Transcriptional Regulation

futsch expression was analyzed in mutant backgrounds that specifically alter the 22C10 expression pattern. The Zn finger transcription factor Tramtrack acts as a repressor of 22C10 antigen expression, and in tramtrack mutant embryos, high levels of 22C10 antigen are found in the mesoderm (Giesen, 1997). Consistent with Futsch being the 22C10 antigen, the expression of futsch mRNA is also drastically increased in tramtrack mutant embryos. In a second experiment, a ftz-GAL4 driver line and EP(X)1419 were used to drive ectopic futsch expression in a pair-rule expression pattern. Ectopic expression of the 22C10 antigen correlates with ectopic expression of the deduced futsch transcript, further supporting the possibility that futsch encodes the 22C10 antigen (Hummel, 2000).

Nerfin-1 is a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. The neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development or in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch<, genes whose encoded proteins participate in these early navigational events (Kuzin, 2005).

Given the axon guidance defects in nerfin-1null embryos and the fact that Nerfin-1 is a Zn-finger nuclear protein, it was hypothesized that Nerfin-1 may be required for the correct expression of genes involved in axon guidance. Accordingly, the embryonic expression profiles of over 35 genes that have been shown to play important roles in axon guidance were examined. Included in the candidate screen were genes encoding transcription factors, RNA-binding proteins, cell surface receptor proteins, their ligands, signal transduction proteins, and components of the cytoskeleton. Homozygous nerfin-1null embryos were identified by the absence of Nerfin-1 immunoreactivity. Whole-mount in situ hybridization and/or protein immunostaining for altered spatial or temporal expression in nerfin-1null embryos identified six genes that require nerfin-1 function to achieve full wild-type expression levels (Kuzin, 2005).

Two genes involved in anterior vs. posterior commissure choice, those encoding the receptor tyrosine kinase Derailed, and its ligand Wnt5, both required nerfin-1 for full expression. In the absence of nerfin-1, ventral cord expression levels of Robo and Robo3 were unaffected; however, Robo2 expression levels were significantly reduced. Expression of Slit, the ligand for Robo receptors, and Commissureless, a factor responsible for clearing Robo receptors from commissural axons, was unaffected in nerfin-1null embryos (Kuzin, 2005).

Loss of nerfin-1 function also significantly delayed and/or reduced the early expression of the neuron-specific microtubule-associated MAP1B-like gene futsch. futsch expression is normally activated in newborn neurons starting at stage 11; however, in nerfin-1null embryos expression is first detected only at the stage 13. Not until embryonic stage 15 did the level of futsch expression in mutant embryos approach that of wild type. Reduced mRNA steady state levels for the genes encoding Leukocyte-antigen-related-like (Lar), another receptor tyrosine kinase, and G-oα47A gene, which encodes an alpha subunit of heterotrimeric G proteins, were also detected in nerfin-1null embryos. The reduced level of gene expression in mutant embryos was nervous system specific. For example, G-oα47A gene expression in mesodermal derived tissues was not altered in nerfin-1null embryos (Kuzin, 2005).

Post-transcriptional Regulation

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

Evidence is presented that Fmr1 negatively regulates Futsch expression. (1) Fmr1 associates with Futsch mRNA. This interaction is specific, since Fmr1 fails to bind other targets such as alpha-tubulin mRNAs and the interaction is missing in Fmr1 null mutants. (2) In Fmr1 null mutants, Futsch protein level in the nervous system is increased and Fmr1 neuronal overexpression causes Futsch expression to be reduced. These results show that the level of Futsch in the nervous system is inversely regulated by the level of Fmr1. Taken together, the biochemical association between Fmr1 protein and Futsch mRNA and the inverse regulation of Futsch expression by Fmr1 strongly support a hypothesis that Fmr1 acts as a negative regulator of Futsch translation (Zhang, 2001).
Futsch appears to be the major target for Fmr1 in the regulation of synaptic structure and function. Structurally, futsch hypomorphs display fewer and enlarged NMJ synaptic boutons with dispersed, punctate anti-Futsch immunoreactivity, a phenotype indistinguishable from that caused by overexpression of Fmr in dfxrNOE. However, futschNOE causes synaptic overgrowth, a phenotype similar to Fmr1 null mutants. Functionally, all four genotypes (loss and overexpression of either Fmr1 or Futsch) enhance neurotransmission at the larval NMJ, and all four genotypes impair neurotransmission in the adult eye. Thus, the expression alterations of Futsch are sufficient to explain the synaptic phenotypes of Fmr1 mutants (Zhang, 2001).

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

TDP-43 regulates Drosophila neuromuscular junctions growth by modulating Futsch/MAP1B levels and synaptic microtubules organization

TDP-43 is an evolutionarily conserved RNA binding protein recently associated with the pathogenesis of different neurological diseases. At the moment, neither its physiological role in vivo nor the mechanisms that may lead to neurodegeneration are well known. Previous studies have shown that TDP-43 mutant flies presented locomotive alterations and structural defects at the neuromuscular junctions (Feiguin, 2009). This study investigated the functional mechanism leading to these phenotypes by screening several factors known to be important for synaptic growth or bouton formation. As a result it was found that alterations in the organization of synaptic microtubules correlate with reduced protein levels in the microtubule associated protein futsch/MAP1B. Moreover, TDP-43 was found to physically interact with futsch mRNA and that its RNA binding capacity is required to prevent futsch down-regulation and synaptic defects (Godena, 2011).

TDP-43 is an RNA binding protein of 43 kDa that belongs to the hnRNP family and plays numerous roles in mRNA metabolism such as transcription, pre-mRNA splicing, mRNA stability, microRNA biogenesis, transport and translation. TDP-43 is very well conserved during the evolution, especially with regards to the two RNA-recognition motifs (RRMs), the first (RRM1) being responsible for the binding of TDP-43 with UG rich RNA. In consonance with these described functions, TDP-43 prevalently resides in the cell nucleus where it co-localizes with other members of the RNA processing machinery. Nevertheless, in pathological conditions such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), TDP-43 appears in the form of large insoluble protein aggregates redistributed within the cytoplasm. At the moment, however, it is not clear how these alterations may lead to neurodegeneration. In theory, the cytosolic accumulation of TDP-43 may induce a toxic, gain of function effect on motoneurons while the exclusion of TDP-43 from the cell nucleus may lead to neurodegeneration due to a loss of function mechanism. At present, several lines of evidence mainly from different cellular and animal models support either view suggesting that both models may be acting to lead the disease condition. Recently, to determine the physiological role of TDP-43 in vivo it has been reported that the flies which lack the TDP-43 homologue (TBPH) closely reproduce many of the phenotypes observed in ALS patients, such as progressive defects in the animal locomotion and reduced life span. Moreover, it was observed that loss of TDP-43 function in Drosophila results in reduced number of motoneurons terminal branches and synaptic boutons at neuromuscular junctions (NMJs), indicating TDP-43 may regulate the assembly and organization of these structures. In coincidence with that, it should be noted that overexpression of TDP-43 in Drosophila has been reported to increase dendritic branching (Lu, 2009), lead to motor dysfunction and reduced life span (Hanson, 2010), axon loss and neuronal death (Li, 2010), is generally toxic regardless of inclusion formations (Miguel, 2010), and at least in part is the cause behind the degeneration associated with TER94 mutations which is the Drosophila homologue of the VCP protein (Ritson, 2010). Taken together, and in consideration that Drosophila TDP-43 (TBPH) can functionally substitute for human TDP-43 in functional splicing assays (D'Ambrogio, 2009), these reports confirm that Drosophila may represent a highly suitable animal model to investigate TDP-43 functions both in normal and disease conditions (Godena, 2011).

Drosophila larval NMJ is a well-characterized system to analyze the cellular and molecular events that are involved in synapse development and plasticity. Synaptic growth during larval development is expanded according to muscle size and is accomplished by the addition of new boutons to the existing presynaptic terminals. Typically, defects in synapse formation and synaptic growth are linked to cytoskeleton abnormalities, since the synaptic boutons and the newly formed buds require the underlying presynaptic microtubules to maintain their structural organization and plasticity inside the innervated muscles. Thus, to determine the physiological role of TDP-43 in vivo and the pathological consequences of its altered function, the molecular organization of Drosophila NMJs during larval development was examined in TDP-43 minus flies (Godena, 2011).

This work, shows that in TBPH minus Drosophila model the changes observed at the level of NMJs and synaptic boutons formation can be explained by defects at the cytoskeleton level, which in turn are mediated by a down-regulation of the futsch protein (but not mRNA). These results provide additional insight with regards to potential disease mechanisms mediated by TDP-43 and considerably extend knowledge with regards to defining the basic molecular functions of this protein. Future work will be aimed at better characterizing more in depth the functional mechanism through which TBPH regulates futsch protein levels and how these results can be extended to the human disease model (Godena, 2011).

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

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

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

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

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

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

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

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

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

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

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

Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction

Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. This study shows that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR were used to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1 (RhoGAP100F), neurexin-1, futsch, highwire, discs large, and alpha-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. These results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. It is proposed that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function (McDermott, 2014).

Localized translation is a widespread and evolutionarily ancient strategy used to temporally and spatially restrict specific proteins to their site of function and has been extensively studied during early development and in polarized cells in a variety of model systems. It is thought to be of particular importance in the regulation of neuronal development and in the plastic changes at neuronal synapses that underlie memory and learning, allowing rapid local changes in gene expression to occur independently of new transcriptional programs. The Drosophila neuromuscular junction (NMJ) is an excellent model system for studying the general molecular principles of the regulation of synaptic development and plasticity. Genetic or activity-based manipulations of synaptic translation at the NMJ has previously been shown to affect the morphological and electrophysiological plasticity of NMJ synapses. However, neither the mRNA targets nor the molecular mechanism by which such translational regulation occurs are fully understood (McDermott, 2014).

Previously work identified CG17838, the fly homolog of the mammalian RNA binding protein SYNCRIP/hnRNPQ, which was named Syncrip (Syp). Mammalian SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that contain CamKIIα, Arc, and IP3R1 mRNAs and is thought to regulate translation via an interaction with the noncoding RNA BC200/BC1, itself a translational repressor. Moreover, SYNCRIP/hnRNPQ competes with poly(A) binding proteins to inhibit translation in vitro and regulates dendritic morphology (Chen, 2012) via association with, and localization of, mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex. Drosophila Syp has a domain structure similar to its mammalian homolog, containing RRM RNA binding domains and nuclear localization signal(s), as well as encoding a number of protein isoforms. It was previously shown that Syp binds specifically to the gurken (grk) mRNA localization signal together with a number of factors previously shown to be required for grk mRNA localization and translational regulation (McDermott, 2012). Furthermore, syp loss-of-function alleles lead to patterning defects indicating that syp is required for grk and oskar (osk) mRNA localization and translational regulation in the Drosophila oocyte (McDermott, 2014).

This study shows that Syp is detected in the Drosophila third instar larval muscle nuclei and also postsynaptically at the NMJ. Syp is required for proper synaptic morphology at the NMJ, as syp loss-of-function mutants show a synaptic overgrowth phenotype, while overexpression of Syp in the muscle can suppress NMJ growth. Syp protein associates with a number of mRNAs encoding proteins with key roles in synaptic growth and function including, msp-300, syd-1, neurexin-1 (nrx-1), futsch, highwire (hiw), discs large 1 (dlg1), and α-spectrin (α-spec). The protein levels of a number of these mRNA targets, including msp-300 and dlg1, are significantly affected in syp null mutants. Furthermore, in addition to regulating MSP-300 protein levels, Syp is required for correct MSP-300 protein localization, and syp null mutants have defects in myonuclear distribution and morphology that resemble those observed in msp-300 mutants. It is proposed that Syp coordinates the protein levels from a number of transcripts with key roles in synaptic growth and is a mediator of synaptic morphology and growth at the Drosophila NMJ (McDermott, 2014).

The results demonstrate that Syp is required for the appropriate branching of the motoneurons and the number of synapses they make at the muscle. These observations are potentially explained by the finding that Syp is also required for the correct level of expression of msp-300, dlg1 and other mRNA targets. Given that it was previously shown that Syp regulates mRNA localization and localized translation in the Drosophila oocyte, and studies by others have shown that mammalian SYNCRIP/hnRNPQ inhibits translation initiation by competitively binding poly(A) sequences (Svitkin, 2013), these functions of Syp as occurring at the level of translational regulation of the mRNAs to which Syp binds. Our data are also consistent with other work in mammals showing that SYNCRIP/hnRNPQ is a component of neuronal RNA transport granulesthat can regulate dendritic morphology via the localized expression of mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex (McDermott, 2014 and references therein).

Translation at the Drosophila NMJ is thought to provide a mechanism for the rapid assembly of synaptic components and synaptic growth during larval development, in response to rapid increases in the surface area of body wall muscles or in response to changes in larval locomotion. The phenotypes observed in this study resemble, and are comparable to, those seen when subsynaptic translation is altered genetically or by increased locomotor activity. In syp null mutants, NMJ synaptic terminals are overgrown, containing more branches and synaptic boutons. Similarly, bouton numbers are increased by knocking down Syp in the muscle using RNAi. In contrast, overexpression of Syp in the muscle has the opposite phenotype, resulting in an inhibition of synaptic growth and branching. Furthermore, expressing RNAi against syp in motoneurons alone does not result in a change in NMJ morphology, indicating that Syp acts postsynaptically in muscle, but not presynaptically at the NMJ to regulate morphology. Interestingly, pan-neuronal syp knockdown or overexpression using Elav-GAL4 also results in NMJ growth defects, revealing that some of the defects observed in the syp null mutant may be attributed to Syp function in neuronal cell types other than the motoneurons, such as glial cells, which are known to influence NMJ morphology. Finally, while Syp is not required in the motoneuron to regulate synapse growth and is not detected in the motoneuron, the possibility cannot be excluded that Syp is present at low levels in the presynapse and regulates processes independent of synapse morphology. A further detailed characterization of the cell types and developmental stages in which Syp is expressed and functions is required to better understand the complex phenotypes that were observe (McDermott, 2014).

RNA binding proteins have emerged as critical regulators of both neuronal morphology and synaptic transmision, suggesting that protein production modulates synapse efficacy. Consistent with this, it has been shown in a parallel study that Syp is required for proper synaptic transmission and vesicle release and regulates the presynapse through expression of retrograde Bone Morphogenesis Protein (BMP) signals in the postsynapse. In this role, Syp may coordinate postsynaptic translation with presynaptic neurotransmitter release. These observations provide a good explanation for how Syp influences the presynapse despite being only detectable in the postsynapse. This study has shown that Syp associates with a large number of mRNAs within third instar larvae, many of which encode proteins with key roles in synaptic growth and function. Syp mRNA targets include msp-300, syd-1, nrx-1, futsch, hiw, dlg1, and α-spec. Syp negatively regulates Syd-1, Hiw, and DLG protein levels in the larval body wall but positively regulates MSP-300 and Nrx-1 protein levels. Dysregulation of these multiple mRNA targets likely accounts for the phenotypes that were observed. Postsynaptically expressed targets with key synaptic roles that could explain the synaptic phenotypes that were observed in syp alleles include MSP-300, α-Spec, and DLG. For example, mutants in dlg1 and mutants where postsynaptic DLG is destabilized or delocalized have NMJ morphology phenotypes similar to those observed upon overexpression of Syp in the muscle. Presynaptically expressed targets include syd-1, nrx-1, and hiw. However, this study has shown that syp knockdown in presynaptic motoneurons does not result in any defects in NMJ morphology. The RIP-Seq experiments were carried out using whole larvae and will, therefore, identify Syp targets in a range of different tissues and cells, the regulation of which may or may not contribute to the phenotype that were observed in syp mutants. It is, therefore, possible that Syp associates with these presynaptic targets in other neuronal cell types such as the DA neurons of the larval peripheral nervous system. It is also possible that Nrx-1 or Hiw are expressed and required postsynaptically in the muscle, but this has not been definitively determined. syp alleles may provide useful tools to examine where key synaptic genes are expressed and how they are regulated (McDermott, 2014).

The identity of localized mRNAs and the mechanism of localized translation at the NMJ are major outstanding questions in the field. To date, studies have shown that GluRIIA mRNA aggregates are distributed throughout the muscle. The Syp targets identified in this study, such as msp-300, hiw, nrx-1, α-spec, and dlg1, are now excellent candidates for localized expression at the NMJ. Ultimately, conclusive demonstration of localized translation will involve the visualization of new protein synthesis of targets during activity-dependent synaptic plasticity. Biochemical experiments will also be required to establish the precise mode of binding of Syp to its downstream mRNA targets, the basis for interaction specificity, and the molecular mechanism by which Syp differentially regulates the protein levels of its mRNA targets at the Drosophila NMJ. Despite the fact that mammalian SYNCRIP is known to associate with poly(A) tails, this study and other published work have revealed that Syp can associate with specific transcripts. How Syp associates with specific mRNAs is unknown, and future studies are needed to uncover whether the interaction of Syp with specific transcripts is dictated by direct binding of the three Syp RRM RNA binding domains or by binding to other specific mRNA binding proteins. It is also possible that specific mRNA stem–loops, similar to the gurken localization signal, are required for Syp to bind to its mRNA targets (McDermott, 2014).

This study shows that msp-300 (also known as Nesprin) is the most significant mRNA target of Syp. MSP-300 is the Drosophila ortholog of human Nesprin proteins. These proteins have been genetically implicated in various human myopathies. For example, Nesprin/Syne-1 or Nesprin/Syne-2 is associated with Emery-Dreifuss muscular dystrophy (EDMD) as well as severe cardiomyopathies. Moreover, Syp itself is increasingly linked with factors and targets that can cause human neurodegenerative disorders. Recent work has revealed that SYNCRIP/hnRNPQ and Fragile X mental retardation protein (FMRP) are present in the same mRNP granule, and loss of expression of FMRP or the ability of FMRP to interact with mRNA and polysomes can cause cases of Fragile X syndrome. Separate studies have also shown that SYNCRIP interacts with wild-type survival of motor neuron (SMN) protein but not the truncated or mutant forms found to cause spinal muscular atrophy, and Syp genetically interacts with Smn mutations in vivo. Understanding Syp function in the regulation of such diverse and complex targets may, therefore, provide new avenues for understanding the molecular basis of complex disease phenotypes and potentially lead to future therapeutic approaches (McDermott, 2014).

Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of Amyotrophic Lateral Sclerosis

TDP-43 is an RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) that is known to regulate the splicing, transport, and storage of specific mRNAs into stress granules. Although TDP-43 has been shown to interact with translation factors, its role in protein synthesis remains unclear, and no in vivo translation targets have been reported to date. This study provides evidence that Drosophila TDP-43 associates with futsch mRNA in a complex and regulates its expression at the neuromuscular junction (NMJ) in Drosophila. In the context of TDP-43-induced proteinopathy, there is a significant reduction of futsch mRNA at the NMJ compared with motor neuron cell bodies where higher levels of transcript were found compared with controls. TDP-43 also leads to a significant reduction in Futsch protein expression at the NMJ. Polysome fractionations coupled with quantitative PCR experiments indicate that TDP-43 leads to a futsch mRNA shift from actively translating polysomes to nontranslating ribonuclear protein particles, suggesting that in addition to its effect on localization, TDP-43 also regulates the translation of futsch mRNA. futsch overexpression was shown to be neuroprotective by extending life span, reducing TDP-43 aggregation, and suppressing ALS-like locomotor dysfunction as well as NMJ abnormalities linked to microtubule and synaptic stabilization. Furthermore, the localization of MAP1B, the mammalian homolog of Futsch, is altered in ALS spinal cords in a manner similar to these observations in Drosophila motor neurons. Together, these results suggest a microtubule-dependent mechanism in motor neuron disease caused by TDP-43-dependent alterations in futsch mRNA localization and translation in vivo (Coyne, 2014).

TDP-43, an RNA-binding protein linked to a significant fraction of ALS cases, associates with futsch mRNA in a complex in vivo and regulates its localization and translation in Drosophila motor neurons. Using polysome fractionations, wild-type and disease-associated mutant TDP-43 was shown to cofractionate with both the untranslated fractions, namely RNPs and ribosomal subunits, and actively translating polyribosomes. These results add translation regulation to TDP-43's plethora of known roles in RNA processing, such as transcription, splicing, and mRNA transport, and suggest that TDP-43 contributes to the pathophysiology of ALS via multiple RNA-based mechanisms (Coyne, 2014).

These data provide the first in vivo demonstration that TDP-43 associates with polysomes and regulates the translation of futsch mRNA. These findings are consistent with previous reports that futsch mRNA coimmunoprecipitates with Drosophila TBPH, and the mammalian homolog, MAP1B, is a candidate target of TDP-43 in mouse. The results showing a decrease in Futsch levels at the NMJ and an increase in Futsch levels in motor neuron cell bodies suggest a model whereby futsch/MAP1B mRNA may not be properly transported into axons. This is substantiated by qPCR from ventral ganglia where futsch mRNA is found at higher levels than at the NMJ compared with controls. Although it cannot be excluded that TDP-43 regulates futschmRNA stability, given the more pronounced reduction in protein versus transcript levels at the NMJ compared with cell bodies and the shift to untranslated fractions in polysomes, these data suggest TDP-43-dependent defects in futsch/MAP1B mRNA transport and protein expression at the NMJ. Although the underlying cause remains unknown, Futsch protein levels are also reduced at the NMJ in the absence of TBPH (the Drosophila homolog of TDP-43), suggesting a loss-of-function mechanism for the disease. Interestingly, there is a marked increased in futsch mRNA in motor neuron cell bodies in ventral ganglia relative to Futsch protein levels. This suggests that TDP-43 also regulates the expression of Futsch in motor neuron cell bodies possibly through sequestration into RNP complexes. Although cytoplasmic TDP-43 puncta were not seen in vivo, a plausible scenario is that TDP-43-containing complexes are below the limit of resolution of the confocal microscope, as suggested by cellular fractionation experiments indicating the presence of cytoplasmic TDP-43. Since futsch is the Drosophila homolog of MAP1B and MAP1B mRNA has been identified in TDP-43-containing RNP complexes in mouse models, these results predict that MAP1B and microtubule-based processes may also be affected in ALS patient tissues. Indeed, similar to the current results in the fly, immunohistochemistry experiments reveal a significant accumulation of MAP1B in motor neuron cell bodies in ALS spinal cords compared with controls but not in the hippocampus. Although these alterations may be the result of ongoing neurodegeneration, the remarkable similarities with the fly model suggest that comparable defects in transport and translation processes may occur in the human disease. It is interesting to note that spinal cord motor neurons in the patient with the C9ORF72 repeat expansion failed to contain MAP1B immunostaining in the cell body, suggesting altered pathogenic mechanisms from sporadic ALS patients, though further studies with an increased number of patients within this repeat expansion are required to confirm this finding (Coyne, 2014).

Interestingly, Futsch protein expression is similarly inhibited by wild-type or mutant TDP-43, supporting a scenario in which MAP1B dysregulation may be a shared feature of ALS cases with TDP-43-positive pathology, regardless of etiology. It is possible that other targets, which remain to be identified, are regulated in a variant-dependent manner. Indeed, RNA sequencing experiments from wild-type and mutant polysome fractions indicate several distinct, up- and down-translated mRNAs. Perhaps TDP-43's role in translation is not surprising as it has previously been shown to associate with stress granules (SGs), which are known to sequester mRNAs and inhibit their translation during environmental stress. Although TDP-43 does not seem to be required for SG formation, ALS-linked mutations in TDP-43 were shown to alter the dynamics of SG assembly and disassembly. This is consistent with previous findings that the molecular mobility of wild-type TDP-43 differs from that of the mutant variants in primary motor neurons. Furthermore, TDP-43-containing cytoplasmic aggregates can 'evolve' from paraquat-induced SG. Together, these findings suggest a scenario whereby, in response to stress, possibly caused by aging or environmental factors, TDP-43 localizes to cytoplasmic SGs that in turn lead to altered ribostasis, including abnormal futsch mRNA expression as demonstrated by these experiments (Coyne, 2014).

Using genetic interaction approaches, this study shows that futsch is a physiologically significant RNA target of TDP-43 and can alleviate locomotor dysfunction and increase life span. At the structural level, Futsch protein shares homology with mammalian MAP1B as well as neurofilaments, which do not exist per se in Drosophila. Interestingly, TDP-43 was shown to bind, transport, and sequester neurofilament light (NEFL) mRNA into SG, which contributes to ALS-like phenotypes in motor neurons. Given Futsch's known requirement in axonal and dendritic development and the organization of microtubules at the synapse, the current findings suggest that these processes may be involved in the pathophysiology of ALS. Indeed, recent studies in SOD1- and TDP-43-based models of ALS demonstrate an impairment in microtubule-based axonal transport (Coyne, 2014).

Consistent with previous studies in which tubulin acetylation has been shown to rescue transport defects in neurodegeneration, these data show that TDP-43 leads to reduced levels of acetylated tubulin, and this is rescued by futsch overexpression. Other TDP-43 targets such as HDAC6, which is regulated by TDP-43 at the level of transcription, are also linked to microtubule stability, providing additional support to the notion that microtubule stability is an important factor mediating TDP-43 toxicity. It is possible that microtubule stability is regulated locally by an interplay between Futsch and HDAC6 at the NMJ. Additionally, microtubule integrity and stress granules have been intricately linked. It has been demonstrated that microtubule integrity is important for the transportation and disassembly of stress granules. Also, SGs can be cleared by autophagy, and microtubules are used to transport autophagosomes to the microtubule organizing center where they fuse with lysosomes. Notably, microtubule stability can promote transport by association of motors that preferentially bind acetylated microtubules. Futsch is often associated with the stabilization of microtubules at the synapse through the formation of loop structures as this study has seen in the context of TDP-43. Therefore, restoration of Futsch levels and microtubule stability may mitigate TDP-43 toxicity through multiple mechanisms, including restoring transport of target mRNAs essential for synaptic stability and transport, disassembly, and/or clearance of stress granules, which in turn may interfere with aggregate formation as suggested by the observed decrease in insoluble TDP-43. In conclusion, futsch has been identified as a disease-relevant and functionally significant post-transcriptional target of TDP-43. Given the role of futsch/MAP1B in microtubule and synaptic stabilization, the current findings point to microtubule-based processes as targets for the development of therapeutic strategies for TDP-43 proteinopathies (Coyne, 2014).

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

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

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

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

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

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

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

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

Protein Interactions

LRRK2 kinase regulates synaptic morphology through distinct substrates at the presynaptic and postsynaptic compartments of the Drosophila neuromuscular junction

Mutations in leucine-rich repeat kinase 2 (LRRK2) are linked to familial as well as sporadic forms of Parkinson's disease (PD), a neurodegenerative disease characterized by dysfunction and degeneration of dopaminergic and other types of neurons. The molecular and cellular mechanisms underlying LRRK2 action remain poorly defined. This study shows that LRRK2 controls synaptic morphogenesis at the Drosophila neuromuscular junction. Loss of Drosophila LRRK2 results in synaptic overgrowth, whereas overexpression of Drosophila LRRK or human LRRK2 has opposite effects. Alteration of LRRK2 activity also affects neurotransmission. LRRK2 exerts its effects on synaptic morphology by interacting with distinct downstream effectors at the presynaptic and postsynaptic compartments. At the postsynapse, LRRK2 interacts with the previously characterized substrate 4E-BP (Imai, 2008), an inhibitor of protein synthesis. At the presynapse, LRRK2 phosphorylates and negatively regulates the microtubule (MT)-binding protein Futsch. These results implicate synaptic dysfunction caused by deregulated protein synthesis and aberrant MT dynamics in LRRK2 pathogenesis and offer a new paradigm for understanding and ultimately treating PD (Lee, 2010).

Parkinson's disease (PD) is one of the most common neurodegenerative diseases and is characterized by locomotor abnormalities as a result of the dysfunction and eventual loss of dopaminergic (DA) neurons. Most PD cases are sporadic with no known cause. Recent advances in PD genetics have led to the identification of familial PD (FPD) genes. It is anticipated that understanding the disease mechanisms of the FPD cases will provide insights into PD pathogenesis in general. Despite intensive studies of the FPD gene products at the biochemical and cell biological levels, understanding of their physiological function and the molecular and cellular pathways underlying disease pathogenesis is still fragmentary. Of all FPD genes, leucine-rich repeat kinase 2 (LRRK2) is the most frequently mutated. LRRK2 encodes a large serine/threonine kinase with multiple other domains. Some pathogenic mutations in LRRK2, such as the I2020T and G2019S substitutions in the kinase domain and R1441C substitution in the ROC domain, appear to augment kinase activity. In Drosophila and mouse models, pathogenic human (hLRRK2) or Drosophila (dLRRK) LRRK2 induce parkinsonian phenotypes in an age-dependent manner. A number of LRRK2-interacting proteins and substrates have been identified through in vitro studies, which implicate diverse biological functions for LRRK2 in translational control, vesicular trafficking, and cytoskeletal regulation. The physiological relevance of these interacting proteins and substrates remain to be established (Lee, 2010).

Actin and microtubule (MT) cytoskeleton dynamics play a crucial role in the formation of the nervous system, regulating such fundamental processes as axonal guidance and synaptogenesis. Dynamic modulation of synaptic structure and function is fundamental to neural network formation during development and is the molecular basis of learning and memory. Synaptic dysfunction is tightly linked to the pathogenesis of major neurodegenerative diseases such as Alzheimer's disease, and its role in PD is beginning to be appreciated. In Drosophila, the MT-associated protein 1B (MAP1B) homolog Futsch is required for axonal and dendritic growth during embryogenesis and for synaptic morphogenesis during larval neuromuscular junction (NMJ) development. This study shows that dLRRK phosphorylates and negatively regulates Futsch function at the presynapse. The previously characterized dLRRK substrate 4E-BP functionally interacts with LRRK2 at the postsynapse. These results implicate defects in presynaptic MT cytoskeleton dynamics and postsynaptic protein synthesis in LRRK2 pathogenesis (Lee, 2010).

Genetic mutations in LRRK2 are frequently found in familial and sporadic PD cases. Understanding the physiological function of LRRK2 will therefore offer insights into PD pathogenesis in general. This study reveals a new physiological function of LRRK2 and offers molecular mechanisms underlying such function. The key findings from this study are that LRRK2 plays an important role in regulating synaptic morphogenesis and that it does so through distinct substrate proteins at the presynaptic and postsynaptic compartments. The results also show that the precise level of LRRK2 activity is important for synaptic morphogenesis and neurotransmission, but the regulation of these two synaptic processes likely involve different mechanisms and players. Given the similarity of Drosophila NMJ synapse to mammalian excitatory glutamatergic synapses, it is possible that the findings reported here are relevant to mammalian systems (Lee, 2010).

Synaptic loss is a major neurobiological substrate of cognitive dysfunction in a number of neurological diseases. Extensive studies in patients and animal models have documented that synaptic failure is one of the earliest events in the pathogenesis of Alzheimer's disease. Interestingly, neurotransmission defects have been repeatedly observed in rodent FPD models, including the LRRK2 model, although no obvious signs of neurodegeneration accompany the electrophysiological defects. These results suggest that synaptic dysfunction is a primary effect of FPD gene mutations and that synaptic failure is intimately involved in PD pathogenesis. The molecular mechanisms underlying these synaptic transmission defects, however, remain elusive. This study of the LOF and GOF models of LRRK2 in Drosophila provides mechanistic insights into the possible cause of synaptic dysfunction in LRRK2-associated PD. It was found that LRRK2 regulates synaptic morphogenesis at the presynaptic and postsynaptic compartments through distinct substrates (Lee, 2010).

In the presynaptic side, LRRK2 forms a complex with tubulin and the MT-binding protein Futsch. Furthermore, LRRK2 phosphorylates Futsch and negatively regulates the presynaptic function of Futsch in controlling MT dynamics. MT cytoskeleton is critical for the generation and maintenance of neuronal axons and dendrites, transport of synaptic vesicles and organelles along the processes, and the initiation and maintenance of synaptic transmission. Disrupted MT dynamics in neuronal synapses has been implicated as an underlying cause for several neurological diseases, including hereditary spastic paraplegia and fragile X syndrome. LRRK2-associated PD may share this feature with the aforementioned diseases. Disrupted MT dynamics could be responsible for the presynaptic effects observed in LRRK2 LOF and GOF mutants, such as aberrant mitochondria distribution. The synaptic vesicle transport phenotypes seen in Caenorhabditis elegans LRK-1 mutant could also be attributable to altered MT dynamics. These could all contribute to synaptic dysfunction. Futsch/MAP1B proteins are large multidomain proteins that are phosphorylated by multiple kinases, including Sgg/GSK-3β, PAR-1/MARK, and Cdk5, which also phosphorylate tau and are implicated in tau pathology. Tau-related pathology has been observed in LRRK2 transgenic animals. It would be interesting to test for possible interplay between LRRK2 and these other kinases in regulating MT-binding proteins and MT dynamics. In mammalian hippocampal neurons, overexpression of pathogenic hLRRK2 led to reduced neurite length and branching, whereas deficiency of LRRK2 had opposite effects. Whether MT dynamics regulated by Futsch/MAP1B is contributing to this LRRK2 effect in mammals will await additional investigation (Lee, 2010).

This study also showed that LRRK2 interacts with 4E-BP at the postsynapse. 4E-BP acts as a negative regulator of the translational initiation factor eIF4E through direct binding and sequestration. Phosphorylation of 4E-BP by LRRK2 weakens 4E-BP binding to eIF4E (Imai, 2008), therefore releasing the inhibitory effect of 4E-BP on eIF4E. Previous studies have demonstrated an important postsynaptic role for eIF4E-mediated protein synthesis in activity-dependent synaptic growth at the Drosophila NMJ (Sigrist, 2000). Genetic interaction studies demonstrate a functional role for 4E-BP in mediating the synaptic effects of LRRK2. However, the exact roles of 4E-BP and LRRK2 in this process appear complex. For example, (1) despite the prominent effects of 4E-BP overexpression on NMJ development, its loss of function has no obvious effect. One would expect a gain of eIF4E function in the absence of 4E-BP and therefore a synaptic-overgrowth phenotype. (2) 4E-BP activity is predicted to be high in dLRRK mutant and low in LRRK2 overexpression condition, but this study observed synapse phenotypes opposite of what is predicted based on the presumed roles of eIF4E and 4E-BP on Drosophila NMJ morphogenesis. One possible explanation of these seemingly disparate results is that phospho-4E-BP, the product of LRRK2-mediated phosphorylation of 4E-BP, instead of being inactive and inert, may actually perform some new synaptic function at the NMJ. In this scenario, loss of 4E-BP function in d4E-BP mutant would not show the same phenotype as LRRK2 overexpression, which produces more phospho-4E-BP. Recent studies in Drosophila dopaminergic neurons suggest a functional role for phospho-4E-BP in vivo. Alternately, effectors other than 4E-BP may also mediate the effects of LRRK2 on NMJ development and neurotransmission. Although 4E-BP overexpression might have masked the effects of these other effectors, in dLRRK mutant background, the functional roles of these other effectors might manifest themselves. A similar situation was observed in pumillo mutant, in which a synapse-loss phenotype was observed despite the upregulation of eIF4E activity in this mutant attributable to the derepression of translational inhibition of eIF4E, which would have resulted in a predicted synapse-overgrowth phenotype. Involvement of other effectors in mediating the effects of LRRK2 on NMJ development and neurotransmission, and possibly different effectors for NMJ development versus neurotransmission, could also explain the complex electrophysiological phenotypes of dLRRK mutant and LRRK2 overexpression animals, as well as the uncoupling of the effects on synaptic differentiation and neurotransmission by the various genetic manipulations. Future studies will test these possibilities as well as the relevance of the NMJ studies to dopaminergic neuron synapses (Lee, 2010).

In addition to LRRK2, the TOR pathway also regulates 4E-BP function through phosphorylation. This pathway primarily regulates cell and organism growth through diverse outputs, including protein synthesis, cytoskeletal change, autophagy, and cell survival. This study found that treatment of flies with rapamycin, an inhibitor of TOR, has the same effects as 4E-BP overexpression in wild type as well as LRRK2 overexpression backgrounds. Although rapamycin has been extensively studied in the context of autophagy induction and neurodegeneration models, its effect on Drosophila NMJ development is unlikely attributable to autophagy, because the current results show that presynaptic or postsynaptic induction of autophagy through Atg1 overexpression had no obvious effect on synapse number. The similar effects of rapamycin and 4E-BP overexpression on NMJ development support that rapamycin acts via the 4E-BP translational control pathway to impact NMJ development. A recent report showed that either 4E-BP overexpression or 4E-BP activation by rapamycin could suppress the muscle and dopaminergic neurodegeneration phenotypes seen in Drosophila Pink1 and Parkin models of PD. These results suggest that deregulation of protein synthesis could be generally involved in PD pathogenesis and that rapamycin or its analogs could be developed into effective PD therapeutics (Lee, 2010).


In Drosophila, mAb 22C10 has been widely used to label neuronal cells within the PNS and CNS. In all stages of development, 22C10 antigen expression starts in postmitotic neurons shortly before axonogenesis is initiated. In the embryonic nervous system, first mAb 22C10 reactivity is detected in the MP2 neurons. During subsequent development, several interneurons and motoneurons express the 22C10 antigen. Within the developing PNS, neuronal cells express the 22C10 antigen from early stages of neuronal differentiation onward. It is important to note that the 22C10 epitope is found in the growing axon, the cell body, and the dendrite. At the end of embryogenesis, 22C10 expression can be found in all sensory neurons. During late larval development, expression is found in all photoreceptor neurons. In addition to expression in the nervous system, some 22C10 antigen can be detected in the muscle attachment sites of the ventrolateral musculature and the tip cell of the Malphigian tubules. By using confocal microscopy, the 22C10 epitope has been localized in the central axonal compartment of larval neurons associated with the microtubule cytoskeleton (Hummel, 2000 and references therein).

The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. At Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1B-related protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, Baz and Par-6 are also expressed at synapses and their synaptic localization depends on aPKC activity. These findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory (Ruiz-Canada, 2004).

During expansion of the NMJ, parent boutons located at the distal end of a branch give rise to new synaptic boutons by budding. New buds separate from parent boutons by the formation of a neck, and NMJ branches extend by neck elongation and bouton enlargement. Throughout this process, the postsynaptic membrane and underlying cytoskeleton impose a barrier to presynaptic extension, since synaptic boutons and their buds are completely surrounded by the muscle cell membrane and underlying cytoskeleton. During branch elongation, a presynaptic signal may induce the retraction of the postsynaptic cytoskeleton barrier. It is proposed that changes in both the pre- and postsynaptic cytoskeleton during branch elongation mediate these events and that these processes are regulated by aPKC with the collaboration of Baz and Par-6 in both locales (Ruiz-Canada, 2004).

The localization of aPKC to MT-rich domains at the NMJ and the marked reduction in new synaptic bouton formation observed in dapkc mutants prompted an investigation of the cytoskeletal changes during branch elongation. At the presynaptic arbor of wild-type larvae, NMJ expansion is accompanied by the presence of unbundled MTs at the distal tip of NMJ branches and by little if any colocalization between MTs and the MAP1B-related protein Futsch. In vertebrates, MAP1B protects MTs from depolymerization. Similarly in Drosophila, MT integrity in axons and at the NMJ is preserved by Futsch; severe mutations in futsch result in MT fragmentation. This Futsch-dependent protection of MT integrity at presynaptic arbors has an important role during NMJ expansion, since MT fragmentation in futsch and dapkc mutants results in marked reduction in bouton number (Ruiz-Canada, 2004).

In epithelial cells and growth cones, an increase in aPKC activity enhances MT lifetime, although the involvement of MT-associated proteins in this process has not been investigated. The results suggest that aPKC activity enhances MT stability in a process that depends on Futsch. (1) Coimmunoprecipitation experiments indicate that aPKC, Tubulin, and Futsch exist in the same biochemical complex and that the interaction between aPKC and MTs is likely to be through Futsch. (2) Loss of dapkc function results in fragmented MTs at terminal boutons, and an increase in aPKC activity by pre-PKM (PKM is a persistantly active kinase) results in longer than normal MTs and an increased colocalization between MTs and Futsch. (3) Presynaptic expression of PKM in a futsch mutant background results in MT fragmentation similar to futsch mutants alone, suggesting that PKM acts through Futsch to stabilize MTs (Ruiz-Canada, 2004).

Effects of Mutation or Deletion

Three futsch mutations lead to a pronounced reduction in the level of 22C10 antigen expression including futschM455, futschP28, and futschN94. The futschM455 allele leads to a uniform reduction in expression, while both futschP28 and futschN94 also lead to an abnormal subcellular distribution of Futsch. In futschP28, a granular expression in the cell body and dendrites is observed. Interestingly, in futschN94, there is diffuse staining within the nerve terminal that is not observed in wild type. This aspect of the phenotype has been analyzed in the visual system. In third instar wild-type photoreceptor cells, distinct 22C10-positive fibers extend from the cell body into the axon and dendrites, but no staining is observed in the nerve terminals. In futschN94, Futsch protein is distributed uniformly in the nerve terminal. The mislocalization of the Futsch protein can be clearly seen in the nerve terminals in the lamina where the photoreceptor axons of cells R1-R6 terminate as well as in the growth cones of photoreceptor cells R7 and R8 in the medulla. In addition, futschN94 mutant larvae show ectopic Futsch expression in the so-called optic lobe pioneer cells (Hummel, 2000).

In two mutations, futschK68 and futschP158, 22C10 antigen expression is eliminated. Homozygous futschK68 flies are viable, and overall neuronal development proceeds normally in the embryo. In the larvae, a reduced number of synaptic boutons visualized by the antibody nc46 is noted. In futschP158, the lethality and the absence of 22C10 expression could not be separated by recombination. In wild-type embryos, highest levels of mAb 22C10 reactivity are seen in all PNS neurons. Futsch is prominently expressed in the dendrites of the five lateral chordotonal organs. To analyze the morphology of the PNS neurons in mutant futschP158 embryos, the GAL4 driver line P0163 was used to express the transmembrane protein CD2 specifically in PNS neurons, thereby visualizing their axonal and dendritic morphology. In mutant futschP158 larvae, about 97% of the PNS neurons fail to develop their normal dendritic morphology. Occasionally, however, dendritic processes are detected. Axonal growth is impaired as well, and few axons grew normally into the CNS. Axonal pathfinding is not affected, and axons appeared to simply stop growing after some distance.

Within the ventral nerve cord, Futsch is expressed in many inter- and motor-neurons that also express the Fasciclin II protein (Fas II). In stage 16 futschP158 mutant embryos, the longitudinal connectives are reduced. Similarly, disruptions are observed in the motoneuronal connections. In particular, SNb nerves fail to reach muscle fibers 12 and 13. Motoneurons stall and fail to set up the correct innervation pattern in 89% of the segments. To determine the null phenotype of futsch, the CNS defects associated with the deficiencies Df(1)A94 and Df(1)S39 that remove the futsch locus were analyzed. In both cases, a nervous system phenotype very similar to the futschP158 mutant phenotype was observed. Thus, P158 appears to be an amorphic mutation, whereas the K68 is a hypomorphic mutation of the futsch gene. Loss of futsch function leads to disruption of dendritic and axonal growth.


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

date revised: 15 November 2015

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