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).
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).
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).
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).
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).
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).
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).
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)
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 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 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).
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).
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).
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 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).
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).
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