfmr150M mutants compared with the wild-type. These results indicate that the G1/S and G2/M checkpoints are functional in dfmr1 mutants. Following genotoxic stress, however, significantly more mitotic cells were found in the larval brains and wing discs of dfmr150M mutants compared with the wild-type controls, indicating a defect in the G2/M DNA damage checkpoint in dfmr1 mutants. In support of this checkpoint defect, cell cycle profiling of the larval brain cells by flow cytometry demonstrates a similar trend of cell cycle profile between dfmr1 and mei-41 mutants. The study therefore concludes that dfmr1 primarily regulates the G2/M checkpoint in response to genotoxic stress (Liu, 2012).

Cyclins and their CDK partners play an important role in regulating cell cycle progression. Misregulation of these cyclin–CDK complexes causes defective cell cycle progression, especially in the cells with damaged DNA. When DNA damage is inflicted at the G1 stage, the G1/S checkpoint regulator CycE–CDK2 is silenced to arrest the G1 to S transition. Alternatively, when DNA damage occurs at the G2 stage or if DNA damage remains unrepaired from the previous G1 or S phase, the CycB–CDK1 complex (also known as a mitosis promoting factor) is inhibited to arrest cells at the G2/M transition. In light of a report demonstrating that dFMRP suppresses the expression of CycB at the mid-blastula transition during early embryonic development, this study speculated that the G2/M checkpoint defect observed in dfmr1 mutants after genotoxic stress might be due to the altered expression of CycB. Indeed, CycB protein was found to be elevated in dfmr150M brains, while the other two G2/M checkpoint regulators, CycA and CycB3, were unaltered, indicating a specific suppression of CycB by dFMRP. This specific regulation of CycB by dFMRP is further supported by the observation that CycB mRNA is enriched in the dFMRP-mRNA protein complex from larval brains. Moreover, reducing the dose of CycB by half partially rescues the increased mitosis and hypersensitivity of dfmr1 mutants to genotoxic stress. Thus, up-regulation of CycB in dfmr1 mutants accounts, at least partially, for the G2/M checkpoint defect in response to DNA damage. In support of this conclusion, overexpression of the truncated, stable form of CycB is sufficient to induce G2/M transition defect in both eye discs and wing discs after irradiation (Liu, 2012).

The CycB level is tightly regulated during the cell cycle at both the transcriptional and post-translational levels. Among the many regulators of CycB, the transcription factors NF-Y, FoxM1 and B-Myb activate transcription of CycB. These CycB regulators are important for the G2/M progression under both normal and stress conditions. Activation of FoxM1 is critical for the G2/M arrest, whereas B-Myb is required for the recovery of G2/M checkpoint in p53-negative cells. This study reveals a negative regulation of CycB by dFMRP at the post-transcriptional level that controls the G2/M checkpoint under genotoxic stress (Liu, 2012).

In addition to the G2/M cell cycle defect in dfmr1 mutants, an obviously disrupted nucleolar structure in the mutant salivary gland cells was found. As the nucleolus is critical for the DNA damage-induced p53 activation and apoptosis, apoptosis in the wing discs was examined by anti-caspase-3 staining. Spontaneous apoptosis is undetectable in the untreated dfmr1 mutants and wild-types, while genotoxic stress evokes excessive p53-dependent apoptosis in dfmr1 mutants. Overactivation of p53 in the dfmr1 mutants was further confirmed by elevated expressions of the pro-apoptotic hid-rpr-grim genes transcriptionally regulated by p53. It is unknown at present why dfmr1 mutants show increased p53-dependent apoptosis after irradiation. One interpretation for the phenotype is compromised DNA damage repair in dfmr1 mutants. Alternatively, the increased level of CycB in dfmr1 mutants may also lead to elevated apoptosis. It is worth noting that without genotoxic stress, dfmr1 is required for apoptosis and clearance of developmentally transient neurons in the adult brains. On the other hand, overexpression of dFMRP in multiple tissues including the wings and eyes also induces apoptosis, though a possible role of p53 in the process was not examined. Thus, dFMRP can either promote or inhibit apoptosis under different conditions by distinct yet uncharacterized mechanisms (Liu, 2012).

Drosophila dfmr1 null mutants also exhibit decreased genome stability as revealed by the LOH assay. Apoptosis is an endogenous and well conserved program to eliminate cells with severely damaged DNA to avoid propagation of potential mutations. It is conceivable that loss of dfmr1 decreases genome stability, presumably resulting from a DNA repair defect, leading to increased apoptosis and lethality following DNA damage. Further experiments are required to unravel the causal relationships between dFMRP, DNA repair and apoptosis (Liu, 2012). 

It is not known if FXS patients and Fmr1 knockout mice are also hypersensitive to genotoxic stress. It is well established that the fragile sites of chromosomes are more prone to DNA damage and thus more dependent on the integrity of DNA repair mechanisms to maintain chromosomal stability. A study using fibroblasts reported that the DNA damage response is required to maintain the stability of the fragile X site. In addition, mutagen-induced genome instability is observed in the cultured lymphocytes from FXS patients. However, a subsequent study reported that lymphocytes from FXS patients display normal genome stability under genotoxic stress. This discrepancy remains to be resolved. In Drosophila, there is only one FMRP homolog instead of three FMRP family members in mammals. The redundancy of three FMRP members in mammals may make the phenotype of single mutants too weak to be detected. It would be of interest to test if double or triple mouse knockouts of the three FMRP family members exhibit the hypersensitivity to genotoxic stress observed in the Drosophila dfmr1 mutants. Such a result would underscore a novel role for FMRP in maintaining genome stability and cell cycle control to allow for proper neuronal proliferation during brain development (Liu, 2012).

Caprin controls follicle stem cell fate in the Drosophila ovary

Adult stem cells must balance self-renewal and differentiation for tissue homeostasis. The Drosophila ovary has provided a wealth of information about the extrinsic niche signals and intrinsic molecular processes required to ensure appropriate germline stem cell renewal and differentiation. The factors controlling behavior of the more recently identified follicle stem cells of the ovary are less well-understood but equally important for fertility. This study reports that translational regulators play a critical role in controlling these cells. Specifically, the translational regulator Caprin (Capr) is required in the follicle stem cell lineage to ensure maintenance of this stem cell population and proper encapsulation of developing germ cells by follicle stem cell progeny. In addition, reduction of one copy of the gene fmr1, encoding the translational regulator Fragile X Mental Retardation Protein, exacerbates the Capr encapsulation phenotype, suggesting Capr and fmr1 are regulating a common process. Caprin was previously characterized in vertebrates as Cytoplasmic Activation/Proliferation-Associated Protein. Significantly, it was found that loss of Caprin alters the dynamics of the cell cycle, and evidence is presented that misregulation of CycB contributes to the disruption in behavior of follicle stem cell progeny. These findings support the idea that translational regulators may provide a conserved mechanism for oversight of developmentally critical cell cycles such as those in stem cell populations (Reich, 2012).

Distinct stem cell populations within the ovary produce the different cell types that must act coordinately to create a functional egg. The Drosophila ovary has proved an extremely fruitful model system to study this process. Two stem cell populations have been identified: the germline stem cells (GSCs), and the follicle stem cells (FSCs), which reside at the anterior of the ovariole in a structure called the germarium. The GSCs give rise to the invariant 15 nurse cells and single oocyte comprising a cyst. Two FSCs produce all of the different types of somatic cells that surround the cysts and connect the developing egg chambers. During development, a cyst progresses through four morphologically and functionally distinct regions of the germarium: 1, 2a, 2b and 3. Region 1 houses the GSCs and escort cells. Here, GSCs divide to produce another GSC (self renewal) and a cystoblast that undergoes four synchronous divisions to produce a 16-cell cyst. As cysts develop, cellular processes from the escort cells surround them in regions 1 and 2a of the germarium and help move the cysts through this region. Two FSCs reside at the border of regions 2a and 2b and produce the follicle cells, stalk cells, and other somatic cells associated with a developing egg chamber. Once a cyst is encapsulated it buds off from the germarium forming a stage 1 egg chamber. Production of a functional egg requires proper control of proliferation and differentiation of both stem cell populations and their progeny (Reich, 2012).

Stem cell activity is controlled by intrinsic and extrinsic factors, which operate in the context of specialized microenvironments, stem cell niches. Much is known about the molecular mechanisms regulating GSCs and their role in producing a functional egg. For example, GSCs are found in a cellular niche at the anterior of the germarium. They are anchored to the cap cells via DE-cadherin, and loss of this adhesion leads to loss of stem cell properties. In their niche, GSCs receive extrinsic signals, such as Dpp, from cap cells, that maintain their stem cell identity and prevent differentiation. Numerous intrinsic factors have also been identified that control GSC proliferation and differentiation and comprise a variety of molecular mechanisms. Prominent among them are proteins involved in translational regulation such as the eukaryotic initiation factor eIF4A and the translational regulators Pumilio, Nanos, and Vasa. In addition, GSC self-renewal and differentiation rely on chromatin modifiers which influence transcriptional regulation. Both intrinsic and extrinsic factors ensure that GSCs remain in an undifferentiated state while in their niche, yet continue to produce daughter cells that form the invariant 16-germ cells of each cyst (Reich, 2012).

Significantly less is known about the regulation of the FSCs. While FSCs also require cell adhesion proteins to maintain their stem cell identity, in this case DE-cadherin and integrins, the cellular nature of the FSC niche is poorly understood. Recent work has suggested that each FSC may maintain contact with a single escort cell, however, the full complement of cells that comprise the FSC niche remains uncertain. Like GSCs, FSCs also receive extrinsic signals controlling their proliferation and differentiation. These include long-range Hh and Wg signals, which emanate from the cap cells, and short-range signals from escort cells. Proteins modulating chromatin structure also appear to affect FSC self-renewal. To date, however, Dicer-1 is the only translational regulator identified as necessary for FSC maintenance or function. This study reports that the translational regulators Caprin (Capr) and the Drosophila ortholog of Fragile X Mental Retardation Protein (FMRP) function together in regulating the FSC lineage. In addition, it was found that FSC-lineage cells have an altered cell cycle in Capr mutants, further implicating Capr in developmental regulation of the cell cycle (Reich, 2012).

Translational regulation has proven to be of fundamental importance in control of GSC identity and behavior, but surprisingly little is known about the relative importance of this mode of regulation in controlling the fate of FSCs. The translational regulator Capr functions as an intrinsic factor required for the proper maintenance of FSCs, and loss of Capr disrupts cell cycle dynamics within the FSC lineage. It is proposed that Capr is required for proper execution of the cell cycle in the FSC-lineage, in part through modulation of CycB protein levels. In this model misregulation of the Capr-dependent cell cycle leads to defects in somatic cell differentiation, with a concomitant disruption of the ability to correctly package developing cysts into egg chambers. The ability of fmr1 mutation to enhance the encapsulation defects implicates these two translational regulatory factors in coordinate control of this aspect of ovary function (Reich, 2012).

Given the similarities between the Capr mutant phenotype and the mutant phenotype of genes involved in FSC proliferation, maintenance, and differentiation, it is possible that Capr is only required in the FSC lineage. The data, however, cannot rule out the possibility that Capr is additionally required in non-FSC lineage cells to send extrinsic signals that impact the encapsulation process. For example, clonal analysis demonstrated that Capr is not required for GSC maintenance. However this technique cannot rule out a requirement for Capr in the GSC lineage for other functions such as cell-cell communication. Similarly, because Capr protein was barely detectable in the cap cells or the escort cells it seems less likely that Capr has a critical function in these populations, but not impossible. A more appealing candidate population might be the terminal filament cells based on their prominent Capr-containing puncta. Terminal filament cells are known to function along with the cap cells as niche cells for both the GSCs and FSCs. It will be interesting to determine whether the bright puncta of Capr observed in the terminal filament cells represent ribonucleoprotein structures involved in signal-responsive translational regulation similar to the Capr-containing neuronal and stress granules of vertebrates {Solomon, 2007; Shiina, 2010a; Shiina, 2010b). Ultimately, because the clonal removal of Capr specifically from the FSC’s alone disrupted stem cell maintenance, the simplest interpretation of the current data is that an intrinsic role for Capr in the FSC’s can account for all the phenotypes observed. Further study will be required to determine whether Capr has additional roles in other ovarian cells (Reich, 2012).

During Drosophila embryogenesis, Capr is known to functionally collaborate with fmr1 to regulate the timing of the mid-blastula transition (Papoulas, 2010). The functional interaction of these two translational regulators is further supported by evidence that Capr and dFMRP coimmunoprecipitate from Drosophila embryos (Papoulas, 2010) and associate with common ribonucleoprotein structures such as neuronal granules, stress granules, and the 5' cap structure of mRNAs in the ovary. In the ovary Capr and fmr1 are expressed in both the germline and somatic cells. A role for fmr1 in somatic cells and encapsulation was initially considered unlikely because fmr1 mutant egg chambers displaying germ cell proliferation defects are surrounded by apparently normal follicle cells, and are typically flanked by appropriately packaged egg chambers. The maintenance of GSCs, however, relies on fmr1 function outside the GSCs leaving open the possibility that fmr1 functions in the germline cysts and somatic cells of the ovary. The data indicate that fmr1 and Capr genetically interact to regulate cyst encapsulation and female fecundity. One possible interpretation of these data is that Capr and dFMRP co-regulate translation of a set of transcripts in FSCs or their progeny important for cyst encapsulation. Alternatively, Capr and dFMRP could individually regulate distinct transcripts required for proper FSC function. In either case both translational regulators are necessary for proper encapsulation of developing cysts and generation of a functional egg chamber (Reich, 2012).

Intriguingly recent studies have indicated that FMRP is required for normal functioning of the human ovary as well. Although the mechanism has yet to be determined, FMR1 premutation carriers with no neuro/psychiatric symptoms nevertheless show reduced fecundity due to aberrant control of follicular recruitment and ovarian reserves. In addition to its role in the ovary, dFMRP is reported to affect proliferation of Sertoli cells, the niche cells of the male gonad, and to regulate stem cell behavior in the nervous system and it will be interesting to determine whether Capr also participates in these processes (Reich, 2012).

In stem cells control of the cell cycle may be uniquely linked to cell fate. For example, in mouse neuroepithelial cells, simply altering the length of G1 using cyclin-dependent kinase inhibitors induces differentiation. Likewise in the ovary, as cells produced by FSCs proceed through successive divisions they acquire longer S-phases and increased epigenetic stability, conditions which promote the differentiated state. Furthermore, elevated levels of CYCE are required in the FSC’s themselves, to promote the adherence of these stem cells to their niche. It is therefore plausible that even subtle modulation of the cell cycle by Capr could have profound consequences for production of a functional egg chamber (Reich, 2012).

Translational control of cell cycle regulation is a specific mechanism reported to affect behavior of both GSCs and FSCs. Although Capr is reported to be a signal-dependent regulator of translation in the vertebrate nervous system it has been equally implicated in developmental regulation of proliferation: Caprin-1 levels correlate with cell proliferation states in many vertebrate tissues, and caprin-1 deficient cells show a specific delay in G1-S progression. Similarly, FMRP has been predominantly studied because of its role in the nervous system where loss of FMRP causes mental retardation and autism. However, loss of FMRP also generates significant aberrations in proliferation in both the ovary and testis. The encapsulation defects seen in this study, therefore, could be due entirely to a Capr- or Capr and fmr1-dependent alteration of the cell cycle in the FSC lineage (Reich, 2012).

Capr is a sequence-specific RNA-binding protein believed to function by altering translation and/or localization of specific mRNA targets. However, despite genetic evidence that CycB misregulation underlies the defects observed in this study, Capr may regulate other mRNAs, and the phenotype seen could be due to a cumulative misexpression of mRNAs involved in cell cycle control and other processes. In this regard there is still much to learn about how Capr or FMRP achieve temporal and target specificity. For example, both Capr and CycB are expressed in GSCs and numerous other tissues but Capr does not appear to regulate CycB in all of these. Future determination of all relevant mRNA targets in the ovary, and the mechanism for regulating Capr function and specificity would be constructive steps towards understanding the role of translational regulation in the control of stem cell behavior (Reich, 2012).

Learning and memory deficits consequent to reduction of the fragile X mental retardation protein result from metabotropic glutamate receptor-mediated inhibition of cAMP signaling in Drosophila

Loss of the RNA-binding fragile X protein [fragile X mental retardation protein (FMRP)] results in a spectrum of cognitive deficits, the fragile X syndrome (FXS), while aging individuals with decreased protein levels present with a subset of these symptoms and tremor. The broad range of behavioral deficits likely reflects the ubiquitous distribution and multiple functions of the protein. FMRP loss is expected to affect multiple neuronal proteins and intracellular signaling pathways, whose identity and interactions are essential in understanding and ameliorating FXS symptoms. Heterozygous mutants and targeted RNA interference-mediated abrogation were used in Drosophila to uncover molecular pathways affected by FMRP reduction. Evidence that FMRP loss results in excess metabotropic glutamate receptor (mGluR) activity, attributable at least in part to elevation of the protein in affected neurons. Using high-resolution behavioral, genetic, and biochemical analyses, evidence is presented that excess mGluR upon FMRP attenuation is linked to the cAMP decrement reported in patients and models, and underlies olfactory associative learning and memory deficits. Furthermore, the data indicate positive transcriptional regulation of the fly fmr1 gene by cAMP, via protein kinase A, likely through the transcription factor CREB. Because the human Fmr1 gene also contains CREB binding sites, the interaction of mGluR excess and cAMP signaling defects presented in this study suggests novel combinatorial pharmaceutical approaches to symptom amelioration upon FMRP attenuation (Kanellopoulos, 2012).

This study has demonstrated robust learning and LTM deficits associated with 50% reduction in dFMRP and mapped the phenotype to adult MB α/β lobes with spatiotemporally controlled RNAi-mediated abrogation. The learning deficit of dfmr13-null allele heterozygotes was quantitatively similar to that of animals lacking significantly more of the protein due to RNAi-mediated dFMRP abrogation either pan-neuronally or specifically in the MBs. This is surprising given the efficient attenuation of dFMRP with UAS-dfmr-R. Interestingly, a similar learning deficit was independently reported for null homozygotes as well (Kanellopoulos, 2012).

The lack of enhanced learning and memory deficits upon further reduction than 50% in the heterozygotes indicates that the activity of the remaining dFMRP in these animals may be reduced to levels functionally approaching those in null homozygotes. This suggests that an essential posttranslational modification may be suppressed in the heterozygotes, effectively further reducing functional dFMRP. Ser499 phosphorylation by S6K1 is essential for the translational repressor function of vertebrate FMRP and phosphate removal or suppression inactivates it. Moreover, phosphorylation by casein kinase II at dFMRP Ser406 has been reported functionally important. Hence, altered dFMRP phosphorylation in dfmr13/+ is a plausible explanation for the phenotypes in the heterozygotes, and this hypothesis is currently under investigation. Because near-complete abrogation of the protein does not completely eliminate olfactory learning, dFMRP does not appear to play an essential role in all molecular processes engaged within the MBs for associative olfactory learning (Kanellopoulos, 2012).

Processes requiring normal dFMRP function appear to be involved in learning rate, because dfmr13 heterozygotes and animals with attenuated protein in the MBs reach asymptotic performance levels like controls, but require additional US/CS pairings. The aberrantly elevated responses after training with two and four pairings may be akin to the heightened arousal reported to result in increased activity and exaggerated responses during the initial sessions of a learning task in mice lacking the Fmr1 gene. In addition, synaptic hyperexcitability, especially upon high-frequency stimulation, at the neuromuscular junction was reported for dfmr1-null larvae. These phenomena may be related to the elevated emotional reactivity and anxiety often associated with FXS patients. Excess DmGluRA activity appears to be involved in this exaggerated response, because it was eliminated after feeding mutant flies with MPEP. The known anxiolytic and antidepressant properties of Rolipram and other PDE inhibitors support the interpretation that the aberrantly elevated responses after few US/CS pairings may reflect anxiety in flies as in mice. The mechanism of this performance enhancement is unclear at the moment but will be investigated in detail in the future (Kanellopoulos, 2012).

Initially, tests were performed to see whether the mGluR elevation proposed to underlie many FXS behavioral deficits and validated in Drosophila with mutant homozygotes was also applicable when dFMRP was reduced, but not eliminated. The results indicate that, in mutant heterozygotes, it is not solely the activity of the mGluRA receptor that is increased but also the levels of the protein itself. Because no evidence was uncover suggesting increased DmGluRA transcripts, this evidence suggests that it is translation of the receptor likely regulated by dFMRP. In the Drosophila larval NMJ, dFMRP has been reported to regulate the abundance of ionotropic glutamate receptor subclasses, and similarly it may regulate the levels of DmGluRA in the adult CNS. Moreover, a recent report indicated that several mGluRs are targets of FMRP-dependent translational regulation. These results are consistent with the RNAi-mediated attenuation of the receptor, which reversed both learning and memory deficits in animals with abrogated dFMRP. Therefore, dFMRP function appears dosage sensitive since 50% reduction suffices to elevate DmGluRA (Kanellopoulos, 2012).

DmGluRA is present in the MB dendrites. Therefore, dFMRP abrogation in these neurons is expected to result in elevation of the receptor within them. The fact that feeding MPEP reverses the learning deficits of animals with abrogated dFMRP specifically in the MBs strongly suggests that the pharmaceutical reaches these neurons and acts on the locally elevated DmGluRA (Kanellopoulos, 2012).

Interestingly, inhibiting DmGluRA with MPEP or abrogating the receptor in the adult fly CNS rescued the low cAMP levels in dfmr13/+. This indicates that cAMP levels are directly influenced by the level of DmGluRA. Furthermore, cAMP levels appear to lie downstream of the receptor, because elevation of the nucleotide by reducing the dosage of the PDE Dnc, or administration of Rolipram resulted in complete reversal of the learning and LTM phenotypes of dfmr13/+. Consistent with this observation, the mGluR antagonists LY341495, MPPG, and MTPG, previously used to rescue the courtship learning defect of dfmr13 homozygotes, are known to also increase cAMP signaling in Drosophila. Hence, the low cAMP levels reported for fly, mouse, and humans with compromised FMRP function are likely a consequence of enhanced levels of a Gi/o-linked glutamate receptor, which is thought to be the DmGluRA in Drosophila. These data strongly suggest this as the mechanism linking mGluR overactivity and/or levels and the proposed FMRP regulation of cAMP levels. Consistently, use of group II mGluR antagonists rescued the LTD phenotype in a mouse FXS model. Therefore, at least in the Drosophila model and with respect to associative learning and memory, the mGluR theories of accounting for the behavioral deficits of FXS seem to converge and describe different points of the same molecular interaction network (Kanellopoulos, 2012).

The behavioral effects of reducing the dosage of Dnc by 50% are also noteworthy. First, dnc1 heterozygosity precipitates small but significant effects on learning, and much larger effects on LTM, suggesting exaggerated effects of elevated cAMP on consolidated memory. The significance of reestablishing cAMP balance within the MBs is likely reflected in the surprising complete rescue of the dfmr13/+ LTM defect by reducing the dosage of the PDE, thus elevating cAMP and dFMRP levels (Kanellopoulos, 2012).

Interestingly, pharmacological or genetic manipulations that raised cAMP levels in dfmr13/+ animals also resulted in increased levels of dFMRP. This response appears to be mediated entirely by a cAMP-dependent increase in dfmr1 transcription, is apparently transduced via PKA signalin, and likely engages the transcription factor CREB. A correlation between cAMP and FMRP levels had been noted previously with respect to regional variation in brain areas suggestive of interactions in a developmental context. In contrast, the current results demonstrate an acute transcriptional response, as elevated cAMP and dfmr1 transcripts are apparent within a few hours of pharmaceutical administration. Therefore, dFMRP levels respond acutely to cAMP in the fly CNS and appear to reflect the abundance or activity of DmGluRA as a negative-feedback loop. In support of this notion, dFMRP levels were reduced in homozygous mutants for the Rut adenylyl cyclase, a situation where DmGluRA levels are presumed normal. Furthermore, given the function of dFMRP as a translational repressor, it is of interest to consider its elevation in dnc heterozygous and further increase in homozygous mutants. Is then the etiology of the learning and memory deficits in dnc mutants cAMP elevation, or exaggerated translational repression within their MBs because of enhanced dFMRP levels therein (Kanellopoulos, 2012)?

Collectively, these results suggest that pharmaceuticals that modulate cAMP signaling are promising routes to effectively ameliorate behavioral symptoms of 'premutation' carriers. Amelioration due to transcriptional upregulation of FMRP in response to cAMP elevation is not possible in patients and models harboring deletions or functional silencing of the gene. However, point mutations that do not affect transcription of the gene, but rather particular functional domains were identified recently. Because these appear associated with some but not all behavioral deficits, they likely do not represent null but rather hypomorphic alleles. Perhaps Rolipram and MPEP-mediated increases in cAMP levels and FMRP transcription may be beneficial to such patients as well as those presenting FXTAS symptoms. Like in Drosophila, the human gene also appears to contain CREB sites in its putative promoter area, indicating that this approach is at least feasible (Kanellopoulos, 2012).

MicroRNA-277 modulates the neurodegeneration caused by Fragile X premutation rCGG repeats

Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder, has been recognized in older male fragile X premutation carriers and is uncoupled from fragile X syndrome. Using a Drosophila model of FXTAS, it has been shown that transcribed premutation repeats alone are sufficient to cause neurodegeneration. miRNAs are sequence-specific regulators of post-transcriptional gene expression. To determine the role of miRNAs in rCGG repeat-mediated neurodegeneration, miRNA expression was profiled, and selective miRNAs were identified, including mir-277 stem loop, that are altered specifically in Drosophila brains expressing rCGG repeats. Their genetic interactions with rCGG repeats were tested, and it was found that miR-277 can modulate rCGG repeat-mediated neurodegeneration. Furthermore, Drep-2 and Vimar were identified as functional targets of miR-277 that could modulate rCGG repeat-mediated neurodegeneration. Finally, it was found that hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These results suggest that sequestration of specific rCGG repeat-binding proteins could lead to aberrant expression of selective miRNAs, which may modulate the pathogenesis of FXTAS by post-transcriptionally regulating the expression of specific mRNAs involved in FXTAS (Tan, 2012).

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by expansion of the rCGG trinucleotide repeat in the 5' untranslated region (5' UTR) of the fragile X mental retardation 1 (FMR1) gene, which leads to silencing of its transcript and the loss of the encoded fragile X mental retardation protein (FMRP). Most affected individuals have more than 200 rCGG repeats, referred to as full mutation alleles. Fragile X syndrome carriers have FMR1 alleles, called premutations, with an intermediate number of rCGG repeats between patients (>200 repeats) and normal individuals (<60 repeats). Recently, the discovery was made that male and, to a lesser degree, female premutation carriers are at greater risk of developing an age-dependent progressive intention tremor and ataxia syndrome, which is uncoupled from fragile X syndrome and known as fragile X-associated tremor/ataxia syndrome (FXTAS). This is combined with cognitive decline associated with the accumulation of ubiquitin-positive intranuclear inclusions broadly distributed throughout the brain in neurons, astrocytes, and in the spinal column (Tan, 2012).

At the molecular level, the premutation is different from either the normal or full mutation alleles. Based on the observation of significantly elevated levels of rCGG-containing FMR1 mRNA, along with either no detectable change in FMRP or slightly reduced FMRP levels in premutation carriers, an RNA-mediated gain-of-function toxicity model has been proposed for FXTAS. Several lines of evidence in mouse and Drosophila models further support the notion that transcription of the CGG repeats leads to this RNA-mediated neurodegenerative disease. The hypothesis is that specific RNA-binding proteins may be sequestered by overproduced rCGG repeats in FXTAS and become functionally limited, thereby contributing to the pathogenesis of this disorder. There are three RNA-binding proteins found to modulate rCGG-mediated neuronal toxicity: Pur α, hnRNP A2/B1, and CUGBP1, which bind rCGG repeats either directly (Pur α and hnRNP A2/B1) or indirectly (CUGBP1, through the interaction with hnRNP A2/B1) (Tan, 2012).

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression at the post-transcriptional level by targeting mRNAs, leading to translational inhibition, cleavage of the target mRNAs or mRNA decapping/deadenylation. Mounting evidence suggests that miRNAs play essential functions in multiple biological pathways and diseases, from developmental timing, fate determination, apoptosis, and metabolism to immune response and tumorigenesis. Recent studies have shown that miRNAs are highly expressed in the central nervous system (CNS), and some miRNAs have been implicated in neurogenesis and brain development (Tan, 2012).

Interest in the functions of miRNAs in the CNS has recently expanded to encompass their roles in neurodegeneration. Investigators have begun to reveal the influence of miRNAs on both neuronal survival and the accumulation of toxic proteins that are associated with neurodegeneration, and are uncovering clues as to how these toxic proteins can influence miRNA expression. For example, miR-133b is found to regulate the maturation and function of midbrain dopaminergic neurons (DNs) within a negative feedback circuit that includes the homeodomain transcription factor Pitx3 in Parkinson's disease. In addition, reduced miR-29a/b-1-mediated suppression of BACE1 protein expression contributes to Aβ accumulation and Alzheimer's disease pathology. Moreover, the miRNA bantam is found to be a potent modulator of poly-Q- and tau-associated degeneration in Drosophila. Other specific miRNAs have also been linked to other neurodegenerative disorders, such as spinocerebellar ataxia type 1 (SCA1) and Huntington's disease (HD). Therefore, miRNA-mediated gene regulation could be a novel mechanism, adding a new dimension to the pathogenesis of neurodegenerative disorders (Tan, 2012).

This study shows that fragile X premutation rCGG repeats can alter the expression of specific miRNAs, including miR-277, in a FXTAS Drosophila model. miR-277 modulates rCGG-mediated neurodegeneration. Furthermore, Drep-2, which is associated with the chromatin condensation and DNA fragmentation events of apoptosis, and Vimar, a modulator of mitochondrial function, were identified two of the mRNA targets regulated by miR-277. Functionally, Drep-2 and Vimar could modulate the rCGG-mediated neurodegeneration, as well. Finally, hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These data suggest that hnRNP A2/B1 could be involved in the transcriptional regulation of selective miRNAs, and fragile X premutation rCGG repeats could alter the expression of specific miRNAs, potentially contributing to the molecular pathogenesis of FXTAS (Tan, 2012).

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that afflicts fragile X syndrome premutation carriers, with earlier studies pointing to FXTAS as an RNA-mediated neurodegenerative disease. Several lines of evidence suggest that rCGG premutation repeats may sequester specific RNA-binding proteins, namely Pur α, hnRNP A2/B1, and CUGBP1, and reduce their ability to perform their normal cellular functions, thereby contributing significantly to the pathology of this disorder. The miRNA pathway has been implicated in the regulation of neuronal development and neurogenesis. A growing body of evidence has now revealed the role of the miRNA pathway in the molecular pathogenesis of neurodegenerative disorders. This study demonstrates that specific miRNAs can contribute to fragile X rCGG repeat-mediated neurodegeneration by post-transcriptionally regulating target mRNAs that are involved in FXTAS. miR-277 plays a significant role in modulating rCGG repeat-mediated neurodegeneration. Overexpression of miR-277 enhances rCGG repeat-induced neuronal toxicity, whereas blocking miR-277 activity could suppress rCGG repeat-mediated neurodegeneration. Furthermore, Drep-2 and Vimar were identified as the functional miR-277 targets that could modulate rCGG repeat-induced neurodegeneration. Finally, hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These biochemical and genetic studies demonstrate a novel miRNA-mediated mechanism involving miR-277, Drep-2, and Vimar in the regulation of neuronal survival in FXTAS (Tan, 2012).

Several lines of evidence from studies in mouse and Drosophila models strongly support FXTAS as an RNA-mediated neurodegenerative disorder caused by excessive rCGG repeats. The current working model is that specific RNA-binding proteins could be sequestered by overproduced rCGG repeats in FXTAS and become functionally limited, thereby contributing to the pathogenesis of this disorder. Three RNA-binding proteins are known to modulate rCGG-mediated neuronal toxicity: Pur α, hnRNP A2/B1, and CUGBP1, which bind rCGG repeats either directly (Pur α and hnRNP A2/B1) or indirectly (CUGBP1, through the interaction with hnRNP A2/B1); how the depletion of these RNA-binding proteins could alter RNA metabolism and contribute to FXTAS pathogenesis has thus become the focus in the quest to understand the molecular pathogenesis of this disorder. Nevertheless, the data presented in this study suggest that the depletion of hnRNP A2/B1 could also directly impact the transcriptional regulation of specific loci, such as miR-277. It is known that hnRNPs can interact with HP1 to bind to genomic DNA and modulate heterochromatin formation. The results indicate that hnRNP A2/B1 could participate in the transcriptional regulation of miR-277; however, it remains to be determined whether other loci could be directly regulated by hnRNP A2/B1, as well. Identifying those loci will be important to better understand how the depletion of rCGG repeat-binding proteins could lead to neuronal apoptosis (Tan, 2012).

In recent years, several classes of small regulatory RNAs have been identified in a range of tissues and in many species. In particular, miRNAs have been linked to a host of human diseases. Some evidence suggests the involvement of miRNAs in the emergence or progression of neurodegenerative diseases. For example, accumulation of nuclear aggregates that are toxic to neurons have been linked to many neurodegenerative diseases, and miRNAs are known to modulate the accumulation of the toxic proteins by regulating either their mRNAs or the mRNAs of proteins that affect their expression. Moreover, miRNAs might contribute to the pathogenesis of neurodegenerative disease downstream of the accumulation of toxic proteins by altering the expression of other proteins that promote or inhibit cell survival. The current genetic modifier screen revealed that miR-277 could modulate rCGG repeat-mediated neurodegeneration. By combining genetic screen and reporter assays, Drep-2 and Vimar were identified as the functional targets of miR-277 that could modulate rCGG-mediated neurodegeneration. The closest ortholog of miR-277 in human is miR-597 based on the seed sequence. It would be interesting to further examine the role of miR-597 in FXTAS using mammalian model systems (Tan, 2012).

Drep-2 is associated with the chromatin condensation and DNA fragmentation events of apoptosis. Drep-2 is one of four Drosophila DFF (DNA fragmentation factor)-related proteins. While Drep-1 is a Drosophila homolog of DFF45 that can inhibit CIDE-A mediated apoptosis. Drep-2 has been shown to interact with Drep-1 and to regulate its anti-apoptotic activity. Vimar is a Ral GTPase-binding protein that has been shown to regulate mitochondrial function via an increase in citrate synthase activity . In the presence of fragile X premutation rCGG repeats, overexpression of miR-277 will suppress the expression of both Drep-2 and Vimar, thereby altering anti-apoptotic activity as well as mitochondrial functions, which have been linked to neuronal cell death associated with neurodegenerative disorders in general. Interestingly, a significant reduction of Drep-2 mRNA was seen in the flies expressing rCGG repeats, while Vimar mRNA levels remained similar to control flies. This observed difference may be due to the fact that miRNA could be involved in different modes of action, including mRNA cleavage, translational inhibition and mRNA decapping/deadenylation its target mRNAs (Tan, 2012).

In summary, this study provides both biochemical and genetic evidence to support a role for miRNA and its selective mRNA targets in rCGG-mediated neurodegeneration. The results suggest that sequestration of specific rCGG repeat-binding proteins can lead to aberrant expression of selective miRNAs that could modulate the pathogenesis of FXTAS by post-transcriptionally regulating the expression of specific mRNAs involved in this disorder. Identification of these miRNAs and their targets could reveal potential new targets for therapeutic interventions to treat FXTAS, as well as other neurodegenerative disorders (Tan, 2012).

CGG repeat-associated translation mediates neurodegeneration in fragile X tremor ataxia syndrome

Fragile X-associated tremor ataxia syndrome (FXTAS) results from a CGG repeat expansion in the 5′ UTR of FMR1. This repeat is thought to elicit toxicity as RNA, yet disease brains contain ubiquitin-positive neuronal inclusions, a pathologic hallmark of protein-mediated neurodegeneration. This study explains this paradox by demonstrating that CGG repeats trigger repeat-associated non-AUG-initiated (RAN) translation of a cryptic polyglycine-containing protein, FMRpolyG. FMRpolyG accumulates in ubiquitin-positive inclusions in Drosophila, cell culture, mouse disease models, and FXTAS patient brains. CGG RAN translation occurs in at least two of three possible reading frames at repeat sizes ranging from normal (25) to pathogenic (90), but inclusion formation only occurs with expanded repeats. In Drosophila, CGG repeat toxicity is suppressed by eliminating RAN translation and enhanced by increased polyglycine protein production. These studies expand the growing list of nucleotide repeat disorders in which RAN translation occurs and provide evidence that RAN translation contributes to neurodegeneration (Todd, 2013).

This study demonstrates that RAN translation occurs in association with CGG repeats in the neurodegenerative disorder FXTAS, a disease previously thought to result primarily from RNA-mediated toxicity. These findings, along with other reports detailing unconventional translation through CAG repeats in spinocerebellar ataxia type 8 and myotonic dystrophy type I and GGGGCC repeats in C9orf72-associated ALS/FTLD, suggest that RAN translation is a shared pathogenic mechanism in many repeat expansion disorders. It was further demonstrated that production of one particular CGG RAN translation product in FXTAS, FMRpolyG, directly modulates CGG-associated pathology in two distinct model systems. First, the ability to generate the FMRpolyG protein explains a key pathologic discrepancy between two established knockin mouse models. Second, in Drosophila it was shown that CGG repeat-associated neurodegeneration is largely dependent on FMRpolyG production. These results suggest that RAN translation contributes to FXTAS pathogenesis and support an emerging view that nonexonic repetitive elements can trigger toxicity simultaneously as both RNA and protein (Todd, 2013).

The mechanisms underlying RAN translation remain unclear. The unconventional translation described in this study appears to initiate predominantly at a near-AUG codon just 5′ proximal of the repeat. This finding suggests a model wherein a scanning 43S ribosomal preinitiation complex stalls at the CGG repeat, allowing for alternate usage of a near match at the initiation codon. This model is based on the observation that placing a stop codon just proximal to the repeat or shortening the 5′ leader before the repeat impairs RAN translation in this reading frame. In contrast, CGG RAN translation in the other two possible reading frames behaves differently. RAN translation product is not detected from the +0 (CGG, polyArginine) reading frame, and RAN translation in the +2 (GCG, polyAlanine) reading frame is less efficient, occurs when stop codons are inserted 5′ of the repeat, and demonstrates CGG repeat length dependence. Differences in the propensity for translational initiation in different reading frames have also been reported for RAN translation of expanded CAG repeats in SCA 8, in which the surrounding sequence appeared to be an important modulator. Thus, RAN translation may not result from a single mechanism. Rather, each repeat, and indeed each reading frame within each repeat, may have different contextual requirements. These differences notwithstanding, the fact that atypical translation has now been observed independently with four different nucleotide RNA repeats in cell lines, animal models, and human tissues suggests that it is a more widespread biological event than anticipated (Todd, 2013).

An emerging question now is what roles do these translation initiation events play in normal physiology and in disease? Findings from this study support a significant role for the FMRpolyG protein in disease pathogenesis, given the evidence in Drosophila and mammalian cells of enhanced toxicity with increased polyglycine translation and lessened toxicity when translation is reduced. However, numerous published studies support a primary role for CGG RNA in toxicity, leading to the suggestion that, in FXTAS, additive or synergistic toxicity associated with both toxic RNA and toxic proteins may be critical to disease pathogenesis. Though this study focuses on FMRpolyG production, other CGG RAN translation-associated products such as the polyalanine protein can represent additional toxic species. Moreover, if RAN translation occurs with CCG repeats, then production of other homopolymeric proteins from the antisense transcript through the repeat can also be relevant. For all of these potentially toxic entities, it will be important to determine their relative production in patients and relative degree of toxicity in animal models to ascertain their roles in disease pathogenesis (Todd, 2013).

CGG RAN translation may also play a normal physiological role in translational regulation of FMR1 mRNA. FMRP, the protein product of FMR1, critically regulates synaptic function and its loss leads to fragile X syndrome, a common cause of autism and mental retardation. FMR1 mRNA is rapidly translated at synapses in an activity-dependent manner, where it constrains local synaptic protein translation. This study's observations in transfected cells show that RAN translation can occur at normal repeat sizes, with initiation occurring within a narrow region just 5′ of the repeat. Analysis of ribosomal profiling data sets derived from samples with normal CGG repeat sizes demonstrates the presence of assembled ribosomes over these regions in both human and mouse cell lines. Intriguingly, the Drosophila homolog of FMRP, dfxr, is expressed as two isoforms, with the larger isoform initiating translation at a CUG codon upstream of the canonical TIS, indicating that aspects of this process may be evolutionarily conserved. Upstream ORFs are believed to suppress expression from downstream canonical ORFs. In the case of FMR1 mRNA, translation through the repeat may assist RNA unwinding via helicase recruitment, allowing normal scanning by trailing ribosomes and appropriate initiation at the canonical ORF. Alternatively, ribosomes translating through the repeat could terminate translation and reinitiate at the AUG of FMRP, or ribosomes could initiate downstream of the repeat via an internal ribosomal entry site (Todd, 2013).

Variations on the RAN translation described in this study potentially could expand the percentage of the transcriptome encoding for protein, complicating the classical definitions by which we divide “coding” from “noncoding” RNA. Consistent with this, unbiased methods in yeast and mammalian cells reveal that thousands of transcripts initiate translation at non-AUG start sites, often creating upstream ORFs in sequences previously identified as 5′ UTR. Usage of these atypical upstream ORFs is responsive to changes in cell state and external stimuli. Mechanisms similar to those reported in this study may therefore have broader repercussions for the neuronal proteome and global translational regulation (Todd, 2013).

Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila

Fragile X syndrome (FXS), the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1) gene product (FMRP), an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1) null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs): GPI-anchored glypican Dally-like protein (Dlp) and transmembrane Syndecan (Sdc). Earlier work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg) ligand abundance and downstream Frizzled-2 (Fz2) receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb), and downstream ERK phosphorylation (dpERK) are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb) and downstream signaling via phosphorylation of the transcription factor MAD (pMAD) seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1) Wg and Jeb trans-synaptic signaling, and (2) synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state (Friedman, 2013).

FXS is widely considered a disease state arising from synaptic dysfunction, with pre- and postsynaptic defects well characterized in the Drosophila disease model. There has been much work documenting FXS phenotypes in humans as well as in animal models, but there has been less progress on mechanistic underpinnings. This study focuses on the extracellular synaptomatrix in FXS owing to identification of pharmacological and genetic interactions between FMRP and secreted MMPs, a mechanism that is conserved in mammals. Other studies have also highlighted the importance of the synaptomatrix in synaptogenesis, particularly the roles of membrane-anchored HSPGs as co-receptors regulating trans-synaptic signaling. Importantly, it has been shown that FMRP binds HSPG mRNAs, thereby presumably repressing translation. Based on these multiple lines of evidence, this study hypothesized that the FMRP-MMP-HSPG intersection provides a coordinate mechanism for the pre- and postsynaptic defects characterizing the FXS disease state, with trans-synaptic signaling orchestrating synapse maturation across the synaptic cleft (Friedman, 2013).

In testing this hypothesis, a dramatic upregulation of GPI-anchored glypican Dlp and transmembrane Sdc HSPGs was discovered at dfmr1 null NMJ synapses. Indeed, these are among the largest synaptic molecular changes reported in the Drosophila FXS disease model. Importantly, HSPGs have been shown to play key roles in synaptic development. For example, the mammalian HSPG Agrin has long been known to regulate acetylcholine receptors, interconnected with a glycan network modulating trans-synaptic signaling. In Drosophila, Dlp, Sdc and Perlecan HSPGs mediate axon guidance, synapse formation and trans-synaptic signaling. Previous work on dlp mutants reports elevated neurotransmission, paradoxically similar to the Dlp overexpression phenotype shown in this study. However, the previous study does not show Dlp overexpression electrophysiological data, although it does show increased active zone areas consistent with strengthened neurotransmission. The same study reports that Dlp overexpression decreases bouton number on muscle 6/7, which differs from finding in this study of increased bouton number on muscle 4. Because HSPG co-receptors regulate trans-synaptic signaling, dfmr1 mutants were tested for changes in three established pathways at the Drosophila NMJ. Strong alterations in both Wg and Jeb pathways were found, with anterograde signaling being downregulated in both cases. In contrast, no change was found in the retrograde BMP Gbb pathway, suggesting that FMRP plays specific roles in modulating anterograde trans-synaptic signaling during synaptogenesis (Friedman, 2013).

The defect in Jeb signaling seems to be simple to understand, with decreased synaptomatrix ligand abundance coupled to decreased dpERK nuclear localization. However, there is no known link to HSPG co-receptor regulation. It has been shown earlier that Jeb signaling is regulated by another synaptomatrix glycan mechanism, providing a clear precedent for this level of regulation. In contrast, the Wnt pathway exhibits an inverse relationship between Wg ligand abundance (elevated) and Fz2-C nuclear signaling (reduced). This apparent contradiction is explained by the dual activity of the Dlp co-receptor, which stabilizes extracellular Wg to retain it at the membrane, but also competes with the Fz2 receptor. This ‘exchange-factor mechanism’ is competitively dependent on the ratio of Dlp co-receptor to Fz2 receptor, with a higher ratio causing more Wg to be competed away from Fz2. Indeed, it has been demonstrated that the same elevated Wg surface retention couples to decreased downstream Fz2-C signaling in an independent HSPG regulative mechanism at the Drosophila NMJ. This study suggests that in the dfmr1 null synapse, highly elevated Dlp traps Wg, thereby preventing it from binding Fz2 to initiate signaling (Friedman, 2013).

Dysregulation of the Wg nuclear import pathway (FNI) provides a plausible mechanism to explain synapse development defects underlying the FXS disease state, with established roles in activity-dependent modulation of synaptic morphogenesis and neurotransmission. FXS has long been associated with defects in activity-dependent architectural modulation, including postsynaptic spine formation, synapse pruning and functional plasticity. Although it is surely not the only player, aberrant Wg signaling could play a part in these deficiencies. Importantly, it has been shown that the FNI pathway is involved in shuttling large RNA granules out of the postsynaptic nucleus, providing a potential intersection with the FMRP RNA transport mechanism. However, the Wg FNI pathway is not the only Wnt signaling at the Drosophila NMJ, with other outputs including the canonical, divergent canonical and planar cell polarity pathways, which could be dysregulated in dfmr1 nulls. For example, a divergent canonical retrograde pathway proceeds through GSK3β (Shaggy) to alter microtubule assembly, and the FXS disease state is linked to dysregulated GSK3β and microtubule stability misregulation via Drosophila Futsch/mammalian MAP1B. Moreover, it has been shown that the secreted HSPG Perlecan (Drosophila Trol) regulates bidirectional Wnt signaling to affect Drosophila NMJ structure and/or function, via anterograde FNI and retrograde divergent canonical pathways. It is also important to note that previous studies show that a reduction in the FNI pathway, due to decreased Fz2-C trafficking to the nucleus, leads to decreased NMJ bouton number. Future work is needed to fully understand connections between FMRP, HSPGs, the multiple Wnt signaling pathways and the established defects in the synaptic microtubule cytoskeleton in the FXS disease state (Friedman, 2013).

Adding to the complications of FXS trans-synaptic signaling regulation, it was shown that two trans-synaptic signaling pathways are suppressed in parallel: the Wg and Jeb pathways. Possibly even more promising for clinical relevance, it has been established that the Jeb signaling functions as a repressor of neurotransmission strength at the Drosophila NMJ, with jeb and alk mutants presenting increased evoked synaptic transmission. Consistently, loss of FMRP leads to increased EJC amplitudes, which could be due, at least partially, to misregulated Jeb-Alk signaling. Importantly, it has been shown that dfmr1 null neurotransmission defects are due to a combination of pre- and postsynaptic changes, and that there is a non-cell-autonomous requirement for FMRP in the regulation of functional changes in the synaptic vesicle (SV) cycle underlying neurotransmission strength. Additionally, jeb and alk mutants exhibit synaptic structural changes consistent with this FMRP interaction, including a larger NMJ area and synaptic bouton maturation defects, which are markedly similar to the structural overelaboration phenotypes of the FXS disease state. These data together suggest that altered Jeb-Alk trans-synaptic signaling plays a role in the synaptic dysfunction characterizing the dfmr1 null. The study proposes that Wg and Jeb signaling defects likely interact, in synergistic and/or antagonistic ways, to influence the combined pre- and postsynaptic alterations characterizing the FXS disease state (Friedman, 2013).

Although trans-synaptic signaling pathways, and in particular both Wnt and Jeb-Alk pathways, have been proposed to be involved in the manifestation of a number of neurological disorders, this study provides the first evidence that aberrant trans-synaptic signaling is causally involved in an FXS disease model. The study proposes a mechanism in which FMRP acts to regulate trans-synaptic ligands by depressing expression of membrane-anchored HSPG co-receptors. HSPG overexpression alone is sufficient to cause both synaptic structure and function defects characterizing the FXS disease state. Increasing HSPG abundance in the postsynaptic cell is enough to increase the number of presynaptic branches and synaptic boutons, as well as elevate neurotransmission. Correlation with these well-established dfmr1 null synaptic phenotypes suggests that HSPG elevation could be a causal mechanism. Conclusively, reversing HSPG overexpression in the dfmr1 null is sufficient to correct Wnt and Jeb signaling, and to restore normal synaptic structure and function. Because there is no dosage compensation, HSPG heterozygosity offsets the elevation caused by loss of dfmr1. Correcting both Dlp and Sdc HSPGs in the dfmr1 background restores Wg and Jeb signaling to control levels. Correcting Dlp levels by itself restores synaptic architecture, but both Dlp and Sdc have to be corrected to restore normal neurotransmission in dfmr1 null synapses. Taken together, these results from the Drosophila FXS disease model provide exciting new insights into the mechanisms of synaptic phenotypes caused by the loss of FMRP, and promising avenues for new therapeutic treatment strategies (Friedman, 2013).

Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation

Topoisomerases are crucial to solve DNA topological problems, but they have not been linked to RNA metabolism. This study shows that human topoisomerase 3β (Top3β) is an RNA topoisomerase that biochemically and genetically interacts with FMRP, a protein deficient in Fragile X syndrome and known to regulate translation of mRNAs important for neuronal function and autism. Notably, the FMRP-Top3β interaction is abolished by a disease-associated FMRP mutation, suggesting that Top3β may contribute to pathogenesis of mental disorders. Top3β binds multiple mRNAs encoded by genes with neuronal functions related to schizophrenia and autism. Expression of one such gene, ptk2/FAK, is reduced in neuromuscular junctions of Top3β mutant flies. Synapse formation is defective in Top3β mutant flies and mice, as observed in FMRP mutant animals. These findings suggest that Top3β acts as an RNA topoisomerase and works with FMRP to promote expression of mRNAs critical for neurodevelopment and mental health (Xu, 2013).

This study utilized Drosophila to examine functions of the Top3β and its genetic interactions with FMRP. CG13472 was identified as the single Drosophila homologue of TDRD3 (dTDRD3) by homology searches. It was conformed that the endogenous dTop3β biochemically interacts with dFMR1 by IP-Western using Drosophila S2 cells. In S2 cells stably expressing Flag-tagged dTDRD3, all three proteins associate with each other, and the amount of dTop3β associating with dFMR1 is increased. These data support the notion that TDRD3 connects the other two proteins (Xu, 2013).

Ectopic expression of dFMR1 in Drosophila eyes causes a rough (disorganized) eye phenotype with increased necrosis of ommatidia. Modifications of this phenotype have been used to identify factors that genetically interact with dFmr1. Interestingly, ectopic expression of dFMR1 in a dTop3β null mutant background causes a 3-fold increase in necrosis and more disorganized photoreceptor cluster in each ommatidium, when compared to ectopic expression of dFMR1 in wildtype background. The dTop3β mutant itself has normal smooth eyes, with no necrotic ommatidia detected. These data indicate that dTop3β mutation enhances the rough eye phenotype caused by dFMR1 overexpression, and suggest that dTop3β genetically interacts with dFMR1 in an antagonistic manner (Xu, 2013).

In a dTDRD3 reduction-of-function mutant background (which has reduced dTDRD3 mRNA level due to a P-element insertion), ectopic expression of dFMR1 fails to induce significant roughness, evidenced by an almost complete absence of necrotic ommatidia and a better organized photoreceptor cluster in each ommatidia, when compared to ectopic expression of dFMR1 in the wildtype background. The dTDRD3 mutant itself has normal eye morphology. Taken together, these findings suggest that mutation of dTDRD3 suppresses rough eye phenotype caused by ectopic expression of dFMR1, and TDRD3 promotes function of FMRP. The fact that Top3β and TDRD3 have opposite effects on FMRP function (one negative and the other positive) implies that the two proteins may antagonize each other in some situations (Xu, 2013).

A null mutation in Drosophila dFmr1 yields neuronal and behavioral defects similar to those in Fragile X patients and Fmr1 mutant mice. One defect is abnormal neuromuscular junctions (NMJs), exemplified by over-elaboration of synaptic branches and an increased number of synaptic boutons. dTop3β null mutant flies show similar NMJ abnormalities, as their branch and bouton numbers are comparable to those of dFmr1 mutants, and significantly higher than those of the wild type flies. Interestingly, the abnormal phenotype is partially suppressed in the dTop3β−/−;dFmr1−/− double mutant, as the numbers of branches and boutons are fewer in the double mutant than the single mutants. These results are reminiscent of genetic interactions between dAdar and dFmr1: whereas either single mutant exhibits abnormal NMJ phenotypes, reduction of dAdar dosage in dFmr1 mutant background corrects the abnormality. These data suggest that dTop3β and dFMR1 functionally antagonize each other in a common pathway to promote formation of normal NMJs (Xu, 2013).

Drep-2 is a novel synaptic protein important for learning and memory>

CIDE-N domains mediate interactions between the DNase Dff40/CAD and its inhibitor Dff45/ICAD. This study reports that the CIDE-N protein DNA fragmentation factor-related protein 2 (Drep-2) is a novel synaptic protein important for learning and behavioral adaptation. Drep-2 was found at synapses throughout the Drosophila brain and was strongly enriched at mushroom body input synapses. It was required within Kenyon cells for normal olfactory short- and intermediate-term memory. Drep-2 colocalized with metabotropic glutamate receptors (mGluRs). Chronic pharmacological stimulation of mGluRs compensated for drep-2 learning deficits, and drep-2 and mGluR learning phenotypes behaved non-additively, suggesting that Drep 2 might be involved in effective mGluR signaling. In fact, Drosophila fragile X protein mutants, shown to benefit from attenuation of mGluR signaling, profited from the elimination of drep-2. Thus, Drep-2 is a novel regulatory synaptic factor, probably intersecting with metabotropic signaling and translational regulation (Andlauer, 2014: PubMed).

Reduced lateral inhibition impairs olfactory computations and behaviors in a Drosophila model of Fragile X syndrome

Fragile X syndrome (FXS) patients present neuronal alterations that lead to severe intellectual disability, but the underlying neuronal circuit mechanisms are poorly understood. An emerging hypothesis postulates that reduced GABAergic inhibition of excitatory neurons is a key component in the pathophysiology of FXS. This idea was directly tested in a FXS Drosophila model. FXS flies were shown to exhibit strongly impaired olfactory behaviors. In line with this, olfactory representations are less odor specific due to broader response tuning of excitatory projection neurons. Impaired inhibitory interactions were found to underlie reduced specificity in olfactory computations. Finally, defective lateral inhibition across projection neurons was shown to be caused by weaker inhibition from GABAergic interneurons. Direct evidence is provided that deficient inhibition impairs sensory computations and behavior in an in vivo model of FXS. Together with evidence of impaired inhibition in autism and Rett syndrome, these findings suggest a potentially general mechanism for intellectual disability (Franco, 2017).

Fragile X syndrome (FXS) is a common inherited intellectual disability disorder. FXS patients exhibit neurological symptoms that include learning disabilities, social anxiety, attention deficits, hyperarousal, hypersensitivity, autism, and epilepsy. Notwithstanding the complexity of neurophysiological and behavioral alterations, FXS is caused by the silencing, deletion, or loss-of-function mutation of a single gene, FMR1. As a result, FMRP (fragile X mental retardation protein), its protein product, is not expressed in the majority of cases or is non-functional in the rare cases with a point mutation. FMRP is an mRNA-binding protein that regulates several aspects of mRNA metabolism such as nuclear export, transport to synaptic terminals, activity-dependent ribosome stalling and gene expression. Although much of FMRP activity is thought to be related to regulation of synaptic function, little is known about the potential defects in neuronal function caused by the absence of FMRP, in particular how these neurophysiological alterations lead to impairment in neuronal computations and behavior in patients with FXS (Franco, 2017).

Initial studies revealed that dendritic spine number is increased in the cortex of FXS patients. In fact, dendritic abnormalities are the most consistent anatomical correlates of intellectual disability. Studies on animal models of FXS showed that FMRP regulates neuronal branching as well as dendritic spine morphology and density. In addition to defects in synaptic structure and axonal branching, impairments in animal behavior have been observed. However, further studies showed that neuroanatomical and behavioral defects can be genetically uncoupled, suggesting that unknown impairments in neuronal circuit function may underlie behavioral deficits (Franco, 2017).

FMRP regulates translation of mRNAs at synapses, some of which encode proteins involved in synaptic plasticity. Importantly, the absence of FMRP leads to abnormally enhanced group 1 mGluR (metabotropic glutamate receptor) signaling, which results in exaggerated long-term depression, with a net loss of AMPA and NMDA receptors. Additionally, enhanced group 1 mGluR signaling contributes to the elongation of dendritic spines in rodent models of FXS and leads to increased intrinsic neuronal excitability through the downregulation of potassium channels controlling resting membrane potential and action potential afterhyperpolarization. Moreover, FMRP directly influences neuronal excitability by regulating expression of potassium channels and by interacting with potassium channels in a translation-independent manner. Nevertheless, the recent failure of FXS clinical trials targeting group 1 mGluR signaling has led the field to re-examine the group 1 mGluR hypothesis (Franco, 2017).

Loss of FMRP was shown to increase network-level hyperexcitability in the rodent cortex, which has been associated with the symptoms observed in FXS patients, such as hypersensitivity, hyperarousal, hyperactivity, anxiety, and epilepsy. Interestingly, absence of FMRP downregulates GABAA receptor subunits in both mice and flies. Furthermore, the enzymes for GABA synthesis and degradation, GABA membrane transporters, a GABA receptor scaffolding protein, and a protein that regulates GABAB receptor signaling are downregulated in the absence of FMRP. These observations suggest a tantalizing, yet poorly understood, link between GABAergic signaling, network hyperexcitability, and behavioral deficits in FXS models and patients (Franco, 2017).

In contrast to the group 1 mGluR component of FXS, the potential effects of altered synaptic inhibition on neuronal circuit excitability and how these changes might impact sensory computations and animal behavior remain unexplored. This study explored the changes in neuronal circuit function and connectivity underlying FXS by using a combination of behavioral assays, functional brain imaging, optogenetics, and electrophysiology in a fly FXS model. Focus was placed on the Drosophila melanogaster olfactory system, which is a well-understood and genetically tractable neuronal circuit. Specifically, olfactory computations were evaluated in the antennal lobe, a circuit constituted by excitatory projection neurons, which receive synaptic input from their cognate olfactory receptor neurons, as well as inhibitory local interneurons involved in mediating lateral inhibition (Franco, 2017).

The absence of dFMRP, the fly homolog of the human FMRP, was found to result in reduced olfactory attraction and aversion. Calcium imaging data show that antennal lobe projection neurons have broader odor tuning in dfmr1- flies, leading to reduced specificity in odor coding and alterations in olfactory representations. Consistent with these results, lateral inhibition was observed across olfactory glomeruli, as well as the inhibitory connections between local interneurons and projection neurons, are impaired in dfmr1- flies. Finally, downregulation of GABA receptors in projection neurons is sufficient to produce olfactory behavioral defects. It is proposed that absence of dFMRP leads to defective lateral inhibition across olfactory glomeruli, which, in turn, results in impaired odor coding and olfactory behaviors (Franco, 2017).

Since the discovery of reduced GABAA receptor subunit expression in the absence of FMRP, accumulated evidence has pointed toward alterations in GABAergic transmission as a key component in the neurophysiology of FXS. In fact, intracellular recordings on acute brain slices suggested that reduced inhibitory input from interneurons onto pyramidal neurons could result in an excitation/inhibition imbalance. Whether this is true in vivo and how it might impact neuronal circuit function and behavior remained unclear (Franco, 2017).

This was tested using the fruit fly antennal lobe circuit; GABAergic connections established by local interneurons, which mediate lateral inhibition, were found to be impaired in a Drosophila melanogaster model of FXS. Moreover, it was shown that deficits in GABAergic lateral inhibition leads to increased circuit excitability, which results in reduced stimulus selectivity in projection neurons. With lower selectivity comes impaired olfactory computations leading to strong odor discrimination deficits. It is postulated that similar deficits in lateral inhibition impair neuronal computations in other sensory modalities. In consonance with this, it has been reported that circuit hyperexcitability leads to behavioral alterations in tactile, auditory, and olfactory tasks in mouse models of FXS (Franco, 2017).

The results indicate that, in the absence of dFMRP, neuronal computations are impaired in the antennal lobe of Drosophila. Consequently, flies exhibit deficits in olfactory behaviors. This is in apparent contradiction with a previous study showing long-term memory defects in dfmr1- flies and no sensory deficits. Responses to many odors are still elicited in projection neurons of dfmr1- flies. They are just less selective due to reduced lateral inhibition. It is suggested that this difference may be due to a more extensive and quantitative analysis of behavior and physiology in this study that revealed defects that may have not been previously detected. Alternatively, the penetrance and severity of phenotypes in FMR1 mutant animals, both mice and flies, can be sensitive to genetic background. It is possible that the previous study did not account for this. At any rate, both null alleles and RNAi flies analyzed using behavioral, imaging, and electrophysiological approaches revealed that dfmr1 mutants exhibit reduced odor specificity and, thus, deficient olfactory processing (Franco, 2017).

Lateral inhibition across Drosophila olfactory glomeruli has been proposed to be important for increasing contrast among odor representations and, therefore, for discriminating odors. Interestingly, such a mechanism has been suggested to be relevant for other sensory modalities. In this winner-take-all model, glomeruli with most prominent odor responses would strongly activate surrounding interneurons, spreading inhibition to nearby weakly activated glomeruli. The spread of lateral inhibition, in turn, would inhibit the odor responses of weakly activated glomeruli, while strongly activated glomeruli remain as the unique encoder of the particular odor. This model also suggests that the lack of many weakly activated glomeruli, in addition to few strongly responding but very odor-specific glomeruli, enhances the separation of odor response patterns from one another. In line with this model, it was observed that lack of lateral inhibition in the antennal lobe of dfmr1- flies, indeed, leads to an increase in the number of weakly activated and less odor-specific glomeruli. By contrast, WT flies present more inhibitory and less weak excitatory responses, sparing strongly responding olfactory glomeruli that are more odor specific. This is probably a consequence of reduced lateral inhibition, which is important for contrast enhancement of odor representations. Additionally, the slight decrease in lateral excitation observed in dfmr1- flies could result in less strongly represented glomeruli. Importantly, defects in olfactory processing have been observed in other animal models of FXS, as well as in human patients, which display hypersensitivity to smells and to other sensory modalities involving lateral inhibitory mechanisms such as tactility and audition (Franco, 2017).

Beyond the olfactory system, several studies have shown that CNS neurons are hyperexcitable in the absence of FMRP. Since activation of the group 1 mGluR signaling pathway results in increased neuronal excitability, circuit hyperexcitability has been attributed to the constitutively enhanced group 1 mGluR signaling observed in mouse FXS models. This study provides the first direct in vivo evidence showing that defects in lateral GABAergic inhibition significantly contribute to circuit hyperexcitability. This is consistent with downregulation of proteins involved in GABAergic transmission both in fruit flies and in rodents. Thus, reduced inhibition could be a consequence of decreased GABA release from local interneurons, reduced expression of postsynaptic GABA receptors, or both. Further studies of protein expression profiles for the specific neuron types are needed to elucidate this. It is possible that this mechanism might explain phenotypes observed in FXS patients such as hypersensitivity, hyperarousal, hyperactivity, and epilepsy, all of which reflect hyperexcitable brain states (Franco, 2017).

In summary, this study demonstrates that lateral inhibition within the antennal lobe is strongly affected in dfmr1- flies due to impaired inhibitory connections from local interneurons onto projection neurons and other local interneurons. The lack of this lateral inhibition on projection neurons is probably the major cause for their increased excitability and reduced odor specificity. It is proposed that this compromised olfactory coding consequently leads to impaired olfactory behaviors in dfmr1- flies. More generally, this study provides the missing in vivo evidence that the lack of dFMRP has a direct impact on sensory processing and animal behavior through a weakening of lateral inhibitory connections, which broadens response tuning of principal neurons. This mechanism might be ubiquitously present in the brain of FXS patients. For instance, reduced GABAergic inhibition could produce hyperexcitable neuronal circuits in FXS patients, which not only explains symptoms such as hypersensitivity, hyperarousal, or epilepsy but also potentially contributes to the misprocessing of information across the brain, which would have severe effects on human behavior. Finally, given the overlap between the phenotypes of FXS and those of other neurological diseases, such as autism, Rett syndrome, or Dravet syndrome, and their corresponding perturbations in GABAergic transmission, it is possible that similar mechanisms involving reduced lateral inhibition are also present in these neurological syndromes, which are yet to be discovered (Franco, 2017).

Hyperactive locomotion in a Drosophila model is a functional readout for the synaptic abnormalities underlying fragile X syndrome

Fragile X syndrome (FXS) is the most common cause of heritable intellectual disability and autism and affects ~1 in 4000 males and 1 in 8000 females. The discovery of effective treatments for FXS has been hampered by the lack of effective animal models and phenotypic readouts for drug screening. FXS ensues from the epigenetic silencing or loss-of-function mutation of the fragile X mental retardation 1 (FMR1) gene, which encodes an RNA binding protein that associates with and represses the translation of target mRNAs. Previous studies found that the activation of LIM kinase 1 (LIMK1) downstream of augmented synthesis of bone morphogenetic protein (BMP) type 2 receptor (BMPR2) promotes aberrant synaptic development in mouse and Drosophila models of FXS and that these molecular and cellular markers were correlated in patients with FXS. This study reports that larval locomotion is augmented in a Drosophila FXS model. Genetic or pharmacological intervention on the BMPR2-LIMK pathway ameliorated the synaptic abnormality and locomotion phenotypes of FXS larvae, as well as hyperactivity in an FXS mouse model. This study demonstrates that (1) the BMPR2-LIMK pathway is a promising therapeutic target for FXS and (2) the locomotion phenotype of FXS larvae is a quantitative functional readout for the neuromorphological phenotype associated with FXS and is amenable to the screening novel FXS therapeutics (Kashima, 2017).

Behavioral manifestations in the Drosophila FXS model have been reported and include abnormal crawling and locomotion of third-instar larvae. This study developed quantitative behavioral assays that showed that reduction of Wit gene dosage in dFMR1 mutant larvae reverts the locomotion phenotype and that oral administration of LIMK antagonists and a protein synthesis inhibitor restores normal crawling velocity and reduces NMJ bouton numbers. It was also confirmed that administration of a LIMK antagonist in the mouse FXS model rescues the rodent behavioral abnormalities. Thus, this study demonstrates that (1) the locomotion phenotype in dFMR1 mutant larvae serves as a readout of NMJ bouton phenotype; (2) the larval crawling assay system that was developed can be used for the genetic or chemical screening of therapeutic molecules for FXS as well as other synapse formation abnormalities; and (3) targeting the LIMK1 pathway, which is conserved from Drosophila to human, is a potential therapeutic strategy for FXS (Kashima, 2017).

In Drosophila, glass bottom boat (Gbb), which is produced by the postsynaptic muscle, binds to the presynaptic receptor Wit and plays a critical role in modulating neuromuscular synaptic growth, stability, and function. Upon Gbb binding, Wit forms a heteromeric receptor complex with Thickveins (Tkv) and Saxophone (Sax), which then phosphorylate Mothers against decapentaplegic (MAD), a Drosophila homolog of Smad1/5/8. It has been reported that loss of Spartin, a Drosophila homolog of SPG20 that promotes endocytotic degradation of Wit and represses the BMP-Wit signaling pathway, results in an increment of neuromuscular synapses (Nahm, 2013). The result with the dFMR1Δ113/+ mutant is consistent with the Spartin study and confirms the effect of increased Gbb-Wit signal on abnormal synapse development. Loss-of-expression mutants of Spartin develop age-dependent and progressive neuronal defects resembling hereditary spastic paraplegia (HSP). Because frameshift mutations in the SPG20 gene cause a form of HSP known as Troyer syndrome (Online Mendelian Inheritance in Man no. 275900), these results underscore the significance of a presynaptic BMP signal finely tuned by multiple regulatory molecules, including SPG20 and FMRP, for proper motor neuron development and function. Beyond the domain of the NMJ, multiple studies reinforce the notion that the correct intensity and spatiotemporal dynamics of the BMP signaling pathway are critical for axon regeneration upon neuronal and glial injury responses after CNS injury. Furthermore, BMP signaling specifies large and fast-transmitting synapses in the auditory system in a process that largely shares homologies with retrograde BMP signaling in Drosophila neuromuscular synapses. In line with these findings, the current results propose an essential role for the FMRP-BMPR2 axis in the development of the neuropathology of patients with FXS (Kashima, 2017).

The major obstacle against the development of drugs for neurodevelopmental and neurodegenerative diseases is the lack of proper animal models that recapitulate the range of intellectual disability and/or cognitive dysfunction found in human patients. The existing, inadequate models also lack quantitative and reproducible assays to examine cognitive phenotypes. The mouse model of FXS exhibits cognitive and behavioral phenotypes that are both consistent and inconsistent with the symptoms of patients with FXS. For example, a frequent FXS trait is a high-anxiety behavior, whereas the FMR1-KO mice exhibit lower anxiety-like behaviors in the 'light-dark compartment' test. Furthermore, the result of the OFT is confusing because the FMR1-KO mice tend to spend a longer period in the center. The number of crossings and their velocity are both augmented compared to control mice because of their hyperactivity, but this behavior is interpreted as lower anxiety-like. The results of the 'elevated plus maze (EPM)' test, which is frequently used to investigate anxiety-like behaviors in FXS mice, exhibit both a decrease and an increase in anxiety. Furthermore, the tests show great variability and, sometimes, opposing outcomes in behavior depending on the genetic background of the mice, for example, FVB versus C57BL/6J. Considering the variability and lack of reproducibility of behavioral test results in FMR1-KO mice, as well as the concerns of animal welfare and the cost of husbandry, there is a strong need for an animal model and phenotypic readout to screen for FXS drugs (Kashima, 2017).

There are multiple advantages of the Drosophila FXS model over the rodent models. Flies are invertebrates, which are inexpensive and easily cared for. They have a shorter life span and produce numerous externally laid embryos than rodent models. Their genome is small, minimally redundant, and easy to genetically manipulate in a tissue-specific manner. It is easy to orally administer drugs to larvae by adding compounds to the Drosophila medium Formula 4-24 (Carolina Biological Supply Company). Previously, small molecules had to be delivered through conventional fly food that requires boiling followed by the addition of propionic acid, which disables the effect of heat- or acid-sensitive molecules. The use of Formula 4-24, which can be dissolved in water at room temperature and does not require exposure to high temperature nor addition of acid, expands the range of molecules that can be delivered to larvae without loss of activity (Kashima, 2017).

Drosophila has contributed extensively to the discovery and validation of drug targets, as well as to the mechanistic understanding of their genetic cause. In the context of FXS studies, it has been reported that dFMR1 adult mutant flies exhibit defects in learning/memory assays, such as Pavlovian olfactory association and courtship conditioning. These behavioral abnormalities can be restored by various compounds known to target different FMRP targets, including protein synthesis inhibitors, such as puromycin and cycloheximide, the metabotropic glutamate receptor 5 antagonist MPEP, γ-aminobutyric acid agonists, phosphodiesterase-4 inhibitor, and glycogen synthase kinase 3 inhibitor. The current study demonstrates that several dFMR1 mutant larvae exhibit an abnormally high number of NMJ synaptic boutons that correlate with their locomotion abnormality. Both are reversed by LIMK-i treatment, similarly to the effect of this drug in the FMR1-KO mouse. Thus, the crawling assay in dFMR1 mutant Drosophila larvae is proposed as a rapid, quantitative, and reproducible preclinical screening strategy for potential FXS therapies that is alternative to behavioral tests using dFMR1 adult mutant flies or vertebrate FXS models. To facilitate the transition to a high-throughput screen of FXS drugs, the current assay will benefit from an improvement in the number of larvae that can be simultaneously assessed and in the robustness of the phenotype of dFMR1Δ113/+ mutants (Kashima, 2017).

Larval locomotion abnormalities are described in Drosophila models of CNS diseases, such as Alzheimer's and Huntington's. It has been reported that a different strain of dFMR1 mutant Drosophila larvae (dFMR14) exhibits frequent turnings compared to wild-type larvae. This study observed that various dFMR1 mutants, including dFMR1Δ113/+, dFMR1Δ50/+, dFMR13/+, and dFMR1Δ113/3, as well as the dFMR1-RNAi line, crawled from the center to the periphery in a linear manner with an enhanced velocity compared to wild-type larvae. It is speculated that this discrepancy might be due to the different nature of the mutations. For example, dFMR1Δ113 harbors a deletion of the first three exons of the dFMR1 gene, including exon 3 that contains the translation initiation methionine. Consequently, the dFMR1Δ113 allele results in a loss of dFMRP. On the contrary, the dFMR14 allele has a replacement of amino acid 289 with a stop codon; hence, it expresses a partial dFMRP missing the C terminus. Furthermore, in the process of creating the dFMR14 mutant, a Gal4-binding site was inserted into the first intron between exons 1 and 2 of the dFMR1 gene to overexpress a truncated dFMRP upon coexpression of Gal4 transcription factor. These differences might explain the distinct larval crawling behavior of the dFMR1Δ113 and dFMR14 mutants. The homozygous dFMR1Δ50 and homozygous dFMR13 mutants are viable and develop into adulthood similarly to the homozygous dFMR14 mutant. The homozygous dFMR1Δ50 and homozygous dFMR13 mutants exhibited frequent turns in the locomotion assay similar to a previous study of the homozygous dFMR14 mutant. The velocity of dFMR13 and dFMR1Δ50 homozygous mutants was slower than wild-type, presumably because of the frequent changes of direction, unlike the transheterozygous dFMR1Δ113/3 mutant, which crawled in a linear fashion with an augmented velocity. It is speculated that the turning phenotype observed in the homozygous mutants is due to complete loss of dFMRP, affecting the CNS neurons. FMRP activity has already been shown to be important for CNS neuron development and function in Drosophila (Kashima, 2017).

Performing the locomotion assay with larvae instead of adults is beneficial as they present an accessible, anatomically simple, and well-described peripheral nervous system (for example, NMJ boutons), which allows the molecular and biochemical assessment of the mechanism underlying the locomotion dysfunction and the therapeutic effects of drugs. For chemical screens of known pathways or targets, the NMJ synapses of larvae that exhibit an altered crawling phenotype should be subjected to synaptic bouton phenotype analysis as well as biochemical investigation to rapidly validate the 'on-target' and eliminate the 'off-target' effects of the candidate molecules. A Drosophila larvae locomotion assay has been proposed as a way to screen drugs for neurodegenerative diseases, such as Alzheimer's disease. They subjected Drosophila larvae expressing the human three-repeat tau gene in motor neurons to crawling assays, such as a five-lane assay and a four-plate open-field assay, video-recorded the locomotion with an Ikegami digital video camera and a 5-mm digital video camera lens, and analyzed locomotion using EthoVision 3.0 software. In comparison, the advantage of the current strategy is that the assay does not require specialized equipment, but a common video recording device, such as an iPhone camera, and an algorithm that is accessible and free to the scientific community. Furthermore, the system can simultaneously track and assess the crawling activities of multiple larvae through the open-access algorithm LarvaTrack, which was developed to trace and measure larval crawling activity. Up to 15 larvae have been simultaneously assessed using a 15-cm agarose plate. The method can be easily adapted to a larger number of larvae by using a larger agarose plate to avoid larvae to cross paths during crawling. Thus, the semiautomated assay of locomotion behavior described in this study can allow the higher-throughput assay that is essential for the screen of candidate molecules. In conclusion, activation of the FMRP-BMPR2 axis plays a role in synaptic abnormalities in both mouse and Drosophila models of FXS. The larval crawling assay is an easy, fast, and well-suited medium-throughput screen for genetic or chemical modulators of locomotion dysfunction in the Drosophila FXS model, which can be further evaluated in cognitive and behavioral tests using mammalian FXS models (Kashima, 2017).

Fragile X mental retardation protein requirements in activity-dependent critical period neural circuit refinement

Activity-dependent synaptic remodeling occurs during early-use critical periods, when naive juveniles experience sensory input. Fragile X mental retardation protein (FMRP) sculpts synaptic refinement in an activity sensor mechanism based on sensory cues, with FMRP loss causing the most common heritable autism spectrum disorder (ASD), fragile X syndrome (FXS). In the well-mapped Drosophila olfactory circuitry, projection neurons (PNs) relay peripheral sensory information to the central brain mushroom body (MB) learning/memory center. FMRP-null PNs reduce synaptic branching and enlarge boutons, with ultrastructural and synaptic reconstitution MB connectivity defects. Critical period activity modulation via odorant stimuli, optogenetics, and transgenic tetanus toxin neurotransmission block show that elevated PN activity phenocopies FMRP-null defects, whereas PN silencing causes opposing changes. FMRP-null PNs lose activity-dependent synaptic modulation, with impairments restricted to the critical period. It is concluded that FMRP is absolutely required for experience-dependent changes in synaptic connectivity during the developmental critical period of neural circuit optimization for sensory input (Doll, 2017).

Neural circuit remodeling during developmental critical periods requires reception of sensory experience (activity) and the responsive orchestration of synaptic refinement to optimize behavioral performance. FMRP is hypothesized to mediate these activity-dependent critical period processes in an activity sensor mechanism and as an activity-dependent translational regulator. To test these hypotheses, this study dissected FMRP requirements in the well-mapped Drosophila olfactory learning/memory circuit, focusing on projection neurons linking upstream sensory neurons to the downstream central brain mushroom body mediating learning acquisition and memory consolidation. Mushroom body KCs also associate sensory input with a valence signal from dopaminergic neurons, connecting sensory experience to the reward pathway. Null dfmr1 mutants exhibit deficits in olfactory learning and memory, KC architecture, projection neuron dendritic arborization, and activity-dependent calcium signaling. In the FXS condition, transiently altered synaptic connectivity between projection neurons and target KCs profoundly impacts establishment of specific associations between sensory input, learning/memory, and resultant behavioral output. It was predicted that the seemingly ephemeral changes have lasting impacts into maturity, when differences in synaptic architecture are minimal but strong behavior deficits persist. It is hypothesized that subtle differences in circuit connectivity, or consequent functional synaptic deficits arising from transient critical period defects, must be manifest in impairments in emergent circuit properties at maturity that result in persistent behavioral deficits (Doll, 2017).

Synaptic connectivity investigations show two primary defects in FMRP-deficient mPN2 neurons: (1) truncated synaptic branches in the posterior mushroom body calyx and (2) enlarged synaptic boutons on postsynaptic KCs. Importantly, both defects manifest only during the early-use critical period and are not detectably present at maturity, after FMRP expression has precipitously declined. Milder, persistent synaptic architecture defects are detected in some cases, dependent on the genetic background. Null dfmr1 mutant boutons also display a critical-period-restricted reduction in presynaptic active zone scaffold Brp (Drosophila ELKS protein) only during the critical period, showing that FMRP regulates a core organizing component of presynaptic maturation selectively during this transient time window. Using transgenic GFP reconstitution to test synapse connectivity, this study found that FMRP-deficient mPN2 neurons develop impaired synaptic partner interactions with reduced mPN2-KC contacts. GRASP synaptic defects likewise are restricted to the early-use critical period. Electron microscopy during the critical period reveals greatly enlarged synaptic boutons with reduced active zone density in dfmr1-null mutants compared to age-matched controls. These ultrastructural results are consistent with the light microscopy findings, revealing expanded synaptic bouton area coupled with reduced synaptic density during the critical period. Taken together, these combined approaches reveal compromised synaptic connectivity in the Drosophila disease model, consistent with defects in the mouse FXS model, which transiently occur only during the early-use critical period (Doll, 2017).

Next, activity-dependent FMRP roles were explored in the critical period. Critical period exposure to sensory olfactory experience causes dramatic changes in mPN2 mushroom body synaptic connectivity (Figure 5), reminiscent of odorant-induced critical period changes in antennal lobe synaptic glomeruli. Synaptic remodeling is FMRP dependent, and critical period activity phenocopies dfmr1-null defects. Induced changes are specific to the pyrrolidine-sensitive VL1-mPN2 glomerulus, as other odorants (i.e., ethyl acetate) do not alter mPN2 synapses. Importantly, olfactory experience at maturity has no effect on wild-type mPN2s but does cause minor changes in dfmr1-null mPN2s, which supports the 'shifted critical period' Autism spectrum disorder (ASD) hypothesis. FMRP and activity may function in parallel pathways, but the fact that FMRP is activity regulated and mediates activity-dependent processes strongly suggests a direct activity-dependent FMRP mechanism for critical period synaptic refinement. mPN2-targeted optogenetic stimulation during the critical period phenocopies FXS model synaptic defects, with reduced branching and enlarged synaptic boutons, reminiscent of defects in downstream KCs. Similar cell-autonomous optogenetic stimulation causes erroneous axonpathfinding and diminished axon outgrowth. Importantly, both sensory stimulation via peripheral odorant exposure and direct mPN2 stimulation via channelrhodopsin optogenetics phenocopy FXS model defects. All activity-dependent changes require FMRP and are tightly restricted to the early-use critical period. Together, these results support the FXS hyperexcitation theory and highlight a critical period deficit in the suppression of excitatory synapses (Doll, 2017).

In contrast to stimulation paradigms, cell-targeted halorhodopsinsuppression of neuronal activity causes increased mPN2 synaptic branching in the MB calyx. This result demonstrates bidirectional capacity for mPN2 to manifest activity-dependent changes in synaptic connectivity during the early-use critical period. This phenotype is comparable to the overgrown axonal projections that result from developmental application of the GABA antagonist picrotoxin, suggesting that activity normally limits synaptic connectivity. Surprisingly, hyperpolarization of wild-type mPN2 neurons also caused increased synaptic bouton size at maturity, albeit not during the critical period. It is therefore clear that neuronal hyperpolarization impacts synaptic connectivity and architecture in a distinct mechanism compared to excess excitation. However, it is not clear what role the FMRP activity sensor plays when neuronal activity is dampened. Indeed, it was surprising that halorhodopsin hyperpolarization influences dfmr1-null mPN2 synaptic bouton area, suggesting that neurons lacking FMRP retain some capacity to function in activity-dependent synaptic bouton refinement during critical period development. There is evidence that FXS disease model dysfunction can be alleviated through increased activation of the inhibitory neural circuitry: for example, pharmacological enhancement of GABAergic signaling is sufficient to rescue some FXS hyperexcitation and can rescue biochemical, morphological, and behavioral phenotypes in the Drosophila FXS disease model. Thus, excitation/inhibition balance appears important for sculpting synaptic circuit connectivity during the critical period (Doll, 2017).

The blockade of mPN2 neurotransmission by conditional, targeted expression of the tetanus neurotoxin (TNT) leads to striking synaptic overgrowth in wild-type neurons that represents an opposite extreme in comparison to dfmr1-null phenotypes. Suppressed circuit activity (via both halorhodopsin and tetanus toxin manipulations) may spur increased process exploration or connectivity with potential synaptic targets in the mushroom body calyx, further suggesting that reduced branching in FMRP-deficient mPN2 neurons may stem from excess excitation during critical period development. TNT neurotransmission blockade similarly causes aberrant competition for glomerular space during olfactory circuit targeting and enlarged downstream postsynaptic terminals within motor circuits. In dfmr1-null mutants, neurotransmission blockade has little impact on mPN2 presynaptic architecture, demonstrating yet another level of activity-dependent FMRP requirement. As tools are not yet available to assay mPN2 postsynaptic partners, no insight was gained into postsynaptic KC differentiation downstream of the TNT neurotransmission blockade. Planned future work to manipulate neuronal excitability and neurotransmission strength should provide more precise understanding of FMRP function in limiting excitatory synapse connectivity in the developing brain circuitry. The clear requirement for FMRP in activity-dependent synaptic refinement during the early-use critical period, evidence of temporally shifted critical periods in the FXS condition, and the promise of new paradigms to rebalance excitatory/inhibitory synaptic connectivity all hold tremendous future therapeutic potential for combatting the FXS disease state (Doll, 2017).


Identification, cloning and expression of Fmr1 homologs

Human YAC clones have been identified that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal to the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome (Verkerk, 1991).

Fragile X syndrome is associated with massive expansion of a CGG trinucleotide repeat within the FMR-1 gene and transcriptional silencing of the gene due to abnormal methylation. Partial cDNA sequence of the human FMR-1 has been reported. This study describes the isolation and characterization of cDNA clones encoding the murine homolog, fmr-1, which exhibits marked sequence identity with the human gene, including the conservation of the CGG repeat. A conserved ATG downstream of the CGG repeat in human and mouse and an in-frame stop codon in other human 5' cDNA sequences demarcate the FMR-1 coding region and confine the CGG repeat to the 5' untranslated region. Evidence is presented for alternative splicing of the FMR-1 gene in mouse and human brain; one of these splicing events alters the FMR-1 reading frame, predicting isoforms with novel carboxy termini (Ashley, 1993b).

Monoclonal antibodies specific for the FMR-1 protein have been raised. They detect 4-5 protein bands that appear identical in cells of normal males and of males carrying a premutation, but are absent in affected males with a full mutation. Immunohistochemistry shows a cytoplasmic localization of FMR-1. The highest levels are observed in neurons, while glial cells contain very low levels. In epithelial tissues, levels of FMR-1 are higher in dividing layers. In adult testis, FMR-1 is detected only in spermatogonia. FMR-1 is not detected in dermis and cardiac muscle except under pathological conditions (Devys, 1993).

Lack of expression of the fragile X mental retardation protein (FMRP) results in mental retardation and macroorchidism, seen as the major pathological symptoms in fragile X patients. FMRP is a cytoplasmic RNA-binding protein that cosediments with the 60S ribosomal subunit. Recently, two proteins homologous to FMRP were discovered: Fmr11 and Fmr12. These novel proteins interact with FMRP and with each other and they are also associated with the 60S ribosomal subunit. The expression pattern of the three proteins in brain and testis has been studied by immunohistochemistry. In adult brain, FMR1, Fmr11 and Fmr12 proteins are coexpressed in the cytoplasm of specific differentiated neurons only. However, a different expression pattern is observed in fetal brain as well as in adult and fetal testis, suggesting independent functions for the three proteins in those tissues during embryonic development and adult life (Tamanini, 1997).

The structure of the N-terminal domain of the fragile X mental retardation protein: a platform for protein-protein interaction

FMRP, whose lack of expression causes the X-linked fragile X syndrome, is a modular RNA binding protein thought to be involved in posttranslational regulation. The structure in solution of the N-terminal domain of FMRP (NDF), a functionally important region involved in multiple interactions, is now know to consist of a composite fold comprising two repeats of a Tudor motif followed by a short alpha helix. The interactions between the three structural elements are essential for the stability of the NDF fold. Although structurally similar, the two repeats have different dynamic and functional properties. The second, more flexible repeat is responsible for interacting both with methylated lysine and with 82-FIP, one of the FMRP nuclear partners. NDF contains a 3D nucleolar localization signal, since destabilization of its fold leads to altered nucleolar localization of FMRP. It is suggested that the NDF composite fold determines an allosteric mechanism that regulates the FMRP functions (Ramos, 2008).

Fragile X mental retardation protein regulates translation by binding directly to the ribosome

Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused by loss of function of the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is involved in the translational regulation of several neuronal mRNAs. However, the precise mechanism of translational inhibition by FMRP is unknown. This study shows that FMRP inhibits translation by binding directly to the L5 protein on the 80S ribosome. Furthermore, cryoelectron microscopic reconstruction of the 80S ribosomeFMRP complex shows that FMRP binds within the intersubunit space of the ribosome such that it would preclude the binding of tRNA and translation elongation factors on the ribosome. These findings suggest that FMRP inhibits translation by blocking the essential components of the translational machinery from binding to the ribosome (Chen, 2014).

FMR1 encodes an RNA binding protein, fragile X mental retardation protein (FMRP) that is highly expressed in the brain and FMRP appears to regulate the expression of many proteins throughout the brain. FMRP has three RNA-binding domains: one RGG domain that is rich in arginines and glycines and two hnRNP K homology domains (KH domains). Consistent with its proposed role in regulating protein synthesis, the majority of FMRP in the cell is associated with polyribosomes. Interestingly, a missense mutation in the KH2 domain (Ile304Asn of human FMRP) abolishes the binding of FMRP to polyribosomes and causes an aggravated form of FXS in humans. This suggests that RNA binding by FMRP plays a key functional role in the brain. In vitro selection experiments identify a G-quadruplex structure and a pseudoknot structure as the potential RNA ligands for the RGG and KH2 domains, respectively. Based on these results it is proposed that FMRP may bind to mRNAs that possess G-quadruplex- or pseudoknot-forming sequences and repress their translation. Additionally, many proteins, microRNAs and noncoding RNAs have been proposed to be important for translational repression by FMRP (Chen, 2014).

To understand the mechanism of translational inhibition by FMRP, this study used the Drosophila FMRP (dFMRP) homolog, in which the RNA-binding domains are nearly 75% identical to human FMRP. Both the full-length and a N-terminally truncated dFMRP (NT-dFMRP) were purified and an in vitro translation system (IVTS) made from Drosophila embryo extract was used to test the activity of dFMRP. Renilla luciferase mRNA was used as the reporter for protein synthesis because it has three G-rich sequences that potentially form G-quadruplex structures, and additionally has 7 ACUK and 6 WGGA sequences. The time course of protein synthesis was monitored by bioluminescence. The addition of dFMRP or NT-dFMRP to the IVTS inhibits the synthesis of luciferase. NT-dFMRP was used in further studies because it is equally active in inhibiting translation as the full-length protein and easier to purify than the full-length dFMRP (Chen, 2014).

Titration experiments show that the inhibition of translation depends on the concentration of NT-dFMRP added to the IVTS. NT-dFMRP also inhibits the translation of luciferase mRNAs that do not have a N7-methyl guanosine cap at the 5′ end or a 3′ poly(A) tail, indicating that translation inhibition is 5′ cap and poly(A) tail independent. To confirm that the inhibition of translation by NT-dFMRP is 5′ cap-independent, uncapped luciferase mRNA with an internal ribosome entry site (IRES) was synthesized from the Reaper mRNA at the 5′ end. IRES-dependent translation of luciferase mRNA is as efficient as the translation with the 5′ capped mRNA. NT-dFMRP inhibits the translation of luciferase mRNA having the IRES element, confirming that the 5′ cap is not essential for inhibition. These results also suggest that FMRP does not affect the initiation step of protein synthesis (Chen, 2014).

Previous studies suggest that FMRP associates directly with the ribosome. However, other reports show that FMRP binds to the ribosome via the mRNA or as an mRNP complex. It is not clear whether mRNA or other components are required for the association of FMRP with the ribosome. By using gel filtration chromatography and SDS-PAGE, it was shown that NT-dFMRP can indeed bind directly to the ribosome in the absence of mRNA. Furthermore, the binding of NT-dFMRP to the ribosome is stoichiometric even though excess amount of NT-dFMRP is present in the binding reaction. Next, the binding of NT-dFMRP with functionally relevant mutations in the KH1 (I244N) or KH2 (I307N) domains was tested. The KH1 mutant shows a 2-fold reduced binding to the 80S ribosome, while the KH2 mutant binds to a similar extent as NT-dFMRP. The binding results are consistent with functional data, which show that the KH1 domain is important for translational inhibition by NT-dFMRP (Chen, 2014).

A cryo-EM map of the Drosophila 80S ribosome•NT-dFMRP complex was obtained to determine the three-dimensional (3D) binding position of NT-dFMRP on the ribosome. Subtraction of the 3D map of the control Drosophila 80S ribosome from that of the 80S ribosome•NT-dFMRP complex shows an elongated mass of density, within the ribosomal inter-subunit space, that spans from central protuberance (CP) to α-sarcin/ricin stem-loop (SRL) region of the 60S subunit. One end of the elongated difference mass interacts with the CP and A-site finger (ASF) of the 60S subunit, while its other end is situated between the protein S12 region of the small (40S) subunit and SRL region of the 60S subunit. Cross-linking data suggests that the N-terminus of the NT-dFMRP construct interacts with the CP protein L5; consequently, that portion of the difference map is assigned to the N-terminus, and the portion between S12 and SRL is tentatively assigned to the C-terminus domain of FMRP. Both the crosslinking and cryo-EM results agree with a previous tandem affinity purification analysis of dFMRP from a cytoplasmic lysate, which showed that FMRP could interact with ribosomal proteins L5 and L18, both located in the CP of the 60S subunit. Indeed, a direct interaction of NT-dFMRP is observed with protein L5. The previous interaction reported with protein L18 could involve the N-terminus of the full-length FMRP that is absent in the construct used in this study. Docking of an I-TASSER homology model of NT-dFMRP into the corresponding cryo-EM map density tentatively places its KH1 and KH2 domains interacting with the CP and ASF, respectively, of the 60S subunit. This region of the 60S subunit would normally be occupied by a tRNA in the peptidyl site (P site) during protein synthesis. Superimposition of the ribosome-bound FMRP and previously known binding position of the tRNA at the ribosomal P site indicates that the KH1 and KH2 domains of FMRP would partially overlap with the anticodon arm of the tRNA. However, future structural studies with a translationally inhibited ribosome•FMRP complex carrying a tRNA in the P site will be essential to understand if and how both the P-site tRNA and FMRP would be accommodated simultaneously on the ribosome (Chen, 2014).

Fmr1 homologs bind to RNA and acts as a negative regulator of translation

Fragile X syndrome is the result of transcriptional suppression of the gene FMR1 as a result of a trinucleotide repeat expansion mutation. The normal function of the FMR1 protein (FMRP) and the mechanism by which its absence leads to mental retardation are unknown. Ribonucleoprotein particle (RNP) domains have been identified within FMRP, and RNA has been shown to bind in stoichiometric ratios, which suggests that there are two RNA binding sites per FMRP molecule. FMRP is able to bind to its own message with high affinity (dissociation constant = 5.7 nM) and interact with approximately 4 percent of human fetal brain messages. The absence of the normal interaction of FMRP with a subset of RNA molecules might result in the pleiotropic phenotype associated with fragile X syndrome (Ashley, 1993a).

FMR1 contains two types of sequence motifs recently found in RNA-binding proteins: an RGG box and two heterogeneous nuclear RNP K homology domains. FMR1 binds RNA in vitro. Using antibodies to FMR1, its expression is detected in divergent organisms and in cells of unaffected humans, but fragile X-affected patients express little or no FMR1. These findings demonstrate that FMR1 expression is directly correlated with the fragile X syndrome and suggest that anti-FMR1 antibodies will be important for diagnosis of fragile X syndrome. Furthermore, the RNA binding activity of FMR1 opens the way to understanding the function of FMR1 (Siomi, 1993).

Fmrp has been found to be a nucleocytoplasmic RNA-binding protein that contains both KH domains and RGG boxes; it associates with polyribosomes as a ribonucleoprotein particle. RNA binding has previously been demonstrated with in vitro-translated Fmrp; however, it remains uncertain whether the selective RNA binding observed is an intrinsic property of Fmrp or requires an associated protein(s). Here, baculovirus-expressed and affinity-purified FLAG-tagged murine Fmrp was shown to bind directly to both ribonucleotide homopolymers and human brain mRNA. FLAG-Fmrp exhibits selectivity for binding poly(G) > poly(U) >> poly(C) or poly(A). Moreover, purified FLAG-Fmrp binds to only a subset of brain mRNA, including the 3' untranslated regions of myelin basic protein message and its own message. Recombinant isoform 4, lacking the RGG boxes but maintaining both KH domains, was also purified and was found to only weakly interact with RNA. FLAG-purified I304N Fmrp, harboring the mutation of severe fragile X syndrome, demonstrates RNA binding. These data demonstrate the intrinsic property of Fmrp to selectively bind RNA and show FLAG-Fmrp as a suitable reagent for structural characterization and identification of cognate RNA ligands (Brown, 1998).

The identification and characterization are reported of a specific and high affinity binding site for FMRP in the RGG-coding region of its own mRNA. This site contains a purine quartet motif that is essential for FMRP binding and can be substituted by a heterologous quartet-forming motif. The specific binding of FMRP to its target site was confirmed further in a reticulocyte lysate through its ability to repress translation of a reporter gene harboring the RNA target site in the 5'-untranslated region. These data address interesting questions concerning the role of FMRP in the post-transcriptional control of its own gene and possibly other target genes (Schaeffer, 2001).

Since FMRP is an RNA-binding protein that associates with polyribosomes, it had been proposed to function as a regulator of gene expression at the post-transcriptional level. FMRP strongly inhibits translation of various mRNAs at nanomolar concentrations in both rabbit reticulocyte lysate and microinjected Xenopus laevis oocytes. This effect is specific for FMRP, since other proteins with similar RNA-binding domains, including the autosomal homologs of FMRP, Fmr11 and Fmr12, fail to suppress translation in the same concentration range. Strikingly, a disease-causing Ile-->Asn substitution at amino acid position 304 (I304N) renders FMRP incapable of interfering with translation in both test systems. Initial studies addressing the underlying mechanism of inhibition suggest that FMRP inhibits the assembly of 80S ribosomes on the target mRNAs. The failure of FMRP I304N to suppress translation is not due to its reduced affinity for mRNA or its interacting proteins Fmr11 and Fmr12. Instead, the I304N point mutation severely impairs homo-oligomerization of FMRP. These data support the notion that inhibition of translation may be a function of FMRP in vivo. It is further suggested that the failure of FMRP to oligomerize, caused by the I304N mutation, may contribute to the pathophysiological events leading to fragile X syndrome (Laggerbauer, 2001).

Microarray analysis of FMRP-associated brain mRNAs is reported in this study. mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. This approach has identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some of these mRNAs to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. It is concluded that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and candidate genes relevant to this phenotype were identified (Brown, 2001).

FMRP is a selective RNA-binding protein associated with polyribosomes. Purified recombinant FMRP causes a dose-dependent translational inhibition of brain poly(A) RNA in rabbit reticulocyte lysate without accelerated mRNA degradation. FMRP interacts with other messenger ribonucleoproteins and pre-exposure of FMRP to mRNA significantly increases the potency of FMRP as a translation inhibitor. Translation suppression by FMRP is reversed in a trans-acting manner by the 3'-untranslated portion of the Fmr1 message, which binds FMRP, suggesting that FMRP inhibits translation via interacting with mRNA. Consistently FMRP suppresses translation of the parathyroid hormone transcript, which binds FMRP, but not the beta-globin transcript, which does not bind FMRP. Moreover, removing the FMRP-binding site on a translation template abolishes the inhibitory effect of FMRP. Taken together, these results support the hypothesis that FMRP inhibits translation via interactions with the translation template (Li, 2001).

Subcellular localization of Fmr1 homologs

The fragile X syndrome results from amplification of the CGG repeat found in the FMR-1 gene. This CGG repeat shows length variation in normal individuals and is increased significantly in both carriers and patients; it is located 250 base pairs distal to a CpG island that is hypermethylated in fragile X patients. The methylation probably results in downregulation of FMR-1 gene expression. This study investigates the nature and function of the protein encoded by the FMR-1 gene using polyclonal antibodies raised against the predicted amino-acid sequences. Four different protein products, possibly resulting from alternative splicing, have been identified by immunoblotting in lymphoblastoid cell lines of healthy individuals. All these proteins were missing in cell lines from patients not expressing FMR-1 messenger RNA. The intracellular localization of the FMR-1 gene products was investigated by transient expression in COS-1 cells and found to be cytoplasmic. Localization was also predominantly cytoplasmic in the epithelium of the oesophagus, but in some cells was obviously nuclear (Verheij, 1993).

To gain insight into FMRP function, immunolocalization analysis of FMRP truncation and fusion constructs has been performed; this reveals a nuclear localization signal (NLS) in the amino terminus of FMRP as well as a nuclear export signal (NES) encoded by exon 14. A 17 amino acid peptide containing the FMRP NES, which closely resembles the NES motifs recently described for HIV-1 Rev and PKI, is sufficient to direct nuclear export of a microinjected protein conjugate. Sucrose gradient analysis shows that FMRP ribosome association is RNA-dependent and FMRP is found in ribonucleoprotein (RNP) particles following EDTA treatment. These data are consistent with nascent FMRP entering the nucleus to assemble into mRNP particles prior to export back into the cytoplasm and suggest that fragile X syndrome may result from altered translation of transcripts which normally bind to FMRP (Eberhart, 1996).

Local translation of proteins in distal dendrites is thought to support synaptic structural plasticity. Metabotropic glutamate receptor (mGluR1) stimulation initiates a phosphorylation cascade, triggering rapid association of some mRNAs with translation machinery near synapses, and leading to protein synthesis. To determine the identity of these mRNAs, a cDNA library produced from distal nerve processes was used to screen synaptic polyribosome-associated mRNA. mRNA has been identified for the fragile X mental retardation protein (FMRP) in these processes by use of synaptic subcellular fractions, termed synaptoneurosomes. This mRNA associates with translational complexes in synaptoneurosomes within 1-2 min after mGluR1 stimulation of this preparation: increased expression of FMRP is observed after mGluR1 stimulation. In addition, FMRP is associated with polyribosomal complexes in these fractions. In vivo, FMRP immunoreactivity is observed in spines, dendrites, and somata of the developing rat brain, but not in nuclei or axons. It is suggested that rapid production of FMRP near synapses in response to activation may be important for normal maturation of synaptic connections (Weiler, 1997).

A detailed cellular localization study of FMRP has been carried out using both biochemical and immunocytochemical approaches. FMRP is highly expressed in neurons but not glia throughout the rat brain, as detected by light microscopy. Although certain structures, such as hippocampus, reveal a strong signal, the regional variation in staining intensity appears to be related to neuron size and density. In human cell lines and mouse brain, FMRP co-fractionates primarily with polysomes and rough endoplasmic reticulum. Ultrastructural studies in rat brain have revealed high levels of FMRP immunoreactivity in neuronal perikarya, where FMRP is concentrated in regions rich in ribosomes, particularly near or between rough endoplasmic reticulum cisternae. Immunogold studies has provides evidence of nucleocytoplasmic shuttling of FMRP, which was localized in neuronal nucleoplasm and within nuclear pores. Moreover, labeling is observed in large- and small-caliber dendrites, in dendritic branch points, at the origins of spine necks, and in spine heads, all known locations of neuronal polysomes. Dendritic localization, which was confirmed by co-fractionation of FMRP with synaptosomal ribosomes, suggests a possible role of FMRP in the translation of proteins involved in dendritic structure or function and relevant for the mental retardation occurring in fragile X syndrome (Feng, 1997b).

Targets of Fragile X mental retardation protein

Loss of fragile X mental retardation protein (FMRP) function causes the fragile X mental retardation syndrome. FMRP harbors three RNA binding domains, associates with polysomes, and is thought to regulate mRNA translation and/or localization, but the RNAs to which it binds are unknown. RNA selection was used to demonstrate that the FMRP RGG box binds intramolecular G quartets. These data allowed the identification of mRNAs encoding proteins involved in synaptic or developmental neurobiology that harbor FMRP binding elements. The majority of these mRNAs have an altered polysome association in fragile X patient cells. These data demonstrate that G quartets serve as physiologically relevant targets for FMRP and identify mRNAs whose dysregulation may underlie human mental retardation (Darnell, 2001).

The Fragile X mental retardation-1 (Fmr1) gene encodes a multifunctional protein, FMRP, with intrinsic RNA binding activity. Antibody-positioned RNA amplification (APRA) has been developed to identify the RNA cargoes associated with the in vivo configured FMRP messenger ribonucleoprotein (mRNP) complex. Using APRA as a primary screen, putative FMRP RNA cargoes were assayed for their ability to bind directly to FMRP, using traditional methods of assessing RNA-protein interactions, including UV-crosslinking and filter binding assays. Approximately 60% of the APRA-defined mRNAs directly associate with FMRP. By examining a subset of these mRNAs and their encoded proteins in brain tissue from Fmr1 knockout mice, it has been observed that some of these cargoes as well as the proteins they encode show discrete changes in abundance and/or differential subcellular distribution. These data are consistent with spatially selective regulation of multiple biological pathways by FMRP (Miyashiro, 2003).

The Fragile X syndrome, which results from the absence of functional FMRP protein, is the most common heritable form of mental retardation. FMRP acts as a translational repressor of specific mRNAs at synapses. Interestingly, FMRP associates not only with these target mRNAs, but also with the dendritic, non-translatable RNA BC1. Blocking of BC1 inhibits the interaction of FMRP with its target mRNAs. Furthermore, BC1 binds directly to FMRP and can also associate, in the absence of any protein, with the mRNAs regulated by FMRP. This suggests a mechanism where BC1 could determine the specificity of FMRP function by linking the regulated mRNAs and FMRP. Thus, when FMRP is not present, loss of translational repression of specific mRNAs at synapses results in synaptic dysfunction phenotype of Fragile X patients (Zalfa, 2003).

Fragile X syndrome, the most common form of inherited mental retardation, is caused by absence of FMRP, an RNA-binding protein implicated in regulation of mRNA translation and/or transport. dFMR1, the Drosophila ortholog of FMRP, is genetically linked to the dRac1 GTPase, a key player in actin cytoskeleton remodeling. This study demonstrates that FMRP and the Rac1 pathway are connected in a model of murine fibroblasts. Rac1 activation induces relocalization of four FMRP partners to actin ring areas. Moreover, Rac1-induced actin remodeling is altered in fibroblasts lacking FMRP or carrying a point-mutation in the KH1 or in the KH2 RNA-binding domain. In absence of wild-type FMRP, it was found that phospho-ADF/Cofilin (P-Cofilin) level, a major mediator of Rac1 signaling, is lowered, whereas the level of protein phosphatase 2A catalytic subunit (PP2Ac), a P-Cofilin phosphatase, is increased. FMRP binds with high affinity to the 5'-UTR of pp2acbeta mRNA and is thus a likely negative regulator of its translation. The molecular mechanism unraveled here points to a role for FMRP in modulation of actin dynamics, which is a key process in morphogenesis of dendritic spines, synaptic structures abnormally developed in Fragile X syndrome patient's brain (Castets, 2005).

Binding of Fmr1 homologs to ribosomes

The FMR1 transcript is alternatively spliced and generates different splice variants coding for FMR1 proteins (FMRP) with a predicted molecular mass of 70-80 kDa. FMRP is widely expressed and localized in the cytoplasm. To study a possible interaction with other cellular components, FMRP was isolated and characterized under non-denaturing conditions. Under physiological salt conditions FMRP appears to have a molecular mass of > 600 kDa, indicating a binding to other cellular components. This interaction is disrupted in the presence of high salt concentrations. The dissociation conditions to free FMRP from the complex are similar to the dissociation of FMRP from RNA as shown before. The binding of FMRP from the complex is also disrupted by RNAse treatment. That the association of FMRP to a high molecular weight complex possibly occurs via RNA, is further supported by the observation that the binding of FMRP, containing an lle304Asn substitution, to the high molecular weight complex is reduced. An equal reduced binding of mutated FMRP to RNA in vitro was observed before under the same conditions. The reduced binding of FMRP with the lle304Asn substitution further indicates that the interaction to the complex indeed occurs via FMRP and not via other RNA binding proteins. In a reconstitution experiment where the low molecular mass FMRP (70-80 kDa) was mixed with a reticulocyte lysate (enriched in ribosomes) it was shown that FMRP can associate to ribosomes and that this binding most likely occurs via RNA (Tamanini, 1996).

Kissing complex RNAs mediate interaction between the Fragile-X mental retardation protein KH2 domain and brain polyribosomes

Fragile-X mental retardation is caused by loss of function of a single gene encoding the Fragile-X mental retardation protein, FMRP, an RNA-binding protein that harbors two KH-type and one RGG-type RNA-binding domains. Previous studies identified intramolecular G-quartet RNAs as high-affinity targets for the RGG box, but the relationship of RNA binding to FMRP function and mental retardation remains unclear. One severely affected patient harbors a missense mutation (I304N) within the second KH domain (KH2), and some evidence suggests this domain may be involved in the proposed role of FMRP in translational regulation. The RNA target for the KH2 domain has been identied as a sequence-specific element within a complex tertiary structure termed the FMRP kissing complex (kc). This study demonstrates that the association of FMRP with brain polyribosomes is abrogated by competition with the FMRP kissing complex RNA, but not by high-affinity G-quartet RNAs. It is concluded that mental retardation associated with the I304N mutation, and likely the Fragile-X syndrome more generally, may relate to a crucial role for RNAs harboring the kissing complex motif as targets for FMRP translational regulation (Darnell, 2005).

Prior structural studies have demonstrated that KH domains bind to single-stranded regions of RNA, and these may be presented in the context of stem-loop structure. To further explore the structure of the kc RNA and its relationship to KH2 binding, positions in the RNA accessible to chemical modification were examined, using reagents that modify unpaired nucleotides and detecting modifications by stops in primer extension. Since KH2:kc RNA binding requires the presence of Mg+2, results obtained in the presence of Mg+2 versus EDTA were compared. DMS, CMCT, and kethoxal (which modify unpaired N3-C and N1-A, N3-U and N1-G, and N1-G and N2-G, respectively) were examined for chemical modification. It was found that kc RNA was modified in EDTA, but was protected in Mg+2 in several regions. The data suggest the presence of an unanticipated 4-bp duplex forming between the two loops (a conserved 5'-C48UGG51-3' in the 3' loop and a 5'-C11CAG14-3' present in the fixed sequence of the 5' loop). In addition, an A20-U45 pair previously found to covary with G20-C45 was also protected in the presence of Mg+2, suggesting formation of a Watson-Crick base pair. These experiments also provide evidence that the stem of the 5' stem-loop predicted by mfold (5'-UUCC-3' with 5'-GGAG-3') folds in a Mg+2-dependent manner. The stem of the 3' stem-loop predicted by mfold is base-paired in a Mg+2-independent manner. This finding, that there is a 4-bp interaction between the loops and that the R20 and Y45 nucleotides base pair with each other, provides strong evidence that the two loops form a stable loop-loop pseudoknot, or kissing complex (Darnell, 2005).

The mechanism by which FMRP might interact with polyribosomes has not been well understood. FMRP has been reported to interact with a number of RNA-binding proteins, including FXR1P/FXR2P, YB-1, Staufen, and nucleolin, consistent with data suggesting that FMRP may be part of large mRNPs, some of which may be associated with polyribosomes. In addition, FMRP copurifies with the RISC complex in Drosophila and coimmunoprecipitates with the dAgo-1 homolog, a protein component of the RISC complex in mammalian cells in culture. These observations have suggested that FMRP might bind to polyribosomes indirectly, via protein-RNA networks, and/or RISC complex-targeting of miRNAs to mRNAs. For example, it has been suggested that FMRP might 'scan' mRNAs for G-quartet elements that then become associated with RISC complex proteins in a translationally regulated polyribosome complex. The current data indicate that this model needs revision. G-quartet RNAs may be involved in RNA binding, translational control, and even polyribosome association in the brain. However, G-quartets do not compete FMRP off polyribosomes, and deletion of the RGG-box does not affect FMRP polyribosome association, suggesting that G-quartets are not required for polyribosome association. Instead, the data suggest that direct association of FMRP with RNAs harboring kissing complex motifs fitting the consensus described in this study mediate FMRP association with brain polyribosomes (Darnell, 2005).

The association of FMRP with brain polyribosomes is specifically disrupted by competition with kissing complex RNAs fitting the consensus described here. Furthermore, the FMRP:kissing complex interaction is mediated by KH2 and is sensitive to the I304N mutation. Taken together, these findings indicate that these kissing complex RNAs may provide a crucial link between the association of FMRP in brain polyribosomes, its proposed role in regulating mRNA translation, and neurologic dysfunction in the Fragile-X syndrome. Redirecting the ongoing search for FMRP RNA targets to those RNAs harboring kissing complex motifs may be of importance in identifying RNA ligands central to the Fragile-X syndrome (Darnell, 2005).

Effects of fragile X mutation

Fragile X [fraX] syndrome is a common hereditary disorder associated with a fragile site marker at Xq27.3 that clinically presents as a form of mental retardation (MR). Postmortem investigation of 3 fraX positive males with mild to moderate MR did not document any gross neuropathological changes. Golgi analysis of neocortical dendritic spine morphology extended previous observations of immature, long, tortuous spines in one adult case of fraX to 2 new cases. Evidence for similar dendritic spine abnormalities was found, although Golgi analysis was less than optimal because of incomplete dendritic stain impregnation. Neocortical intra-layer cell density was also investigated in all 3 cases. Cresyl violet stained neurons were counted in 10 randomly selected fields in neocortical layers II-VI of cingulate and temporal association areas (Brodmann's areas 23 and 38). Neuron counts in fraX and control neocortex showed no significant differences. Thus, abnormal dendritic spine morphology with preservation of neuronal density appears to characterize the neocortex in individuals with this common form of mental retardation (Hinton, 1991).

A missense mutation in FMRP, I304N, has been found to result in an unusually severe phenotype. Normal FMRP associates with elongating polyribosomes via large mRNP particles. Despite normal expression and cytoplasmic mRNA association, the I304N FMRP is incorporated into abnormal mRNP particles that are not associated with polyribosomes. These data indicate that association of FMRP with polyribosomes must be functionally important and imply that the mechanism of the severe phenotype in the I304N patient lies in the sequestration of bound mRNAs in nontranslatable mRNP particles. In the absence of FMRP, these same mRNAs may be partially translated via alternative mRNPs, although perhaps abnormally localized or regulated, resulting in typical fragile X syndrome (Feng, 1997a).

Golgi-impregnated mature cerebral cortex from fragile X patients exhibits long, thin, tortuous postsynaptic spines resembling spines observed during normal early neocortical development. This study describes dendritic spines in Golgi-impregnated cerebral cortex of transgenic fragile X gene (Fmr1) knockout mice that lack expression of the protein. Dendritic spines on apical dendrites of layer V pyramidal cells in occipital cortex of fragile X knockout mice are longer than those in wild-type mice and are often thin and tortuous, paralleling the human syndrome and suggesting that FMRP expression is required for normal spine morphological development. Moreover, spine density along the apical dendrite is greater in the knockout mice, which may reflect impaired developmental organizational processes of synapse stabilization and elimination or pruning (Comery, 1997).

Qualitative examination of human brain autopsy material has shown that fragile-X patients exhibit abnormal dendritic spine lengths and shapes on parieto-occipital neocortical pyramidal cells. Dendritic spines on layer V pyramidal cells of human temporal and visual cortices stained using the Golgi-Kopsch method were investigated. Quantitative analysis of dendritic spine length, morphology, and number was carried out on patients with fragile-X syndrome and normal age-matched controls. Fragile-X patients exhibit significantly more long dendritic spines and fewer short dendritic spines than do control subjects in both temporal and visual cortical areas. Similarly, fragile-X patients exhibit significantly more dendritic spines with an immature morphology and fewer with a more mature type morphology in both cortical areas. In addition, fragile-X patients had a higher density of dendritic spines than did controls on distal segments of apical and basilar dendrites in both cortical areas. Long dendritic spines with immature morphologies and elevated spine numbers are characteristic of early development or a lack of sensory experience. The fact that these characteristics are found in fragile-X patients throughout multiple cortical areas may suggest a global failure of normal dendritic spine maturation and or pruning during development that persists throughout adulthood (Irwin, 2001).

Reports that patients and adult FMR1 knock-out mice have abnormally long dendritic spines of increased density suggested that Fragile X syndrome might involve abnormal spine development. Because spine length, density, and motility change dramatically in the first postnatal weeks, these properties were analyzed in mutant mice and littermate controls at 1, 2, and 4 weeks of age. To label neurons, a viral vector carrying the enhanced green fluorescent protein gene was injected into the barrel cortex. Layer V neurons were imaged on a two-photon laser scanning microscope in fixed tissue sections. Analysis of >16,000 spines showed clear developmental patterns. Between 1 and 4 weeks of age, spine density increases 2.5-fold, and mean spine length decreased by 17% in normal animals. Early during cortical synaptogenesis, pyramidal cells in mutant mice have longer spines than controls. At 1 week, spine length is 28% greater in mutants than in controls. At 2 weeks, this difference is 10%, and at 4 weeks only 3%. Similarly, spine density is 33% greater in mutants than in controls at 1 week of age. At 2 or 4 weeks of age, differences are not detectable. The spine abnormality is not detected in neocortical organotypic cultures. The transient nature of the spine abnormality in the intact animal suggests that FMRP might play a role in the normal process of dendritic spine growth in coordination with the experience-dependent development of cortical circuits (Nimchinsky, 2001).

Absence of functional FMRP causes Fragile X syndrome. Abnormalities in synaptic processes in the cerebral cortex and hippocampus contribute to cognitive deficits in Fragile X patients. So far, the potential roles of cerebellar deficits have not been investigated. This study demonstrates that both global and Purkinje cell-specific knockouts of Fmr1 show deficits in classical delay eyeblink conditioning in that the percentage of conditioned responses as well as their peak amplitude and peak velocity are reduced. Purkinje cells of these mice show elongated spines and enhanced LTD induction at the parallel fiber synapses that innervate these spines. Moreover, Fragile X patients display the same cerebellar deficits in eyeblink conditioning as the mutant mice. These data indicate that a lack of FMRP leads to cerebellar deficits at both the cellular and behavioral levels and raise the possibility that cerebellar dysfunctions can contribute to motor learning deficits in Fragile X patients (Koekkoek, 2005).

Fragile X syndrome, caused by a mutation in the Fmr1 gene, is characterized by mental retardation. Several studies reported the absence of long-term potentiation (LTP) at neocortical synapses in Fmr1 knockout (FMR1-KO) mice, but underlying cellular mechanisms are unknown. This study found that in the prefrontal cortex (PFC) of FMR1-KO mice, spike-timing-dependent LTP (tLTP) is not so much absent, but rather, the threshold for tLTP induction is increased. Calcium signaling in dendrites and spines is compromised. (1) Dendrites and spines more often fail to show calcium transients. (2) The activity of L-type calcium channels is absent in spines. tLTP could be restored by improving reliability and amplitude of calcium signaling by increasing neuronal activity. In FMR1-KO mice that were raised in enriched environments, tLTP was restored to WT levels. These results show that mechanisms for synaptic plasticity are in place in the FMR1-KO mouse PFC, but require stronger neuronal activity to be triggered (Meredith, 2007).

The function of local protein synthesis in synaptic plasticity and its dysregulation in fragile X syndrome (FXS) is well studied, however the contribution of regulated mRNA transport to this function remains unclear. This study reports a function for the fragile X mental retardation protein (FMRP) in the rapid, activity-regulated transport of mRNAs important for synaptogenesis and plasticity. mRNAs are deficient in glutamatergic signaling-induced dendritic localization in neurons from Fmr1 KO mice, and single mRNA particle dynamics in live neurons revealed diminished kinesis. Motor-dependent translocation of FMRP and cognate mRNAs involved the C terminus of FMRP and kinesin light chain, and KO brain showed reduced kinesin-associated mRNAs. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity (Dictenberg, 2008).

Protein Interactions of Fmr1 homologs

The absence of the fragile X mental retardation protein (FMRP), encoded by the FMR1 gene, is responsible for pathologic manifestations in the Fragile X Syndrome, the most frequent cause of inherited mental retardation. FMRP is an RNA-binding protein associated with polysomes as part of a messenger ribonucleoprotein (mRNP) complex. Although its function is poorly understood, various observations suggest a role in local protein translation at neuronal dendrites and in dendritic spine maturation. CYFIP1/2 (Cytoplasmic FMRP Interacting Proteins) have been identified as FMRP interactors. CYFIP1/2 share 88% amino acid sequence identity and represent the two members in humans of a highly conserved protein family. Remarkably, whereas CYFIP2 also interacts with the FMRP-related proteins FXR1P/2P, CYFIP1 interacts exclusively with FMRP. FMRP--CYFIP interaction involves the domain of FMRP also mediating homo- and hetero-merization, thus suggesting a competition between interaction among the FXR proteins and interaction with CYFIP. CYFIP1/2 are proteins of unknown function, but CYFIP1 has been shown to interact with the small GTPase Rac1, which is implicated in development and maintenance of neuronal structures. Consistent with FMRP and Rac1 localization in dendritic fine structures, CYFIP1/2 are present in synaptosomal extracts (Schenck, 2001).

FMR protein and the microRNA pathway

Fragile X syndrome is caused by a loss of expression of the fragile X mental retardation protein (FMRP). FMRP is a selective RNA-binding protein which forms a messenger ribonucleoprotein (mRNP) complex that associates with polyribosomes. Recently, mRNA ligands associated with FMRP have been identified. However, the mechanism by which FMRP regulates the translation of its mRNA ligands remains unclear. MicroRNAs are small noncoding RNAs involved in translational control. Mammalian FMRP is show to interact in vitro with microRNAs and the components of the microRNA pathways including Dicer and the mammalian ortholog of Argonaute 1 (AGO1). Using two different Drosophila melanogaster models, it is shown that AGO1 is critical for FMRP function in neural development and synaptogenesis. These results suggest that FMRP may regulate neuronal translation via microRNAs and links microRNAs with human disease (Jin, 2004).

A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response

Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. This study identified FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, FMRP was shown to bind chromatin through its tandem Tudor (Agenet) domain in vitro, and it associates with chromatin in vivo. FMRP was also shown to participate in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome (Alpatov, 2014).

Gene therapy and fragile X syndrome

As a step toward understanding the function of FMR1 and the determination of the potential for therapeutic approaches to fragile X syndrome, yeast artificial chromosome (YAC) transgenic mice were generated in order to determine whether the Fmr1 knockout mouse phenotype could be rescued. Several transgenic lines were generated that carried the entire FMR1 locus with extensive amounts of flanking sequence. The YAC transgene supports production of the human protein (FMRP) which was present at levels 10 to 15 times that of endogenous protein and was expressed in a cell- and tissue-specific manner. Macro-orchidism is absent in knockout mice carrying the YAC transgene indicating functional rescue by the human protein. Given the complex behavioral phenotype in fragile X patients and the mild phenotype previously reported for the Fmr1 knockout mouse, a more thorough evaluation of the Fmr1 knockout phenotype was performed using additional behavioral assays that had not previously been reported for this animal model. The mouse displays reduced anxiety-related responses with increased exploratory behavior. FMR1 YAC transgenic mice overexpressing the human protein produce opposing behavioral responses and additional abnormal behaviors were also observed. These findings have significant implications for gene therapy for fragile X syndrome since overexpression of the gene may harbor its own phenotype (Peier, 2000).

The cyclic AMP cascade is altered in the fragile X nervous system

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP) cascade. This study tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. These results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling (Kelley, 2007).

FMRP acts as a key messenger for dopamine modulation in the forebrain

The fragile X mental retardation protein (FMRP) is an RNA-binding protein that controls translational efficiency and regulates synaptic plasticity. FMRP is involved in dopamine (DA) modulation of synaptic potentiation. AMPA glutamate receptor subtype 1 (GluR1) surface expression and phosphorylation in response to D1 receptor stimulation were reduced in cultured Fmr1−/− prefrontal cortex (PFC) neurons. Furthermore, D1 receptor signaling was impaired, accompanied by D1 receptor hyperphosphorylation at serine sites and subcellular redistribution of G protein-coupled receptor kinase 2 (GRK2) in both PFC and striatum of Fmr1−/− mice. FMRP interacted with GRK2, and pharmacological inhibition of GRK2 rescued D1 receptor signaling in Fmr1−/− neurons. Finally, D1 receptor agonist partially rescued hyperactivity and enhanced the motor function of Fmr1−/− mice. This study has identified FMRP as a key messenger for DA modulation in the forebrain and may provide insights into the cellular and molecular mechanisms underlying fragile X syndrome (Wang, 2008).

Dephosphorylation-induced ubiquitination and degradation of FMRP in dendrites: a role in immediate early mGluR-stimulated translation

Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP), which represses and reversibly regulates the translation of a subset of mRNAs in dendrites. Protein synthesis can be rapidly stimulated by mGluR-induced and protein phosphatase 2a (PP2A)-mediated dephosphorylation of FMRP, which is coupled to the dissociation of FMRP and target mRNAs from miRNA-induced silencing complexes. This study reports the rapid ubiquitination and ubiquitin proteasome system (UPS)-mediated degradation of FMRP in dendrites upon DHPG (3,5-dihydroxyphenylglycine) stimulation in cultured rat neurons. Using inhibitors to PP2A and FMRP phosphomutants, degradation of FMRP was observed to depend on its prior dephosphorylation. Translational induction of an FMRP target, postsynaptic density-95 mRNA, required both PP2A and UPS. Thus, control of FMRP levels at the synapse by dephosphorylation-induced and UPS-mediated degradation provides a mode to regulate protein synthesis (Nalavadi, 2012).

Short- and long-term memory are modulated by multiple isoforms of the fragile X mental retardation protein

The diversity of protein isoforms arising from alternative splicing is thought to modulate fine-tuning of synaptic plasticity. Fragile X mental retardation protein (FMRP), a neuronal RNA binding protein, exists in isoforms as a result of alternative splicing, but the contribution of these isoforms to neural plasticity are not well understood. This study shows that two isoforms of Drosophila melanogaster FMRP (dFMR1) have differential roles in mediating neural development and behavior functions conferred by the dfmr1 gene. These isoforms differ in the presence of a protein interaction module that is related to prion domains and is functionally conserved between FMRPs. Expression of both isoforms is necessary for optimal performance in tests of short- and long-term memory of courtship training. The presence or absence of the protein interaction domain may govern the types of ribonucleoprotein (RNP) complexes dFMR1 assembles into, with different RNPs regulating gene expression in a manner necessary for establishing distinct phases of memory formation (Banerjee, 2010).

Pharmacological rescue of Ras signaling, GluA1-dependent synaptic plasticity, and learning deficits in a fragile X model

Fragile X syndrome, caused by the loss of Fmr1 gene function, is the most common form of inherited mental retardation, with no effective treatment. Using a tractable animal model, this study investigated mechanisms of action of a few FDA-approved psychoactive drugs that modestly benefit the cognitive performance in fragile X patients. Compounds activating serotonin (5HT) subtype 2B receptors (5HT2B-Rs) or dopamine (DA) subtype 1-like receptors (D1-Rs) and/or those inhibiting 5HT2A-Rs or D2-Rs were shown to moderately enhance Ras-PI3K/PKB signaling input, GluA1-dependent synaptic plasticity, and learning in Fmr1 knockout mice. Unexpectedly, combinations of these 5HT and DA compounds at low doses synergistically stimulate Ras-PI3K/PKB signal transduction and GluA1-dependent synaptic plasticity and remarkably restore normal learning in Fmr1 knockout mice without causing anxiety-related side effects. These findings suggest that properly dosed and combined FDA-approved psychoactive drugs may effectively treat the cognitive impairment associated with fragile X syndrome (Lim, 2014).

Dynamic mRNA transport and local translation in radial glial progenitors of the developing brain

In the developing brain, neurons are produced from neural stem cells termed radial glia. Radial glial progenitors span the neuroepithelium, extending long basal processes to form endfeet hundreds of micrometers away from the soma. Basal structures influence neuronal migration, tissue integrity, and proliferation. Yet, despite the significance of these distal structures, their cell biology remains poorly characterized, impeding understanding of how basal processes and endfeet influence neurogenesis. This study used live imaging of embryonic brain tissue to visualize, for the first time, rapid mRNA transport in radial glia, revealing that the basal process is a highway for directed molecular transport. RNA- and mRNA-binding proteins, including the syndromic autism protein FMRP (see Drosophila Fmr1), move in basal processes at velocities consistent with microtubule-based transport, accumulating in endfeet. An ex vivo tissue preparation was developed to mechanically isolate radial glia endfeet from the soma, and photoconvertible proteins were used to demonstrate that mRNA is locally translated. Using RNA immunoprecipitation and microarray analyses of endfeet, FMRP-bound transcripts, which encode signaling and cytoskeletal regulators, were discovered including many implicated in autism and neurogenesis. FMRP controls transport and localization of one target, Kif26a. These discoveries reveal a rich, regulated local transcriptome in radial glia, far from the soma, and establish a tractable mammalian model for studying mRNA transport and local translation in vivo. It is concluded that cytoskeletal and signaling events at endfeet may be controlled through translation of specific mRNAs transported from the soma, exposing new mechanistic layers within stem cells of the developing brain (Pilz, 2016).


Search PubMed for articles about Drosophila Fmr1

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Ashley C. T. Jr., et al. (1993b). Human and murine FMR-1: alternative splicing and translational initiation downstream of the CGG-repeat. Nat. Genet. 4: 244-251. 8358432

Banerjee, P., Schoenfeld, B. P., Bell, A. J., Choi, C. H., Bradley, M. P., Hinchey, P., Kollaros, M., Park, J. H., McBride, S. M. and Dockendorff, T. C. (2010). Short- and long-term memory are modulated by multiple isoforms of the fragile X mental retardation protein. J Neurosci 30: 6782-6792. PubMed ID: 20463240

Barbee, S. A., et al. (2006). Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies. Neuron 52(6): 997-1009. Medline abstract: 17178403

Bardoni, B., et al. (2003). 82-FIP, a novel FMRP (fragile X mental retardation protein) interacting protein, shows a cell cycle-dependent intracellular localization. Hum. Mol. Genet. 12: 1689-1698. PubMed Citation: 12837692

Bhogal, B., et al. (2011). Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein. Nat. Neurosci. 14(12): 1517-24. PubMed Citation: 22037499

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Bozzetti, M.P., Specchia, V., Cattenoz, P.B., Laneve, P., Geusa, A., Sahin, H.B., Di Tommaso, S., Friscini, A., Massari, S., Diebold, C. and Giangrande, A. (2015). The Drosophila fragile X mental retardation protein participates in the piRNA pathway. J Cell Sci 128: 2070-2084. PubMed ID: 25908854

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Brown, V., et al. (2001). Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107(4): 477-87. 11719188

Bulgari, D., Zhou, C., Hewes, R. S., Deitcher, D. L. and Levitan, E. S. (2014). Vesicle capture, not delivery, scales up neuropeptide storage in neuroendocrine terminals. Proc Natl Acad Sci U S A 111(9): 3597-3601. PubMed ID: 24550480

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Cavolo, S. L., Bulgari, D., Deitcher, D. L. and Levitan, E. S. (2016). Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles. J Neurosci 36(46): 11781-11787. PubMed ID: 27852784

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Coyne, A. N., Yamada, S. B., Siddegowda, B. B., Estes, P. S., Zaepfel, B. L., Johannesmeyer, J. S., Lockwood, D. B., Pham, L. T., Hart, M. P., Cassel, J. A., Freibaum, B., Boehringer, A. V., Taylor, J. P., Reitz, A. B., Gitler, A. D. and Zarnescu, D. C. (2015). Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26385636

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Biological Overview
22 December 2017

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