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Amyotrophic Lateral Sclerosis
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Drosophila genes associated with
Amyotrophic Lateral Sclerosis
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Neuromuscular junction
Relevant studies of ALS

Romano, G., Appocher, C., Scorzeto, M., Klima, R., Baralle, F.E., Megighian, A. and Feiguin, F. (2015). Glial TDP-43 regulates axon wrapping, GluRIIA clustering and fly motility by autonomous and non-autonomous mechanisms. Hum Mol Genet 24: 6134-6145. PubMed ID: 26276811

Alterations in the glial function of TDP-43 are becoming increasingly associated with the neurological symptoms observed in Amyotrophic Lateral Sclerosis (ALS), however, the physiological role of this protein in the glia or the mechanisms that may lead to neurodegeneration are unknown. To address these issues, this study modulated the expression levels of TDP-43 in the Drosophila glia and found that the protein was required to regulate the subcellular wrapping of motoneuron axons, promote synaptic growth and the formation of glutamate receptor clusters at the neuromuscular junctions. Interestingly, it was determined that the glutamate transporter EAAT1 mediates the regulatory functions of TDP-43 in the glia and that genetic or pharmacological compensations of EAAT1 activity is sufficient to modulate glutamate receptor clustering and locomotive behaviors in flies. The data uncovers autonomous and non-autonomous functions of TDP-43 in the glia and suggests new experimentally based therapeutic strategies in ALS (Romano, 2015).


  • Loss of endogenous glial TDP-43 protein (TBPH) provokes wrapping defects in motor axons.
  • The glial role of TBPH promotes synaptic growth and glutamate receptor clustering.
  • The formation of GluRIIA clusters is early affected by TBPH dysfunctions in glia.
  • dEAAT1 mediates GluRIIA clustering.

In addition to neurons, histological aberrations in the distribution of TDP-43 have also been observed in glial cells suggesting that these tissues might be associated with the neurodegenerative process present in ALS. In coincidence with this idea, this study found that the suppression of TDP-43 in the Drosophila glia provokes serious locomotive defects with functional alterations in synaptic transmission followed by early neurodegeneration and reduced life span but, without affecting the number or the survival rate of glial cells in vivo. More importantly, the acute reduction of TBPH function in the glia of adult flies is sufficient to initiate the typical locomotive problems observed in the disease, demonstrating that TBPH function is permanently required in these tissues to prevent neurodegeneration. Although, the Drosophila glia presents consistent molecular and cytological differences with the mammalian astrocytes, the functional characteristics of these tissues are well conserved. In agreement with these observations, it was found that the expression of the human TDP-43 in the Drosophila glia is sufficient to rescue the motility defects observed in TBPH minus larvae, revealing that the molecular role of this protein is well-preserved and analogous consequences could be expected in TDP-43 affected patients (Romano, 2015).

At the subcellular level, it was observed that the suppression of TBPH provokes evident defects in the anatomical organization of the peripheral glia with morphological alterations in the formation of the cytosolic projections that cover the synaptic surface of the presynaptic terminal axons that constitute the NMJs. Although, it is still unknown how these modifications may lead to neurodegeneration, the non-autonomous defects in the transmission of the evoked potentials described in presynaptic motoneurons coincides with analogous alterations found in different experimental models as well as in patients suffering of ALS, proposing that comparable modifications may autonomously initiate the neurological symptoms of the disease or induce the pathological mechanisms of neurodegeneration (Romano, 2015).

It was also observed that the glial function of TBPH is sufficient to rescue the molecular levels and the wild-type distribution of the GluRIIA clusters at the postsynaptic terminals of TBPH null L3 larvae. These molecular parameters were recovered together with the locomotive abilities both in larvae and adult flies, revealing that the glial function of TBPH may directly influence these genetic traits. In agreement with this view, it was found that the suppression of TBPH in the glia alters the formation of evoked synaptic potentials at the NMJ, without affecting the cycle of vesicle release in the presynaptic membranes. On the contrary, the anatomical distribution of the GluRIIA clusters present at the postsynaptic membranes is highly disturbed in the TBPH depleted glia, suggesting that electrophysiological problems originate from defects in the organization of the postsynaptic membranes. The genetic rescue experiments instead, reveal that the glial function of TBPH is necessary to promote the synaptic growth of the motoneurons terminal axons during larval development. This neurotrophic function of TBPH, nevertheless, is not sufficient to rescue the neuronal levels of the vesicular protein Syx. In addition, the distribution of the postsynaptic protein Dlg, whose localization largely depends on the presynaptic activity of the motoneurons, is not recovered after the activation of TBPH in the glia compared with identical genetic rescue experiments performed expressing TBPH in neurons (17), revealing tissue-specific differences in TBPH function and regulatory mechanisms. Finally, the genetic rescues generated through the late expression of TBPH in mature glia indicate that the pathological defects described in locomotive behaviors and postsynaptic distribution of the GluRIIA clusters are not permanent and can be regenerated (Romano, 2015).

Defects in the regulation of the intracellular levels of the glutamate transporter EAAT1 in the glia have been associated with several neurodegenerative diseases comprising ALS. Indeed, molecular data previously generated shows that EAAT1 and EAAT2 mRNA levels are largely modified in ALS patients and, moreover, these modifications are associated with the increased oxidative stress present in the affected cases. Recent studies performed in Drosophila have also identified that EAAT1 and EAAT2 messengers are modified in TBPH minus flies suggesting that these modifications may play a role in the mechanisms behind these phenotypes despite experimental evidences are not available. This study directly tested this hypothesis and uncovered that EAAT1 has an important role in the phenotypic mechanisms derived from the loss of TBPH function in the glia. The data also strongly suggest that the levels of the extracellular glutamate might be affected in TBPH null flies and responsible for the alterations in the localization of the GluRIIA at the postsynaptic membranes. These evidences, allow the hypothesis that the implementation of pharmacological treatments aimed to enhance the glutamate uptake or counteract the oxidative stress, produced by defects in its intracellular transport, may contribute to improve the symptoms observed in TBPH minus flies. In this direction, it was observed that nordihydroguaiaretic acid (NDGA) significantly improves the locomotive capacities of TBPH-RNAi treated larvae, demonstrating the role of this protein in the organization of the NMJs and proposing that analogous situations could be expected in human pathologies associated with TDP-43 dysfunctions like ALS or frontotemporal lobar degeneration (FTLD) (Romano, 2015).

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Coyne, A.N., Yamada, S.B., Siddegowda, B.B., Estes, P.S., Zaepfel, B.L., Johannesmeyer, J.S., Lockwood, D.B., Pham, L.T., Hart, M.P., Cassel, J.A., Freibaum, B., Boehringer, A.V., Taylor, J.P., Reitz, A.B., Gitler, A.D. and Zarnescu, D.C. (2015). Fragile X protein mitigates TDP-43 toxicity by remodeling RNA granules and restoring translation. Hum Mol Genet 24: 6886-6898. PubMed ID: 26385636

RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). This study identifies Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. It was found that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduces 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 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 OE also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP OE 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 in motor neurons (Coyne, 2015).


  • dFMRP is a potent modifier of TDP-43 neurotoxicity in vivo.
  • TDP-43 and dFMRP colocalize with PABP in neuronal RNA granules.
  • FMRP forms a complex with TDP-43 in vivo.
  • dFMRP modulates TDP-43 solubility and molecular mobility.
  • dFMRP OE restores the translation of futsch mRNA.
  • dFMRP OE restores Futsch dependent neuromuscular junction phenotypes.

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 that was observed in the context of dFMRP OE. 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).

It has been previously 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, 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. Taken together, these findings and published data suggest 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).

Results from this study 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. Results from this study support this model and provide evidence that mitigating translational repression can suppress disease phenotypes. 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 (Coyne, 2015).

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 OE alters RNP complexes in the TDP-43 model of ALS used in this study. Taken together, these data 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. Further, targeting RNP remodeling or translation restoration may prove useful as therapeutic strategies (Coyne, 2015).

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Zhang, K., Donnelly, C.J., Haeusler, A.R., Grima, J.C., Machamer, J.B., Steinwald, P., Daley, E.L., Miller, S.J., Cunningham, K.M., Vidensky, S., Gupta, S., Thomas, M.A., Hong, I., Chiu, S.L., Huganir, R.L., Ostrow, L.W., Matunis, M.J., Wang, J., Sattler, R., Lloyd, T.E. and Rothstein, J.D. (2015). The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525: 56-61. PubMed ID: 26308891

The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and protein interactors for the G4C2 RNA including RanGAP1 have been previously identified. This study performed a candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats which identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention (Zhang, 2015).


  • RanGAP suppresses HRE-mediated toxicity in Drosophila.
  • Nucleocytoplasmic transport modulates G4C2 toxicity.
  • G4C2 repeats bind RanGAP and cause NPC pathology.
  • The Ran gradient is disrupted by the C9orf72 HRE.
  • The C9orf72 HRE inhibits import of nuclear proteins.
  • Rescue of HRE-mediated neurodegeneration.

Data from this study demonstrates that the G4C2 repeat expansion disrupts nucleocytoplasmic transport in a fly model and in human cells. While it was found that RanGAP is a key target of the G4C2 repeat expansion, other members of the NPC may also interact directly or indirectly with G4C2. Several human genetic studies have implicated nuclear transport deficits as the cause of a rare fetal motor neuron disease and infrequent cases of ALS, including studies on the role of the nucleoporin GLE1 implicated in mRNA export. In addition, irregularities of the nuclear membrane and distribution of nuclear pore proteins were recently noted in sporadic ALS tissue. A recent study has independently identified additional components of the NPC and nucleocytoplasmic trafficking pathways as dominant modifiers of G4C2 HRE toxicity in another C9-ALS fly model. Importantly, the observed NPC and nucleocytoplasmic trafficking defects in both iPS-cell-derived neurons and motor neurons in this study are relevant to both ALS and FTD. Taken together, these studies suggest that products of the C9orf72 HRE disrupt nucleocytoplasmic transport at the NPC and are a fundamental mechanism for inducing cellular injury in ALS and FTD. These defects may account for the nuclear depletion and cytoplasmic accumulation of TDP-43 widely seen in C9-ALS and FTD (Zhang, 2015).

Although the data only demonstrate a role for disruption of nuclear import in C9-ALS pathogenesis, the robust nuclear pore pathology that was detected suggests that both nuclear import and export may be affected. It is enticing to speculate that NPC dysfunction leads to age-related neurodegeneration, since many of the NPC components, including Nup205, are extremely long-lived, and NPC integrity is lost during normal ageing (Zhang, 2015).

The sense strand appears to be the cause of the described nucleocytoplasmic trafficking deficits in the human and fly model systems, as small molecules targeting the sense RNA suppress the nuclear import phenotypes, and neurodegeneration is caused by expression of G4C2 repeat RNA in C9orf72 iPSC neurons or Drosophila. While DPRs cannot be excluded as a contributor to nucleocytoplasmic trafficking defects, data in multiple model systems are most consistent with an RNA-mediated mechanism. Future studies will be required to determine the contribution of RanGAP disruption in C9-ALS pathogenesis compared with other pathogenic mechanisms implicated in C9-ALS such as nucleolar stress, which could act independently or in conjunction with nucleocytoplasmic transport disruption (Zhang, 2015).

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Machamer, J.B., Collins, S.E., Lloyd, T.E. (2014). The ALS gene FUS regulates synaptic transmission at the Drosophila neuromuscular junction. Hum Mol Genet. 23: 3810-3822. PubMed ID: 24569165

Mutations in the RNA binding protein Fused in sarcoma (FUS) are estimated to account for 5-10% of all inherited cases of amyotrophic lateral sclerosis (ALS), but the function of FUS in motor neurons is poorly understood. This study investigates the early functional consequences of overexpressing wild-type or ALS-associated mutant FUS proteins in Drosophila motor neurons, and compares them to phenotypes arising from loss of the Drosophila homolog of FUS, Cabeza (Caz). It is found that lethality and locomotor phenotypes correlate with levels of FUS transgene expression, indicating that toxicity in developing motor neurons is largely independent of ALS-linked mutations. At the neuromuscular junction (NMJ), overexpression of either wild-type or mutant FUS results in decreased number of presynaptic active zones and altered postsynaptic glutamate receptor subunit composition, coinciding with a reduction in synaptic transmission as a result of both reduced quantal size and quantal content. Interestingly, expression of human FUS downregulates endogenous Caz levels, demonstrating that FUS autoregulation occurs in motor neurons in vivo. However, loss of Caz from motor neurons increases synaptic transmission as a result of increased quantal size, suggesting that the loss of Caz in animals expressing FUS does not contribute to motor deficits. These data demonstrate that FUS/Caz regulates NMJ development and plays an evolutionarily conserved role in modulating the strength of synaptic transmission in motor neurons (Machamer, 2014).


  • Lethality and locomotor deficits correlate with FUS expression levels.
  • FUS overexpression depletes nuclear Caz.
  • FUS overexpression results in loss of presynaptic active zones and postsynaptic Discs large (Dlg).
  • FUS overexpression impairs synaptic transmission.
  • Presynaptic FUS overexpression disrupts postsynaptic glutamate receptor subunit composition.
  • Loss of Caz enhances synaptic transmission.

A previous study concluded that ALS-linked mutations in FUS resulted in a toxic gain-of-function in Drosophila, and this conclusion was based on the finding that the wild-type and mutant UAS-HA-FUS transgenic lines expressed equivalent levels of protein in the eye. However, this study's analysis of these same UAS-HA-FUS lines demonstrated that when expressed in motor neurons, message and protein expression were 3- to 4-fold higher in the R521C mutant line than the wild-type line. Thus, these results suggest that the increased severity of phenotypes seen in the mutant line relative to wild-type is due to increased expression level rather than mutation-specific toxicity. Therefore, evidence for a gain-of-function effect of ALS-associated mutations in FUS is not found in this model (Machamer, 2014).

There are two non-mutually exclusive explanations for these dramatic differences in relative transgene expression levels observed in different tissues. First, given that HA-FUSR521C overexpression in eyes caused degeneration, the simplest explanation is that HA-FUSR521C lines express at higher levels than wild-type in the eyes during development, and this leads to cell loss or dysfunction that causes a reduction in protein expression in the adult, whereas expression in glutamatergic neurons with OK371-GAL4 does not have these effects. Indeed, morphological analysis of GMR>FUSR521C fly eyes demonstrated severe cell loss. An alternative possibility is that tissue-specific enhancers may differentially regulate gene expression from P-elements located at different genomic loci, as these position effects are well known in Drosophila (Machamer, 2014).

These observations suggest two important guidelines for analyzing overexpression disease models in Drosophila. First, given the widely available technologies for site-specific transgenesis, comparisons of gain-of-function phenotypes between wild-type and mutant proteins should utilize transgenic lines inserted at identical genomic sites. Indeed, when the lines were generated in this manner (FL-FUS) and analyzed, identical expression levels between the wild-type and mutant proteins in motor neurons were observed. Second, when comparing protein expression levels between wild-type and mutant transgenic lines, expression levels should be compared in the absence of cell loss and in the tissue most relevant to the disease (Machamer, 2014).

Recent data from mammalian cell culture suggests that disruption of FUS autoregulation leading to overexpression of wild-type FUS is sufficient to cause disease. In this study, it was shown that wild-type or mutant FUS overexpression in Drosophila motor neurons lead to severe downregulation of fly FUS. This suggests that an autoregulatory mechanism is conserved in Drosophila and occurs in motor neurons in vivo. This autoregulation likely explains why in many cases, stronger phenotypes in higher-expressing transgenic lines were not seen (Machamer, 2014).

Whether disease-associated mutations in FUS and other ALS genes cause disease through a gain- or loss-of-function is a matter of debate. Simple genetic model systems such as Drosophila are ideal for interpreting the effect of mutations on protein function; however, one must be cautious in interpreting results gained from overexpression studies, particularly when overexpressing human proteins in the fly. However, because low-level expression of human FL-FUS in neurons rescued phenotypes caused by loss of Drosophila FUS (i.e. Caz), this strongly argues for evolutionary conservation of FUS as well as a requirement for FUS in neurons. Furthermore, because FUSP525L failed to rescue Caz phenotypes, this strongly argues that the P525L mutation causes a loss-of-function (Machamer, 2014).

Consistent with this interpretation, analyses of disease-associated mutations in FUS was found to be most consistent with a partial loss-of-function, given that (a) low-level FUSWT but not FUSP525L expression caused increased larval and adult locomotor activity, and (b) expressing higher level FUSR521C in some instances caused less severe phenotypes than lower level FUSWT expression (e.g. Caz downregulation), altered EJP rise time, and GluRIIA upregulation. Nonetheless, the possibility that FUS mutations do exhibit some gain-of-toxic effects could not be excluded, particularly given that there was a greater reduction in GluRIIB levels with mutant FUS expression than with wild-type expression. Since mutations in the 3'UTR autoregulatory domain are sufficient to cause ALS in patients by upregulating wild-type FUS protein, ALS may be caused by increased levels of wild-type protein. In this context, low-level overexpression of FUS or Caz in aging flies may be a reasonable way to model the disease (Machamer, 2014).

FUS has been implicated in a wide range of processes in many cell types both within the nucleus and cytoplasm. Since this study was unable to detect significant wild-type FUS or Caz protein within motor axons or at the NMJ, it was postulated that FUS/Caz normally functions in the nucleus to regulate the expression of genes that modulate synapse function. Importantly, FUS/Caz appeared to be required within neurons to regulate synaptic transmission, as caz1 loss-of-function phenotypes were mimicked by presynaptic caz knockdown, and overexpression of FUS in motor neurons lead to altered synaptic transmission (Machamer, 2014).

The study showed that FUS expression inhibited evoked release due to both a reduction of quantal size (mEJP amplitude) and quantal content (number of quanta released per stimulus). A reduction in mEJP amplitude could be due to a decrease in synaptic vesicle size, glutamate concentration or postsynaptic currents due to alterations in glutamate receptor levels, localization or composition. It was postulated that the reduction in quantal size was due to a disruption of the spatial coupling of synaptic vesicle release sites with glutamate receptors given the disruption in the number and morphology of active zones and the postsynaptic density. Importantly, reduced GluR levels were not responsible for the decrease in mEJP amplitude observed in FUS overexpressing animals, but rather that GluR clustering at active zones might be altered. Furthermore, there was an increase in relative expression of A-type GluRs which would be expected to have the opposite effect on mEJP amplitude. This increase in ratio of A- to B-type GluRs was seen when glutamate release was blocked at larval NMJs and was a homeostatic response to reduction in glutamate-mediated synaptic transmission (Machamer, 2014).

There was also a striking reduction in the number of active zones in FUS-expressing animals. These changes likely contributed to the reduction in quantal content, as bruchpilot mutations in Drosophila have reduced synaptic transmission due to a reduction in the readily releasable pool. The relatively subtle reduction in quantal content in FUS-expressing animals might be due to a homeostatic increase in the probability of vesicle fusion at any given active zone. Surprisingly, the reduction in active zone number was associated with a marked increase in the frequency of spontaneous release, suggesting that the remaining active zones had a marked increase in release probability. Consistent with this hypothesis, superresolution microscopic imaging of Brp demonstrated abnormal morphology of active zones in FUS-expressing animals (Machamer, 2014).

The results of this study contrast with those of a recent report analyzing larval NMJ physiology of HA-FUSR521C-expressing and caz1 animals using discontinuous single electrode voltage clamp recordings. This study did not observe a physiologic phenotype with FUSWT overexpression, and a reduction in evoked release in caz1 animals was observed; these findings were consistent with findings in zebrafish with overexpression of mutant FUS and with morpholino-mediated knockdown of wild-type FUS. Since FUS protein redistributes to cytoplasmic stress granules with various stressors, one possibility is that alterations in animal rearing or recording conditions may have large effects (Machamer, 2014).

The study also reported the occurrence of cell nonautonomous loss of Dlg from muscle postsynaptic compartments and alterations in GluRII subunit composition at the NMJ as a result of FUS overexpression in motor neurons. The mechanism of Dlg loss induced by FUS overexpression is unclear, and it is unknown whether the reduction in Dlg expression is accompanied by morphological alterations in the subsynaptic reticulum of muscle cells. Although the increase in the ratio of A- to B-type GluRs was consistent with homeostatic compensation for impaired synaptic transmission, Dlg is not known to be regulated by homeostatic feedback at the Drosophila NMJ. Thus, it is speculated that non-cell autonomous effects on Dlg are mediated through alterations in transsynaptic adhesion molecules. This notion was supported by the altered kinetics of EJP rise and decay times and disrupted apposition of GluRIIB/IIC with the active zone (Machamer, 2014).

Furthermore, several of the morphological and electrophysiological phenotypes caused by FUS overexpression overlapped with loss-of-function alleles of the neurexin (nrx1)/neuroligin (nlg1 and nlg2) transsynaptic adhesion complex in Drosophila. For example, both nrx1 and nlg1 animals showed expanded interbouton regions that lacked post-synaptic markers, a phenotype observed with FUS overexpression. nrx1 mutants showed decreased quantal content, increased mEJP frequency, and an increase in ratio of A to B-type glutamate receptors, phenotypes that were also observed with FUS overexpression (Machamer, 2014).

As the best described function of FUS is regulation of transcription and splicing, alterations in transcription and/or splicing may underlie the changes seen in synaptic function. Many cell adhesion molecules are highly alternatively spliced, and this splicing alters their function in synaptic development and differentiation. For example, mutations in beag alter splicing of fasciclin II (fasII), the Drosophila homologue of neural cell adhesion molecule (NCAM), and altered splicing leads to fewer synaptic boutons and decreased neurotransmitter release. Thus, this study hypothesizes that the changes in synaptic structure and function may be partially explained by altered expression and/or splicing of transsynaptic adhesion molecules (Machamer, 2014).

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Chou, C. C., Alexeeva, O. M., Yamada, S., Pribadi, A., Zhang, Y., Mo, B., Williams, K. R., Zarnescu, D. C. and Rossoll, W. (2015). PABPN1 suppresses TDP-43 toxicity in ALS disease models. Hum Mol Genet [Epub ahead of print]. PubMed ID: 26130692

TAR DNA-binding protein 43 (TDP-43; see Drosophila TDP-43) is a major disease protein in amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases. Both the cytoplasmic accumulation of toxic ubiquitinated and hyperphosphorylated TDP-43 fragments and the loss of normal TDP-43 from the nucleus may contribute to the disease progression by impairing normal RNA and protein homeostasis. Therefore, both the removal of pathological protein and the rescue of TDP-43 mislocalization may be critical for halting or reversing TDP-43 proteinopathies. This study reports poly(A)-binding protein nuclear 1 (PABPN1) as a novel TDP-43 interaction partner that acts as a potent suppressor of TDP-43 toxicity. Overexpression of full-length PABPN1 but not a truncated version lacking the nuclear localization signal protects from pathogenic TDP-43-mediated toxicity, promotes the degradation of pathological TDP-43 and restores normal solubility and nuclear localization of endogenous TDP-43. Reduced levels of PABPN1 enhances the phenotypes in several cell culture and Drosophila models of ALS and results in the cytoplasmic mislocalization of TDP-43. Moreover, PABPN1 rescues the dysregulated stress granule (SG) dynamics and facilitates the removal of persistent SGs in TDP-43-mediated disease conditions. These findings demonstrate a role for PABPN1 in rescuing several cytopathological features of TDP-43 proteinopathy by increasing the turnover of pathologic proteins (Chou, 2015).

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Hautbergue, G. M., et al. (2017). SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits. Nat Commun 8: 16063. PubMed ID: 28677678


Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. This study shows that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. it was further demonstrated that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, this study shows that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection (Hautbergue, 2017).

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M'Angale, P. G. and Staveley, B. E. (2017). A loss of Pdxk model of Parkinson disease in Drosophila can be suppressed by Buffy. BMC Res Notes 10(1): 205. PubMed ID: 28606139


The identification of a DNA variant in pyridoxal kinase (Pdxk) associated with increased risk to Parkinson disease (PD) gene has led to a study the inhibition of this gene in the Dopa decarboxylase (Ddc)-expressing neurons of Drosophila. The multitude of biological functions attributable to the vitamers of vitamin B6 catalysed by this kinase reveal an overabundance of possible links to PD, that include dopamine synthesis, antioxidant activity and mitochondrial function. Drosophila possesses a single homologue of Pdxk, and this study used RNAi to inhibit the activity of this kinase in the Ddc-Gal4-expressing neurons. Any association was further investigated between this enhanced disease risk gene with the established PD model induced by expression of alpha-synuclein in the same neurons. The pro-survival functions of Buffy, an anti-apoptotic Bcl-2 homologue, were relied on to rescue the Pdxk-induced phenotypes. Ddc-Gal4, which drives expression in both dopaminergic and serotonergic neurons, was used to drive the expression of Pdxk RNA interference in DA neurons of Drosophila. The inhibition of Pdxk in the alpha-synuclein-induced Drosophila model of PD did not alter longevity and climbing ability of these flies. It has been previously shown that deficiency in vitamers lead to mitochondrial dysfunction and neuronal decay, therefore, co-expression of Pdxk-RNAi with the sole pro-survival Bcl-2 homologue Buffy in the Ddc-Gal4-expressing neurons, resulted in increased survival and a restored climbing ability. In a similar manner, when Pdxk was inhibited in the developing eye using GMR-Gal4, it was found that there was a decrease in the number of ommatidia and the disruption of the ommatidial array was more pronounced. When Pdxk was inhibited with the alpha-synuclein-induced developmental eye defects, the eye phenotypes were unaltered. Interestingly co-expression with Buffy restored ommatidia number and decreased the severity of disruption of the ommatidial array. It is concluded that though Pdxk is not a confirmed Parkinson disease gene, the inhibition of this kinase recapitulated the PD-like symptoms of decreased lifespan and loss of locomotor function, possibly producing a new model of PD (M'Angale, 2017).

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Coyne, A. N., Lorenzini, I., Chou, C. C., Torvund, M., Rogers, R. S., Starr, A., Zaepfel, B. L., Levy, J., Johannesmeyer, J., Schwartz, J. C., Nishimune, H., Zinsmaier, K., Rossoll, W., Sattler, R. and Zarnescu, D. C. (2017). Post-transcriptional inhibition of Hsc70-4/HSPA8 expression leads to synaptic vesicle cycling defects in multiple models of ALS. Cell Rep 21(1): 110-125. PubMed ID: 28978466


Amyotrophic lateral sclerosis (ALS) is a synaptopathy accompanied by the presence of cytoplasmic aggregates containing TDP-43, an RNA-binding protein linked to approximately 97% of ALS cases. Using a Drosophila model of ALS, this study shows that TDP-43 overexpression (OE) in motor neurons results in decreased expression of the Hsc70-4 chaperone at the neuromuscular junction (NMJ). Mechanistically, mutant TDP-43 sequesters hsc70-4 mRNA and impairs its translation. Expression of the Hsc70-4 ortholog, HSPA8, is also reduced in primary motor neurons and NMJs of mice expressing mutant TDP-43. Electrophysiology, imaging, and These deficits can be partially restored by OE of Hsc70-4, cysteine-string protein (Csp), or dynamin. This suggests that TDP-43 toxicity results in part from impaired activity of the synaptic CSP/Hsc70 chaperone complex impacting dynamin function. Finally, Hsc70-4/HSPA8 expression is also post-transcriptionally reduced in fly and human induced pluripotent stem cell (iPSC) C9orf72 models, suggesting a common disease pathomechanism (Coyne, 2017).

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Byrne, D. J., Harmon, M. J., Simpson, J. C., Blackstone, C. and O'Sullivan, N. C. (2017). Roles for the VCP co-factors Npl4 and Ufd1 in neuronal function in Drosophila melanogaster. J Genet Genomics 44(10): 493-501. PubMed ID: 29037990


The VCP-Ufd1-Npl4 complex regulates proteasomal processing within cells by delivering ubiquitinated proteins to the proteasome for degradation. Mutations in VCP are associated with two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD). Extensive study has revealed crucial functions of VCP within neurons. By contrast, little is known about the functions of Npl4 or Ufd1 in vivo. Using neuronal-specific knockdown of Npl4 or Ufd1 in Drosophila melanogaster, it is inferred that Npl4 contributes to microtubule organization within developing motor neurons. Moreover, Npl4 RNAi flies present with neurodegenerative phenotypes including progressive locomotor deficits, reduced lifespan and increased accumulation of TAR DNA-binding protein-43 homolog (TBPH). Knockdown, but not overexpression, of TBPH also exacerbates Npl4 RNAi-associated adult-onset neurodegenerative phenotypes. In contrast, this study finds that neuronal knockdown of Ufd1 has little effect on neuromuscular junction (NMJ) organization, TBPH accumulation or adult behaviour. These findings suggest the differing neuronal functions of Npl4 and Ufd1 in vivo (Byrne, 2017).

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Berson, A., Sartoris, A., Nativio, R., Van Deerlin, V., Toledo, J. B., Porta, S., Liu, S., Chung, C. Y., Garcia, B. A., Lee, V. M., Trojanowski, J. Q., Johnson, F. B., Berger, S. L. and Bonini, N. M. (2017). TDP-43 promotes neurodegeneration by impairing chromatin remodeling. Curr Biol 27(23):3579-3590. PubMed ID: 29153328


Regulation of chromatin structure is critical for brain development and function. However, the involvement of chromatin dynamics in neurodegeneration is less well understood. This study found, launching from Drosophila models of amyotrophic lateral sclerosis and frontotemporal dementia, that TDP-43 impairs the induction of multiple key stress genes required to protect from disease by reducing the recruitment of the chromatin remodeler Chd1 to chromatin. Chd1 depletion robustly enhances TDP-43-mediated neurodegeneration and promotes the formation of stress granules. Conversely, upregulation of Chd1 restores nucleosomal dynamics, promotes normal induction of protective stress genes, and rescues stress sensitivity of TDP-43-expressing animals. TDP-43-mediated impairments are conserved in mammalian cells, and, importantly, the human ortholog CHD2 physically interacts with TDP-43 and is strikingly reduced in level in temporal cortex of human patient tissue. These findings indicate that TDP-43-mediated neurodegeneration causes impaired chromatin dynamics that prevents appropriate expression of protective genes through compromised function of the chromatin remodeler Chd1/CHD2. Enhancing chromatin dynamics may be a treatment approach to amyotrophic lateral scleorosis (ALS)/frontotemporal dementia (Berson, 2017).

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Wu, C. H., Giampetruzzi, A., Tran, H., Fallini, C., Gao, F. B. and Landers, J. E. (2017). A Drosophila model of ALS reveals a partial loss of function of causative human PFN1 mutants. Hum Mol Genet. PubMed ID: 28379367


Mutations in the profilin 1 (PFN1) gene are causative for familial amyotrophic lateral sclerosis (fALS). However, it is still not fully understood how these mutations lead to neurodegeneration. To address this question, a novel Drosophila model was generated expressing human wild-type and ALS-causative PFN1 mutants. At larval neuromuscular junctions (NMJ), motor neuron expression of wild-type human PFN1 increases the number of ghost boutons, active zone density, F-actin content, and the formation of filopodia. In contrast, the expression of ALS-causative human PFN1 mutants causes a less pronounced phenotype, suggesting a loss of function of these mutants in promoting NMJ remodeling. Importantly, expression of human PFN1 in motor neurons results in progressive locomotion defects and shorter lifespan in adult flies, while ALS-causative PFN1 mutants display a less toxic effect. In summary, this study provides evidence that PFN1 is important in regulating NMJ morphology and influences survival and locomotion in Drosophila. Furthermore, the results suggest ALS-causative human PFN1 mutants display a partial loss-of-function relative to wild-type hPFN1 that may contribute to human disease pathogenesis (Wu, 2017).

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Feuillette, S., Delarue, M., Riou, G., Gaffuri, A.L., Wu, J., Lenkei, Z., Boyer, O., Frébourg, T., Campion, D. and Lecourtois, M. (2017). Neuron-to-Neuron Transfer of FUS in Drosophila primary neuronal culture Is enhanced by ALS-associated mutations. J Mol Neurosci [Epub ahead of print]. PubMed ID: 28429234


The DNA- and RNA-binding protein fused in sarcoma (FUS; see Drosophila Cabeza) has been pathologically and genetically linked to amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Cytoplasmic FUS-positive inclusions have been identified in the brain and spinal cord of a subset of patients suffering with ALS/FTLD. An increasing number of reports suggest that FUS protein can behave in a prion-like manner. However, no neuropathological studies or experimental data are available regarding cell-to-cell spread of these pathological protein assemblies. This study investigated the ability of wild-type and mutant forms of FUS to transfer between neuronal cells. The study combined the use of Drosophila models for FUS proteinopathies with that of the primary neuronal cultures to address neuron-to-neuron transfer of FUS proteins. Using conditional co-culture models and an optimized flow cytometry-based methodology, it was demonstrated that ALS-mutant forms of FUS proteins can transfer between well-differentiated mature Drosophila neurons. These new observations support that a propagating mechanism could be applicable to FUS, leading to the sequential dissemination of pathological proteins over years (Feuillette, 2017).

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Khalil, B., Cabirol-Pol, M. J., Miguel, L., Whitworth, A. J., Lecourtois, M. and Lievens, J. C. (2017). Enhancing Mitofusin/Marf ameliorates neuromuscular dysfunction in Drosophila models of TDP-43 proteinopathies. Neurobiol Aging 54: 71-83. PubMed ID: 28324764


Transactive response DNA-binding protein 43 kDa (TDP-43) is considered a major pathological protein in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The precise mechanisms by which TDP-43 dysregulation leads to toxicity in neurons are not fully understood. Using TDP-43-expressing Drosophila, this study examined whether mitochondrial dysfunction is a central determinant in TDP-43 pathogenesis. Expression of human wild-type TDP-43 in Drosophila neurons results in abnormally small mitochondria. The mitochondrial fragmentation is correlated with a specific decrease in the mRNA and protein levels of the Drosophila profusion gene mitofusin/marf. Importantly, overexpression of Marf ameliorates defects in spontaneous walking activity and startle-induced climbing response of TDP-43-expressing flies. Partial inactivation of the mitochondrial profission factor, dynamin-related protein 1, also mitigates TDP-43-induced locomotor deficits. Expression of TDP-43 impairs neuromuscular junction transmission upon repetitive stimulation of the giant fiber circuit that controls flight muscles, which is also ameliorated by Marf overexpression. Enhancing the profusion gene mitofusin/marf is shown to be beneficial in an in vivo model of TDP-43 proteinopathies, serving as a potential therapeutic target (Khalil, 2017).

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Krug, L., Chatterjee, N., Borges-Monroy, R., Hearn, S., Liao, W. W., Morrill, K., Prazak, L., Rozhkov, N., Theodorou, D., Hammell, M. and Dubnau, J. (2017). Retrotransposon activation contributes to neurodegeneration in a Drosophila TDP-43 model of ALS. PLoS Genet 13(3): e1006635. PubMed ID: 28301478


Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43; see Drosophila TBPH), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. This study expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. Such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. Gypsy causes the degenerative phenotypes in these flies because it was possilble to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, evidence is provided that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. These findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD (Krug, 2017).

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Şahin, A., Held, A., Bredvik, K., Major, P., Achilli, T.M., Kerson, A.G., Wharton, K., Stilwell, G. and Reenan, R. (2016). The chaperone HSPB8 reduces the accumulation of truncated TDP-43 species in cells and protects against TDP-43-mediated toxicity. Hum Mol Genet [Epub ahead of print]. PubMed ID: Human SOD1 ALS mutations in a Drosophila knock-in model cause severe phenotypes and reveal dosage-sensitive gain and loss of function components. Genetics [Epub ahead of print]. PubMed ID: 27974499

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc Superoxide Dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, this study genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical and behavioral phenotypes. Contrary to previous transgenic models, ALS-like phenotypes recapitulate without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R and H71Y) exhibits neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) is phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes are recessive, a gain of function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which fail to show severe locomotor defects or nerve degeneration. The study concludes that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies (Şahin, A., 2016).

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De Rose, F., Marotta, R., Talani, G., Catelani, T., Solari, P., Poddighe, S., Borghero, G., Marrosu, F., Sanna, E., Kasture, S., Acquas, E. and Liscia, A.(2017). Differential effects of phytotherapic preparations in the hSOD1 Drosophila melanogaster model of ALS. Sci Rep 7: 41059. PubMed ID: 28102336


Anti-inflammatory extracts of Withania somnifera (Wse) and Mucuna pruriens (Mpe) were tested on a Drosophila model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse- but not in Mpe-treated hSOD1 mutants. These results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptoms (De Rose, 2017).

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Crippa, V., et al. (2016). The chaperone HSPB8 reduces the accumulation of truncated TDP-43 species in cells and protects against TDP-43-mediated toxicity. Hum Mol Genet [Epub ahead of print]. PubMed ID: 27466192

Aggregation of TAR-DNA binding protein 43 (TDP-43; see Drosophila TBPH) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragment of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. This study reports that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in Drosophila melanogaster. Overexpression of HSP67Bc, the Drosophila functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, a novel Drosophila model expressing TDP-35 is reported, and it was shown that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively these data demonstrate that HSPB8 upregulation mitigates TDP fragment mediated toxicity, in mammalian neuronal cells and flies (Crippa, 2016).

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Lee, K. H., et al. (2016). C9orf72 dipeptide repeats impair the assembly, dynamics, and function of membrane-less organelles. Cell 167: 774-788 e717. PubMed ID: 27768896


Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. This study identified the interactome of all DPRs and found that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, it was shown that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles (Lee, 2016).

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Kramer, N. J., et al. (2016). Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts. Science 353: 708-712. PubMed ID: 27516603


An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. This study found that targeting the transcription elongation factor Spt4 (see Drosophila Spt4) selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. In Drosophila, Spt4 RNAi partially suppressed the degenerative phenotype of the external and internal eye in (GGGGCC)49-expressing flies and almost completely suppressed the retinal thinning normally observed in (GGGGCC)29-expressing flies. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately (Kramer, 2016).

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Matsukawa, K., Hashimoto, T., Matsumoto, T., Ihara, R., Chihara, T., Miura, M., Wakabayashi, T. and Iwatsubo, T. (2016). Familial ALS-linked mutations in Profilin 1 exacerbate TDP-43-induced degeneration in the retina of Drosophila melanogaster through an increase in the cytoplasmic localization of TDP-43. J Biol Chem [Epub ahead of print]. PubMed ID: 27634045


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive and selective loss of motor neurons. Causative genes for familial ALS (fALS) include mutations within profilin 1 (PFN1; see Drosophila Chickadee) have recently been identified in ALS18. Transgenic Drosophila melanogaster were generated overexpressing human PFN1 in the retinal photoreceptor neurons. Overexpression of wild-type or fALS mutant PFN1 caused no degenerative phenotypes in the retina. Double overexpression of fALS mutant PFN1 and human TDP-43 (see Drosophila TDP-43) markedly exacerbated the TDP-43-induced retinal degeneration, i.e., vacuolation and thinning of the retina, whereas co-expression of wild-type PFN1 did not aggravate the degenerative phenotype. Notably, co-expression of TDP-43 with fALS mutant PFN1 increased the cytoplasmic localization of TDP-43, the latter being remained in nuclei upon co-expression with wild-type PFN1, whereas co-expression of TDP-43 lacking the nuclear localization signal with fALS mutant PFN1 did not aggravate the retinal degeneration. Knockdown of endogenous Drosophila PFN1 did not alter the degenerative phenotypes of the retina in flies overexpressing wild-type TDP-43. These data suggest that ALS-linked PFN1 mutations exacerbate TDP-43-induced neurodegeneration in a gain-of-function manner, possibly by shifting the localization of TDP-43 from nuclei to cytoplasm (Matsukawa, 2016).

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Deshpande, M., Feiger, Z., Shilton, A. K., Luo, C. C., Silverman, E. and Rodal, A. A. (2016). Role of BMP receptor traffic in synaptic growth defects in an ALS model. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27535427


TAR DNA-binding protein 43 (TDP-43) is genetically and functionally linked to Amyotrophic Lateral Sclerosis (ALS), and regulates transcription, splicing, and transport of thousands of RNA targets that function in diverse cellular pathways. In ALS, pathologically altered TDP-43 is thought to lead to disease by toxic gain-of-function effects on RNA metabolism, as well as by sequestering endogenous TDP-43 and causing its loss of function. However, it remains unclear which of the numerous cellular processes disrupted downstream of TDP-43 dysfunction lead to neurodegeneration. This study found that both loss- and gain-of-function of TDP-43 in Drosophila cause a reduction of synaptic-growth-promoting Bone Morphogenic Protein (BMP) signaling at the neuromuscular junction (NMJ). Further, a shift of BMP receptors from early to recycling endosomes was observed along with increased mobility of BMP receptor-containing compartments at the NMJ. Inhibition of the recycling endosome GTPase Rab11 partially rescued TDP-43-induced defects in BMP receptor dynamics and distribution, and suppressed BMP signaling, synaptic growth, and larval crawling defects. These results indicate that defects in receptor traffic lead to neuronal dysfunction downstream of TDP-43 misregulation, and that rerouting receptor traffic may be a viable strategy for rescuing neurological impairment (Deshpande, 2016).

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Baldwin, K.R., Godena, V.K., Hewitt, V.L. and Whitworth, A.J. (2016). Axonal transport defects are a common phenotype in Drosophila models of ALS. Hum Mol Genet [Epub ahead of print]. PubMed ID: 27056981


Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motor neurons resulting in a catastrophic loss of motor function. Current therapies are severely limited owing to a poor mechanistic understanding of the pathobiology. Mutations in a large number of genes have now been linked to ALS, including SOD1, TARDBP (TDP-43), FUS and C9orf72. Functional analyses of these genes and their pathogenic mutations have provided great insights into the underlying disease mechanisms. Defective axonal transport is hypothesized to be a key factor in the selective vulnerability of motor nerves due to their extraordinary length and evidence that ALS occurs as a distal axonopathy. Axonal transport is seen as an early pathogenic event that precedes cell loss and clinical symptoms and so represents an upstream mechanism for therapeutic targeting. Studies have begun to describe the impact of a few pathogenic mutations on axonal transport but a broad survey across a range of models and cargos is warranted. This study assessed the axonal transport of different cargos in multiple Drosophila models of ALS. It was found that axonal transport defects are common across all models tested, although they often show a differential effect between mitochondria and vesicle cargos. Motor deficits are also common across the models and generally worsen with age, though surprisingly there isn't a clear correlation between the severity of axonal transport defects and motor ability. These results further support defects in axonal transport as a common factor in models of ALS that may contribute to the pathogenic process (Whitworth, 2016).

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Coyne, A.N., Siddegowda, B.B., Estes, P.S., Johannesmeyer, J., Kovalik, T., Daniel, S.G., Pearson, A., Bowser, R. and Zarnescu, D.C. (2014). Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of Amyotrophic Lateral Sclerosis. J Neurosci 34: 15962-15974. PubMed ID: 25429138

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 was 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 lead to a significant reduction in Futsch protein expression at the NMJ. Polysome fractionations coupled with quantitative PCR experiments indicated that TDP-43 lead 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 regulated 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, was 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).


  • futsch mRNA associates with TDP-43 in a complex in vivo.
  • TDP-43 alters futsch mRNA localization and inhibits Futsch expression post-transcriptionally.
  • futsch mRNA translation is inhibited in the context of TDP-43 proteinopathy in motor neurons.
  • Futsch is neuroprotective in the context of TDP-43 overexpression.
  • Futsch mitigates architectural defects at the neuromuscular junction by increasing microtubule stability.
  • TDP-43 aggregates are significantly decreased by futsch overexpression. Futsch/MAP1B localization is altered in ALS spinal cords.

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, this study showed that wild-type and disease-associated mutant TDP-43 co-fractionated 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. These data provide the first in vivo demonstration that TDP-43 associates with polysomes and regulates the translation of futsch mRNA (Coyne, 2014).

A decrease in Futsch levels at the NMJ and an increase in Futsch levels in motor neuron cell bodies was shown that suggested a model whereby futsch/MAP1B mRNA may not be properly transported into axons. This was substantiated by qPCR from ventral ganglia where futsch mRNA was found at higher levels than at the NMJ compared with controls. Although the possibility that TDP-43 regulated futsch mRNA stability could not be excluded, 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, the data suggested TDP-43-dependent defects in futsch/MAP1B mRNA transport and protein expression at the NMJ (Coyne, 2014).

Since futsch is the Drosophila homolog of MAP1B and MAP1B mRNA has been identified in TDP-43-containing RNP complexes in mouse models, it was predicted that MAP1B and microtubule-based processes might also be affected in ALS patient tissues. Indeed, similar to their results in the fly, immunohistochemistry experiments revealed 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. Interestingly, Futsch protein expression was similarly inhibited by wild-type or mutant TDP-43, supporting a scenario in which MAP1B dysregulation might be a shared feature of ALS cases with TDP-43-positive pathology, regardless of etiology (Coyne, 2014).

Using genetic interaction approaches, it was shown that futsch is a physiologically significant RNA target of TDP-43 and can alleviate locomotor dysfunction and increase life span. Given Futsch’s known requirement in axonal and dendritic development and the organization of microtubules at the synapse, it was suggested that these processes may be involved in the pathophysiology of ALS. Consistent with previous studies in which tubulin acetylation was shown to rescue transport defects in neurodegeneration, it was shown that TDP-43 lead to reduced levels of acetylated tubulin, and this was rescued by futsch overexpression. Other TDP-43 targets such as HDAC6, which is regulated by TDP-43 at the level of transcription, were 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 (Coyne, 2014).

In conclusion, this study identifies futsch as a disease-relevant and functionally significant post-transcriptional target of TDP-43. Given the role of futsch/MAP1B in microtubule and synaptic stabilization, this points to microtubule-based processes as targets for the development of therapeutic strategies for TDP-43 proteinopathies (Coyne, 2014).

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Joardar, A., Menzl, J., Podolsky, T. C., Manzo, E., Estes, P. S., Ashford, S. and Zarnescu, D. C. (2014). PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43. Hum Mol Genet. 24: 1741-1754. PubMed ID: 25432537

Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease for which there is no cure. A Drosophila model has been developed of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, a drug screening strategy was designed based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1,200 FDA approved compounds, the PPARgamma agonist pioglitazone was found to be neuroprotective in Drosophila. It was shown that pioglitazone could rescue TDP-43 dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS, it was found that pioglitazone was also neuroprotective when FUS, but not SOD1, was expressed in motor neurons. Interestingly, survival analyses of TDP or FUS models showed no increase in lifespan, which was consistent with recent clinical trials. Using a pharmacogenetic approach, it was shown that the predicted Drosophila PPARgamma homologs, E75 and E78 were in vivo targets of pioglitazone. Finally, using a global metabolomic approach, a set of metabolites was identified that pioglitazone could restore in the context of TDP-43 expression in motor neurons. Taken together, the study provides evidence that modulating PPARgamma activity, although not effective at improving lifespan, provides a molecular target for mitigating locomotor dysfunction in TDP-43 and FUS but not SOD1 models of ALS in Drosophila. Furthermore, it also identify several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials (Joardar, 2014).


  • Drug screening in a Drosophila model of ALS based on TDP-43 identifies pioglitazone, a PPARgamma agonist as neuroprotective.
  • Larval locomotor deficits caused by TDP-43 expression in motor neurons are rescued by pioglitazone.
  • Pioglitazone exerts no protective effect on lifespan in the context of TDP-43 expression in motor neurons.
  • Glial toxicity caused by TDP-43 is partially mitigated by pioglitazone.
  • Locomotor defects caused by TDP-43 in muscles are not rescued by pioglitazone.FUS-dependent toxicity in motor neurons is partially mitigated by pioglitazone.
  • Pioglitazone is not protective in a Drosophila model of ALS based on SOD1.
  • PPARgamma acts as the molecular target of pioglitazone in vivo, in Drosophila.
  • Pioglitazone restores a subset of metabolites dysregulated in the context of TDP-43 proteinopathy.

Using a previously generated Drosophila model of ALS based on TDP-43, this study showed that the antidiabetic drug pioglitazone acted as a neuroprotectant for aspects of TDP-43 proteinopathy by activating the putative Drosophila PPARgamma homologs E75 and E78. Pioglitazone mitigated FUS but not SOD1-dependent toxicity in Drosophila, consistent with previous published work showing that distinct mechanisms were likely at work in the context of these different models of ALS. Interestingly, pioglitazone did not improve, and in some cases worsened, the lifespan of TDP-43-expressing flies, when administered either during development, or after ‘disease onset’, which was consistent with results from recent clinical trials. This apparent disconnect was consistent with the effects of pioglitazone on cellular metabolism. While pioglitazone treatment restored some metabolites altered owing to TDP-43 overexpression in motor neurons, others were unchanged or even worsened. This provided a potential explanation for why some phenotypes but not others were rescued by pioglitazone. Aside from the possibility that different drug concentrations might be needed, it remains unclear why pioglitazone is protective in mouse but not fly SOD1 models and, in retrospect, given the similarities between the effect of pioglitazone in Drosophila models of ALS and humans, the fly appears to be a more accurate predictor of clinical trial outcomes (Joardar, 2014).

It is tempting to speculate that the predictive power of the Drosophila model may lie in the tools that enable motor neuronal versus glial versus muscle-specific expression of the toxic TDP-43 protein. It was shown that pioglitazone mitigated neuronal and glial TDP-43-dependent toxicity but had no effect on the locomotor dysfunction caused by muscle-specific expression of TDP-43. The protective effects of pioglitazone were specific to the nervous system and were not observed in muscles, at least within the limits of experimental conditions (i.e. tissue-specific levels of expression and drug concentration). These findings suggest that future preclinical studies may benefit from testing candidate therapies in multiple disease models in which tissue specificity and several phenotypic outcomes are easily ascertained (Joardar, 2014).

Pioglitazone has been originally developed for the treatment of type 2 diabetes as PPARgamma activation in the liver improves glucose metabolism systemically. In the nervous system, activation of the nuclear hormone receptor PPARgamma has been shown to have anti-inflammatory and neuroprotective effects. In the model used it this study, it was found that pioglitazone could restore a rather limited set of metabolites altered in a TDP-43-dependent manner. Evidence for altered glutamine/glutamate metabolism in TDPWT flies was found, as displayed by elevated levels of N-acetylglutamine, which was restored by pioglitazone. Excessive levels of extracellular glutamate in the central nervous system cause hyperexcitability of neurons, ultimately leading to their death. The glutamate transporter GLT1/EAAT2 plays a major role in maintaining extracellular glutamate levels below the excitotoxic concentrations by efficiently transporting this metabolite. Interestingly, astrocytic GLT1/EAAT2 gene is a target of PPARgamma, leading to neuroprotection by increasing glutamate uptake (Joardar, 2014).

Furthermore, pyruvate, which was significantly high in both TDPWT and TDPG298S, showed a trend toward reduction upon pioglitazone treatment for TDPWT. Pyruvate is a central metabolite that lies at the junction of several intersecting cellular pathways including glucose and fatty acid metabolism. It is converted to oxaloacetate by the enzyme pyruvate carboxylase, which is a key step in lipogenesis. Interestingly, PPARgamma, the target of pioglitazone, is a direct transcriptional modulator of the pyruvate carboxylase gene (Joardar, 2014).

Given the fact that ALS patients suffer from massive weight loss, results from this study provide a possible explanation for the potential protective effects of pioglitazone through increased lipogenesis. Taken together, the metabolomics approach of this study provides useful insights for understanding the molecular mechanisms underlying ALS pathophysiology. Notably, the fly model used in this study also showed signs of hypermetabolism including an increase in pyruvate, a key metabolite linking glucose metabolism to the TCA cycle. Additionally, the ketone body GHB was reduced in the context of TDPWT, consistent with a clinical study showing that a ketogenic diet slowed ALS disease progression. Given the similarities between the metabolic profile of the Drosophila model and human samples, it will be interesting in the future, to design therapeutic approaches aimed at restoring these common metabolic changes using nutritional supplementation (Joardar, 2014).

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He, F., Krans, A., Freibaum, B. D., Taylor, J. P., Todd, P. K. (2014). TDP-43 suppresses CGG repeat-induced neurotoxicity through interactions with HnRNP A2/B1. Hum Mol Genet. 23: 5036-5051. PubMed ID: 24920338

Nucleotide repeat expansions can elicit neurodegeneration as RNA by sequestering specific RNA-binding proteins, preventing them from performing their normal functions. Conversely, mutations in RNA-binding proteins can trigger neurodegeneration at least partly by altering RNA metabolism. In Fragile X-associated tremor/ataxia syndrome (FXTAS), a CGG repeat expansion in the 5'UTR of the fragile X gene (FMR1) leads to progressive neurodegeneration in patients and CGG repeats in isolation elicit toxicity in Drosophila and other animal models. This study identified the amyotrophic lateral sclerosis (ALS)-associated RNA-binding protein TAR DNA-binding protein (TDP-43) as a suppressor of CGG repeat-induced toxicity in a Drosophila model of FXTAS. The rescue appeared specific to TDP-43, as co-expression of another ALS-associated RNA-binding protein, FUS, exacerbated the toxic effects of CGG repeats. Suppression of CGG RNA toxicity was abrogated by disease-associated mutations in TDP-43. TDP-43 did not co-localize with CGG RNA foci and its ability to bind RNA was not required for rescue. TDP-43-dependent rescue did, however, require fly hnRNP A2/B1 homologues Hrb87F and Hrb98DE. Deletions in the C-terminal domain of TDP-43 that precluded interactions with hnRNP A2/B1 abolished TDP-43-dependent rescue of CGG repeat toxicity. In contrast, suppression of CGG repeat toxicity by hnRNP A2/B1 was not affected by RNAi-mediated knockdown of the fly TDP-43 orthologue, TBPH. Lastly, TDP-43 suppressed CGG repeat-triggered mis-splicing of an hnRNP A2/B1-targeted transcript. These data support a model in which TDP-43 suppresses CGG-mediated toxicity through interactions with hnRNP A2/B1 and suggest a convergence of pathogenic cascades between repeat expansion disorders and RNA-binding proteins implicated in neurodegenerative disease (He, 2014).


  • Overexpression of TDP-43 suppresses CGG repeat toxicity.
  • CGG repeat RNA levels and RAN translation are not altered by TDP-43.
  • TDP-43 suppression of CGG repeat toxicity is dependent on hnRNP A2/B1 homologues.
  • Normal TBPH expression is not required for hnRNP A2/B1 suppression of CGG toxicity.
  • TDP-43 restores alternative splicing of EPH, which is perturbed by CGG repeat expression.

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Watson, M. R., Lagow, R. D., Xu, K., Zhang, B., Bonini, N. M. (2008). A Drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1. J Biol Chem. 283: 24972-29481. PubMed ID: 18596033

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease that leads to loss of motor function and early death. About 5% of cases are inherited, with the majority of identified linkages in the gene encoding copper, zinc-superoxide dismutase (SOD1). Strong evidence indicates that the SOD1 mutations confer dominant toxicity on the protein. To provide new insight into mechanisms of ALS, this study generated and characterized a model for familial ALS in Drosophila with transgenic expression of human SOD1. Expression of wild type or disease-linked (A4V, G85R) mutants of human SOD1 selectively in motor neurons induced progressive climbing deficits. These effects were accompanied by defective neural circuit electrophysiology, focal accumulation of human SOD1 protein in motor neurons, and a stress response in surrounding glia. However, toxicity was not associated with oligomerization of SOD1 and did not lead to neuronal loss. These data uncover cell-autonomous injury by SOD1 to motor neurons in vivo, as well as non-autonomous effects on glia, and provide the foundation for new insight into injury and protection of motor neurons in ALS (Watson, 2008).


  • Expression of hSOD1 but not dSOD1 in motor neurons causes progressive motor dysfunction.
  • No apparent loss of motor neurons.
  • Biochemical oligomerization of hSOD1 is not linked to neuronal loss or dysfunction.
  • Synaptic transmission along the giant fiber motor pathway is abnormal.
  • hSOD1 progressively accumulates in motor neuron somata and processes.
  • Expression of hSOD1 in motor neurons produces a stress response in glia.

This study presents a model for SOD-linked fALS in Drosophila that displays motor dysfunction, a defining feature of the human disease. Motor dysfunction in flies was accompanied by failure in high frequency synaptic transmission, focal accumulation of hSOD1 in motor neurons, and up-regulation of heat shock protein in glia. These findings suggest that SOD can cause cell-autonomous damage to motor neurons, and highlight that expression of hSOD1 selectively in motor neurons induces a change in glia (Watson, 2008).

It was shown that a motor neuron-restricted expression pattern conferred behavioral compromise in climbing ability. This suggests that hSOD1 may have an intrinsic toxicity to motor neurons, which can be defined in the Drosophila system. Previous models in mice have demonstrated a dependence of toxicity on widespread tissue expression, specifically with the genes under control of the endogenous hSOD1 enhancer/promoter elements. Several studies with mice have reported no toxicity with neuron-restricted expression using the Thy1 or the neurofilament light chain promoters. This idea was expanded when another study demonstrated in chimeric mice that motor neurons can display ALS-like pathology when they are not expressing the mutant protein themselves but rather are surrounded by other cell types that are expressing the mutant protein. The model presented in this study, on the other hand, provides an approach to define toxic properties of hSOD1 specifically in motor neurons that can lead to a motor deficit (Watson, 2008).

Upon expressing hSOD1 in the fly, deficits were seen with expression restricted to motor neurons which supported a role for cell-autonomous damage to motor neurons by hSOD1. Within motor neurons, there was progressive accumulation of hSOD1, both in the somata surrounding the nucleus, as well as in neurites. These focal accumulations may both cause and result from hindrances in trafficking and axonal transport or insufficient protein degradation. It is known that disruption of anterograde and retrograde axonal movement of synaptic proteins and neurotrophic entities can negatively affect neuronal function. The p150glued mutation in dynactin-1, which severely disrupts axonal transport, causes a progressive, late onset motor phenotype in mice. Mice expressing mutant SOD1 also have compromised axonal transport. The flies displayed electrophysiological defects reflective of impaired motor neuron function, indicating that the fly may provide a sensitive system for the detection of subtle motor neuron defects caused by hSOD1 and disease-linked forms. Despite a progressive motor phenotype, there was no change in numbers of neuronal nuclei, excluding widespread loss of cells. The electrical features of the motor pathway also indicated that it could function fine at low activity levels, suggesting that synapses may be the primary site of dysfunction of SOD1 flies (Watson, 2008).

WT hSOD1 imparted toxicity nearly on a par with either A4V or G85R mutant forms; WT hSOD1 even showed a tendency to accumulate in foci, a feature generally expected of a mutant but not normal hSOD1. It was hypothesized that WT hSOD1 may function as a conformational mutant protein in the context of Drosophila neurons for the following reasons. Toxicity can be conferred onto hSOD1 by any one of more than a hundred distinct amino acid substitutions, which implies an exquisite dependence upon conformation. This raises the possibility that any sequence other than the wild type Drosophila SOD1 conformation in the context of the SOD1 protein may appear abnormal to the fly. Although Drosophila SOD1 and hSOD1 are very similar in sequence, and hSOD1 can even functionally replace the Drosophila gene, the enzymes do differ in many amino acids, including locations where mutations occur that are associated with fALS. Importantly, overexpression of dSOD1 did not mimic the effects of hSOD1 expression in the fly. This finding also fails to support the idea that SOD1 toxicity may be related to dismutase activity of the enzyme as both dSOD1 and hSOD1 would presumably result in the overabundance of hydrogen peroxide, yet there was selective toxicity of hSOD1 (Watson, 2008).

Affected tissues in neurodegenerative diseases often exhibit the induction of a chaperone stress response. The heat shock protein immunoreactivity that was observed in fly thoracic ganglion did not overlap with hSOD1 staining. Rather, it was present exclusively in cells that were positive for the glial-specific marker protein Repo. Thus, in the fly model, the motor neurons contained the toxic protein, but the glia appeared to initiate a stress response. It was unlikely that exogenous SOD1 induced a stress response due to SOD1 expression in glia themselves since the D42 motor neuron driver is specific, and SOD1 was not detected in glia by immunofluorescence using a variety of primary antibodies, despite robust SOD1 levels. Leaky expression due to the genomic insertion sites of the transgenes could result in glial expression of the exogenous proteins, although analysis of flies lacking the GAL4 driver revealed no detectable hSOD1 protein. Furthermore, expanded polyglutamine protein in flies with the same motor neuron driver was only observed in neurons (Watson, 2008).

The glial chaperone up-regulation may be a reaction to the toxic protein or a signal secondary to effects of SOD1 in motor neurons. Motor neuron expression of dSOD1, but not of a pathogenic polyglutamine protein by the same driver, also resulted in a glial response, indicating that the response occurs with SOD1. Flies with greater chaperone induction showed more severe indicators of motor dysfunction. Thus, the degree of stress response in glia may serve as a measure of neuronal dysfunction or a measure of the extent to which glia are attempting to combat problems in motor neurons (Watson, 2008).

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Huang, Y., Wu, Z., Zhou, B. (2015). hSOD1 promotes Tau phosphorylation and toxicity in the Drosophila model. J Alzheimers Dis. 45: 235-244. PubMed ID: 25524953

Tau hyperphosphorylation has been found in several neurodegenerative diseases such as Alzheimer's disease (AD), Down syndrome, and amyotrophic lateral sclerosis (ALS). However, factors affecting tau hyperphosphorylation are not yet clearly understood. SOD1, a Cu/Zn superoxide dismutase whose mutations can cause adult-onset ALS, is believed to be involved in the pathology of Down syndrome. In this work, the model organism Drosophila was used to study the possible link between hSOD1 and tau. It was shown that hSOD1, and to a higher degree hSOD1(A4V), could increase tau toxicity in Drosophila and exacerbate the corresponding neurodegeneration phenotype. The increased tau toxicity appeared to be explainable by elevated tau phosphorylation. Tau(S2A), a tau mutant with impaired phosphorylation capabilities, did not respond to expression of hSOD1 and hSOD1(A4V). The study suggests that increased SOD1 expression can lead to tau hyperphosphorylation, which might serve as an important contributing factor to the etiology of Down syndrome and SOD1-related ALS disease (Huang, 2015).

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Takayama, Y., Itoh, R.E., Tsuyama, T. and Uemura, T. (2014). Age-dependent deterioration of locomotion in Drosophila melanogaster deficient in the homologue of amyotrophic lateral sclerosis 2. Genes Cells: 464-477. PubMed ID: 24702731

Recessive mutations in the amyotrophic lateral sclerosis 2 (ALS2) gene have been linked to juvenile-onset ALS2. Although one of the molecular functions of the ALS2 protein is clearly the activation of Rab5, the mechanisms underlying the selective dysfunction and degeneration of motor neurons in vivo remain to be fully understood. This study focused on the ALS2 homologue of Drosophila melanogaster, isolated two independent deletions, and systematically compared phenotypes of the mutants with those of animals in which Rab5 function in identified neurons was abrogated. In the dALS2 mutant flies, it was found that the stereotypic axonal and dendritic morphologies of neurons shared some features with those in Rab5-deficient flies, but the dALS2 mutant phenotypes were much milder. It was also found that the abrogation of Rab5 function in motor neurons strongly depressed the locomotion activity of adults, resembling the behavior of aged dALS2 mutants. Importantly, this age-dependent locomotion deficit of dALS2 mutants was restored to normal by expressing the dALS2 transgene in a wide range of tissues. This finding provided a platform where particular cell types responsible for the phenotype by tissue-specific rescue experiments coule be potentially identified. The study also discussed the future usage of the dALS2 mutant as a new ALS model (Takayama, 2014).


  • CG7158 encodes a homologue of ALS2.
  • CG7158/dALS2 has a GEF activity for Rab5.
  • dALS2 mutants are viable and fertile.
  • Morphological analysis of axon terminals and dendritic arborization of identified neurons in the dALS2 mutant and in wild-type flies expressing a dominant negative form of Rab5 .
  • Mutant dALS2 adults show a lowered locomotion activity.
  • Locomotion deficit of the dALS2 mutant is rescued by dALS2 transgene expression.

Annotated genes in the genome of Drosophila melanogaster include homologues of four human GEFs and two GTPase activating proteins (GAPs). Among them, the predicted protein product of a single gene CG7158 shows similarities to ALS2/Alsin. This study designated CG7158 and its protein product as dALS2 and dALS2. Both ALS2/Alsin and dALS2 contain four domains in the same order from their amino-terminals, among which the ‘membrane occupation and recognition nexus (MORN)’ motifs and the carboxyl-terminal ‘vacuolar protein sorting 9 (VPS9)’ domain in human ALS2 are necessary for its selective GEF activity for Rab5 in vitro. One distinctive difference between dALS2 and ALS2/Alsin is that dALS2 lacks a DH domain, which is adjacent to the PH domain in human ALS2 and provides a basis of its GEF activity for Rac1 (Kunita et al. 2007). Thus, dALS2 could be a more Rab5-specific GEF compared with ALS2 (Takayama, 2014).

To examine whether dALS2 does possess GEF activity or not, dALS2 and a fluorescence resonance energy transfer (FRET) probe, Raichu-Rab5, were co-expressed in Drosophila S2 cells. The probe comprised Venus (a modified YFP), the amino-terminal Rab5-binding domain of EEA1, SECFP (a modified CFP) and Rab5. In this probe design, the increase in the emission ratio reflected an increase in the active GTP-bound form of Rab5 relative to the inactive GDP-bound form in living cells. The emission ratio was increased significantly when dALS2 was co-expressed compared with the transfection of the vector. This increase in the ratio was indeed dependent on the conversion from the GDP-bound form to the GTP-bound one, as shown by the fact that the emission ratio of Raichu-Rab5[S34N], a constitutive GDP-bound form, was unchanged even in the presence of dALS2. The effects of substitutions of two conserved amino acid residues in the VPS9 domain, which are necessary for full GEF activity of ALS2 in vitro were also studied. The two mutant forms of dALS2 (dALS2[P1425A] and dALS2[L1439A]) increased the FRET efficiency of Raichu-Rab5, but not to the same extent as the wild-type form. Collectively, these results showed that the wild-type form of dALS2 has GEF activity for Rab5 (Takayama, 2014).

To study the in vivo consequences of dALS2 dysfunction, an existing transposable element that was inserted 120-bp upstream of the 1st ATG of the dALS2 coding sequence was mobilized and two independent alleles (Ex44 and Ex54) that delete approximately 30% of the coding sequence, including the start codon and the entire RLD domain, were isolated. Further, precise jumpers, where the transposon excision restored the exact contiguous WT sequence were also obtained, and homozygotes of two of these (Ex101/Ex101 and Ex95/Ex95) were used for subsequent analysis as controls or the wild-type animals. Homozygotes of either Ex44 or Ex54 were viable and fertile; and the adults looked morphologically normal. Thus, dALS2 may be dispensable for viability in flies, as is the case in mice. RT-PCR analysis confirmed the deletion of the amino-terminal coding sequence of dALS2 in adult dALS2−/− flies. To address whether truncated polypeptides might be made by translation initiation from internal ATG codons downstream of the deletion in dALS2−/−, antibodies to the carboxyl-terminal VPS9 domain were generated. Unfortunately however, the antibodies failed to detect endogenous dALS2 with high sensitivity and thus, the possibility of the generation of truncated polypeptides could not be excluded. Nonetheless, it is known that ALS2 without the RLD domain no longer associates with endosomes, so the truncated dALS2 polypeptides, if synthesized from Ex44 and Ex54 alleles, would most likely not be functional (Takayama, 2014).

It was found that Rab5[S34N] expression using OK371-Gal4 in the motor neurons resulted in an increase in the bouton number of presynaptic terminals of motor axons in larvae and adults, and the Rab5[S34N] effect was more dramatic than the phenotype in the dALS2 mutant. In addition to the increase in the number of boutons, each bouton became smaller than that of the control axon terminals at larval NMJs. From an earlier study with Rab5[S34N], it is known that Rab5 controls synaptic transmission at larval NMJs; however, in that study Rab5[S34N] expression was kept low during embryonic and early larval stages so that it did not affect morphological development of NMJs. Further, Rab5[S34N] expression strongly depressed the climbing ability of adults. In the climbing assay with the dALS2−/− mutant adults, a more prominent phenotype, age-dependent locomotion deficit, was observed, which was causally related to loss of dALS2 function (Takayama, 2014).

To realize a broader expression, Ubiquitin (Ubi)-Gal4 was used to drive expression of the wild-type dALS2 transgene in a wide range of tissues. Two-week-old dALS2−/− adults showed lowered climbing ability, compared with wild type, and this phenotype was restored to normal by dALS2 transgene expression in both females and males. These results showed that the age-dependent locomotion deficit is indeed a loss-of-function phenotype of dALS2. This phenotype is reminiscent of the moderate, age-dependent deficit in motor coordination in ALS2-null mice (Takayama, 2014).

The observations from this study can be interpreted in several ways: First, dALS2 is indeed required in the motor neuron; however, the motor neuron GAL4 driver (OK371-Gal4) failed to correct the phenotype significantly because this Gal4-driven expression of dALS2 far exceeds the physiological range and disturbs precise spatial-temporal regulation of Rab5 activity. Second, dALS2 is supplied in the motor neuron at larval stages by Ubi-GAL4 and a portion of the proteins persist and function at the adult stage (Ubi-GAL4 was found to be expressed in the motor neuron in larvae, but not in adults). Third, dALS2 is critically required in cell types other than the motor neuron to prevent the deterioration of locomotion during aging (e.g., the presumptive ‘upper’ motor neuron in flies); and Ubi-GAL4, not OK371-Gal4, is expressed in that cell type. Use of the rich resource of GAL4 stocks and searches for the stocks that realize appropriate expression levels of dALS2 would allow to distinguish these possibilities (Takayama, 2014).

In addition to animal behaviors and neuronal cell morphologies, the absence of dALS2 function could impact synaptic transmission. Control of Rab5 activity is required for normal development of NMJ; in addition, Rab5 regulates the efficacy of the evoked neurotransmitter release once the NMJ is formed. So NMJs in the dALS2 mutant could be a target of physiological and ultrastructural investigations. Other future targets are premotor interneurons that control the neurotransmitter release at NMJ and further upstream neural circuits, which are functional counterparts of UMNs in mammals. Identification of such neurons and technical accessibility to those would allow to readdress whether the markers of neuronal aging and/or the Drosophila homologue of TDP-43 are accumulated in those particular neuronal classes, and this approach may validate Drosophila as a tractable model of not only ALS2 but also other genetic causes of ALS (Takayama, 2014).

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Sanhueza, M., Chai, A., Smith, C., McCray, B.A., Simpson, T.I., Taylor, J.P., Pennetta G., et al. (2015). Network analyses reveal novel aspects of ALS pathogenesis. PLoS Genet. 11: e1005107. PubMed ID: 25826266

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons, muscle atrophy and paralysis. Mutations in the human VAMP-associated protein B (hVAPB) cause a heterogeneous group of motor neuron diseases including ALS8. Despite extensive research, the molecular mechanisms underlying ALS pathogenesis remain largely unknown. Genetic screens for key interactors of hVAPB activity in the intact nervous system, however, represent a fundamental approach towards understanding the in vivo function of hVAPB and its role in ALS pathogenesis. Targeted expression of the disease-causing allele leads to neurodegeneration and progressive decline in motor performance when expressed in the adult Drosophila, eye or in its entire nervous system, respectively. By using these two phenotypic readouts, this study carried out a systematic survey of the Drosophila genome to identify modifiers of hVAPB-induced neurotoxicity. Modifiers clustered in a diverse array of biological functions including processes and genes that had been previously linked to hVAPB function, such as proteolysis and vesicular trafficking. In addition to established mechanisms, the screen identified endocytic trafficking and genes controlling proliferation and apoptosis as potent modifiers of ALS8-mediated defects. Surprisingly, the list of modifiers was mostly enriched for proteins linked to lipid droplet biogenesis and dynamics. Computational analysis revealed that most modifiers could be linked into a complex network of interacting genes, and that the human genes homologous to the Drosophila modifiers could be assembled into an interacting network largely overlapping with that in flies. Identity markers of the endocytic process were also found to abnormally accumulate in ALS patients, further supporting the relevance of the fly data for human biology. Collectively, these results not only lead to a better understanding of hVAPB function but also point to potentially relevant targets for therapeutic intervention (Sanhueza, 2015).


  • A large-scale screen in Drosophila identifies modifiers of the DVAP-P58S-induced eye phenotype.
  • Genetic validation of the identified modifiers.
  • The modifying effect of DVAP-P58S interacting genes is extended to the adult nervous system.
  • Building the human interactome of DVAP-P58S genetic modifiers.
  • Computational and experimental analysis identifies endocytosis as a process implicated in ALS8 pathogenesis.
  • Rab5 accumulates abnormally in motor neurons of patients affected by ALS.

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Deivasigamani, S., Verma, H.K., Ueda, R., Ratnaparkhi, A., Ratnaparkhi, G.S. (2014). A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis. Biol Open. 3: 1127-1138. 25361581

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5-10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions. This study conducted a reverse genetic screen to identify interactors of Drosophila VAPB. By screening 2635 genes, 103 interactors were identified, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci - TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt) and VAP(P58S), but in a contrasting manner. Reversal of VAP(P58S) bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S) expression. The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease (Deivasigamani, 2014).


  • Known ALS loci and physical interactors of VAP act as modifiers.
  • Modifiers identified in screen alter VAP(P58S) induced bouton size.
  • Modulation of TOR pathway components suppresses VAP(P58S) bouton phenotypes.
  • Modulation of Tor pathway components suppresses VAP(wt) bouton phenotypes.
  • Rapamycin feeding mitigates VAP(P58S) phenotype.

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Ambegaokar, S.S., Roy, B., Jackson, G.R. (2010). Neurodegenerative models in Drosophila: polyglutamine disorders, Parkinson disease, and amyotrophic lateral sclerosis. Neurobiol Dis. 40: 29-39. PubMed ID: 20561920

Lloyd, T.E., Taylor, J.P. (2010). Flightless flies: Drosophila models of neuromuscular disease. Ann N Y Acad Sci. 1184: e1-20. PubMed ID: 20329357

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More in IF

The Amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors

hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction

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

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Recent Updates

Yang, D., Abdallah, A., Li, Z., Lu, Y., Almeida, S. and Gao, F.B. (2015). FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions. Acta Neuropathol [Epub ahead of print]. PubMed ID: 26031661

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Sreedharan, J., Neukomm, L.J., Brown, R.H. Jr. and Freeman, M.R. (2015). Age-Dependent TDP-43-Mediated Motor Neuron Degeneration Requires GSK3, hat-trick, and xmas-2. Curr Biol 25: 2130-2136. PubMed ID: 26234214

Cheng, C.W., Lin, M.J. and Shen, C.J. (2015). Rapamycin alleviates pathogenesis of a new Drosophila model of ALS-TDP. J Neurogenet [Epub ahead of print]. PubMed ID: 26219309

Tran, H., Almeida, S., Moore, J., Gendron, T.F., Chalasani, U., Lu, Y., Du, X., Nickerson, J.A., Petrucelli, L., Weng, Z. and Gao, F.B. (2015). Differential toxicity of nuclear RNA foci versus dipeptide repeat proteins in a Drosophila model of C9ORF72 FTD/ALS. Neuron 87: 1207-1214. PubMed ID: 26402604

Di Salvio, M., Piccinni, V., Gerbino, V., Mantoni, F., Camerini, S., Lenzi, J., Rosa, A., Chellini, L., Loreni, F., Carrì, M.T., Bozzoni, I., Cozzolino, M. and Cestra, G. (2015). Pur-alpha functionally interacts with FUS carrying ALS-associated mutations. Cell Death Dis 6: e1943. PubMed ID: 26492376

Cragnaz, L., Klima, R., De Conti, L., Romano, G., Feiguin, F., Buratti, E., Baralle, M. and Baralle, F.E. (2015). An age-related reduction of brain TBPH/TDP-43 levels precedes the onset of locomotion defects in a Drosophila ALS model. Neuroscience 311: 415-421. PubMed ID: 26518462

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Date revised: 4 Sep 2015

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