PIKFYVE inhibition mitigates disease in models of diverse forms of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that results from many diverse genetic causes. Although therapeutics specifically targeting known causal mutations may rescue individual types of ALS, these approaches cannot treat most cases since they have unknown genetic etiology. Thus, there is a pressing need for therapeutic strategies that rescue multiple forms of ALS. Here, we show that pharmacological inhibition of PIKFYVE kinase activates an unconventional protein clearance mechanism involving exocytosis of aggregation-prone proteins. Reducing PIKFYVE activity ameliorates ALS pathology and extends survival of animal models and patient-derived motor neurons representing diverse forms of ALS including C9ORF72, TARDBP, FUS, and sporadic. These findings highlight a potential approach for mitigating ALS pathogenesis that does not require stimulating macroautophagy or the ubiquitin-proteosome system.

Ferrying wingless across the synaptic cleft.

Secreted Wnt morphogens mediate cell-cell communication, but the mechanism of Wnt transfer between cells is unknown. Korkut et al. (2009) report that the transmembrane protein Evi is a versatile carrier that guides Wingless to presynaptic terminals of motor neurons and then escorts it across the synaptic cleft. In postsynaptic muscles, Evi promotes Frizzled-2 trafficking.

The M1311V variant of ATP7A is associated with impaired trafficking and copper homeostasis in models of motor neuron disease.

Disruption in copper homeostasis causes a number of cognitive and motor deficits. Wilson's disease and Menkes disease are neurodevelopmental disorders resulting from mutations in the copper transporters ATP7A and ATP7B, with ATP7A mutations also causing occipital horn syndrome, and distal motor neuropathy. A 65 year old male presenting with brachial amyotrophic diplegia and diagnosed with amyotrophic lateral sclerosis (ALS) was found to harbor a p.Met1311Val (M1311V) substitution variant in ATP7A. ALS is a fatal neurodegenerative disease associated with progressive muscle weakness, synaptic deficits and degeneration of upper and lower motor neurons. To investigate the potential contribution of the ATP7AM1311V variant to neurodegeneration, we obtained and characterized both patient-derived fibroblasts and patient-derived induced pluripotent stem cells differentiated into motor neurons (iPSC-MNs), and compared them to control cell lines. We found reduced localization of ATP7AM1311V to the trans-Golgi network (TGN) at basal copper levels in patient-derived fibroblasts and iPSC-MNs. In addition, redistribution of ATP7AM1311V out of the TGN in response to increased extracellular copper was defective in patient fibroblasts. This manifested in enhanced intracellular copper accumulation and reduced survival of ATP7AM1311V fibroblasts. iPSC-MNs harboring the ATP7AM1311V variant showed decreased dendritic complexity, aberrant spontaneous firing, and decreased survival. Finally, expression of the ATP7AM1311V variant in Drosophila motor neurons resulted in motor deficits. Apilimod, a drug that targets vesicular transport and recently shown to enhance survival of C9orf72-ALS/FTD iPSC-MNs, also increased survival of ATP7AM1311V iPSC-MNs and reduced motor deficits in Drosophila expressing ATP7AM1311V. Taken together, these observations suggest that ATP7AM1311V negatively impacts its role as a copper transporter and impairs several aspects of motor neuron function and morphology.

Increased Cardiac Arrhythmogenesis Associated With Gap Junction Remodeling With Upregulation of RNA-Binding Protein FXR1.

BACKGROUND: Gap junction remodeling is well established as a consistent feature of human heart disease involving spontaneous ventricular arrhythmia. The mechanisms responsible for gap junction remodeling that include alterations in the distribution of, and protein expression within, gap junctions are still debated. Studies reveal that multiple transcriptional and posttranscriptional regulatory pathways are triggered in response to cardiac disease, such as those involving RNA-binding proteins. The expression levels of FXR1 (fragile X mental retardation autosomal homolog 1), an RNA-binding protein, are critical to maintain proper cardiac muscle function; however, the connection between FXR1 and disease is not clear. METHODS: To identify the mechanisms regulating gap junction remodeling in cardiac disease, we sought to identify the functional properties of FXR1 expression, direct targets of FXR1 in human left ventricle dilated cardiomyopathy (DCM) biopsy samples and mouse models of DCM through BioID proximity assay and RNA immunoprecipitation, how FXR1 regulates its targets through RNA stability and luciferase assays, and functional consequences of altering the levels of this important RNA-binding protein through the analysis of cardiac-specific FXR1 knockout mice and mice injected with 3xMyc-FXR1 adeno-associated virus. RESULTS: FXR1 expression is significantly increased in tissue samples from human and mouse models of DCM via Western blot analysis. FXR1 associates with intercalated discs, and integral gap junction proteins Cx43 (connexin 43), Cx45 (connexin 45), and ZO-1 (zonula occludens-1) were identified as novel mRNA targets of FXR1 by using a BioID proximity assay and RNA immunoprecipitation. Our findings show that FXR1 is a multifunctional protein involved in translational regulation and stabilization of its mRNA targets in heart muscle. In addition, introduction of 3xMyc-FXR1 via adeno-associated virus into mice leads to the redistribution of gap junctions and promotes ventricular tachycardia, showing the functional significance of FXR1 upregulation observed in DCM. CONCLUSIONS: In DCM, increased FXR1 expression appears to play an important role in disease progression by regulating gap junction remodeling. Together this study provides a novel function of FXR1, namely, that it directly regulates major gap junction components, contributing to proper cell-cell communication in the heart.

PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease for which there is no cure. We have previously developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, we designed a drug screening strategy based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1200 FDA-approved compounds, we identified the PPARγ agonist pioglitazone as neuroprotective in Drosophila. Here, we show that pioglitazone can rescue TDP-43-dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS, we find that pioglitazone is also neuroprotective when FUS, but not SOD1, is expressed in motor neurons. Interestingly, survival analyses of TDP or FUS models show no increase in lifespan, which is consistent with recent clinical trials. Using a pharmacogenetic approach, we show that the predicted Drosophila PPARγ homologs, E75 and E78, are in vivo targets of pioglitazone. Finally, using a global metabolomic approach, we identify a set of metabolites that pioglitazone can restore in the context of TDP-43 expression in motor neurons. Taken together, our data provide evidence that modulating PPARγ 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, our data also identify several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials.

PABPN1 suppresses TDP-43 toxicity in ALS disease models.

TAR DNA-binding protein 43 (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. Here, we report 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.

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

RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). Here we identify Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. We find that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with polyA binding protein 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. Our 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.

Futsch/MAP1B mRNA is a translational target of TDP-43 and is neuroprotective in a Drosophila model of amyotrophic lateral sclerosis.

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

Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS.

The RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causative locus and as a marker of pathology. With several missense mutations being identified within TDP-43, efforts have been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms. Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapitulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here we report a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts more severe effects on neuromuscular junction architecture, viability and motor neuron loss compared with the A315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indicating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is the sole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and genetic interaction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype and suggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require the cytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 chaperone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypic consequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations are critical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders.

Desmoplakin and talin2 are novel mRNA targets of fragile X-related protein-1 in cardiac muscle.

RATIONALE: The proper function of cardiac muscle requires the precise assembly and interactions of numerous cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and force transmission. Evidence suggests that posttranscriptional regulation is critical for muscle function, but the mechanisms involved remain understudied. OBJECTIVE: To investigate the molecular mechanisms and targets of the muscle-specific fragile X mental retardation, autosomal homolog 1 (FXR1), an RNA binding protein whose loss leads to perinatal lethality in mice and cardiomyopathy in zebrafish. METHODS AND RESULTS: Using RNA immunoprecipitation approaches we found that desmoplakin and talin2 mRNAs associate with FXR1 in a complex. In vitro assays indicate that FXR1 binds these mRNA targets directly and represses their translation. Fxr1 KO hearts exhibit an up-regulation of desmoplakin and talin2 proteins, which is accompanied by severe disruption of desmosome as well as costamere architecture and composition in the heart, as determined by electron microscopy and deconvolution immunofluorescence analysis. CONCLUSIONS: Our findings reveal the first direct mRNA targets of FXR1 in striated muscle and support translational repression as a novel mechanism for regulating heart muscle development and function, in particular the assembly of specialized cytoskeletal structures.

Modelling TDP-43 proteinopathy in Drosophila uncovers shared and neuron-specific targets across ALS and FTD relevant circuits.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comprise a spectrum of neurodegenerative diseases linked to TDP-43 proteinopathy, which at the cellular level, is characterized by loss of nuclear TDP-43 and accumulation of cytoplasmic TDP-43 inclusions that ultimately cause RNA processing defects including dysregulation of splicing, mRNA transport and translation. Complementing our previous work in motor neurons, here we report a novel model of TDP-43 proteinopathy based on overexpression of TDP-43 in a subset of Drosophila Kenyon cells of the mushroom body (MB), a circuit with structural characteristics reminiscent of vertebrate cortical networks. This model recapitulates several aspects of dementia-relevant pathological features including age-dependent neuronal loss, nuclear depletion and cytoplasmic accumulation of TDP-43, and behavioral deficits in working memory and sleep that occur prior to axonal degeneration. RNA immunoprecipitations identify several candidate mRNA targets of TDP-43 in MBs, some of which are unique to the MB circuit and others that are shared with motor neurons. Among the latter is the glypican Dally-like-protein (Dlp), which exhibits significant TDP-43 associated reduction in expression during aging. Using genetic interactions we show that overexpression of Dlp in MBs mitigates TDP-43 dependent working memory deficits, conistent with Dlp acting as a mediator of TDP-43 toxicity. Substantiating our findings in the fly model, we find that the expression of GPC6 mRNA, a human ortholog of dlp, is specifically altered in neurons exhibiting the molecular signature of TDP-43 pathology in FTD patient brains. These findings suggest that circuit-specific Drosophila models provide a platform for uncovering shared or disease-specific molecular mechanisms and vulnerabilities across the spectrum of TDP-43 proteinopathies.

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

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

The cell adhesion molecule Roughest depends on beta(Heavy)-spectrin during eye morphogenesis in Drosophila.

Cell junctions have both structural and morphogenetic roles, and contain complex mixtures of proteins whose interdependencies are still largely unknown. Junctions are also major signaling centers that signify correct integration into a tissue, and modulate cell survival. During Drosophila eye development, the activity of the immunoglobulin cell adhesion molecule Roughest (also known as Irregular chiasm C-roughest protein) mediates interommatidial cell (IOC) reorganization, leading to an apoptotic event that refines the retinal lattice. Roughest and the cadherin-based zonula adherens (ZA) are interdependent and both are modulated by the apical polarity determinant, Crumbs. Here we describe a novel relationship between the Crumbs partner beta(Heavy)-spectrin (beta(H)), the ZA and Roughest. Ectopic expression of the C-terminal segment 33 of beta(H) (betaH33) induces defects in retinal morphogenesis, resulting the preferential loss of IOC. This effect is associated with ZA disruption and Roughest displacement. In addition, loss-of-function karst and roughest mutations interact to cause a synergistic and catastrophic effect on retinal development. Finally, we show that beta(H) coimmunoprecipitates with Roughest and that the distribution of Roughest protein is disrupted in karst mutant tissue. These results suggest that the apical spectrin membrane skeleton helps to coordinate the Cadherin-based ZA with Roughest-based morphogenesis.

Dysregulation of Translation in TDP-43 Proteinopathies: Deficits in the RNA Supply Chain and Local Protein Production.

Local control of gene expression provides critical mechanisms for regulating development, maintenance and plasticity in the nervous system. Among the strategies known to govern gene expression locally, mRNA transport and translation have emerged as essential for a neuron's ability to navigate developmental cues, and to establish, strengthen and remove synaptic connections throughout lifespan. Substantiating the role of RNA processing in the nervous system, several RNA binding proteins have been implicated in both developmental and age dependent neurodegenerative disorders. Of these, TDP-43 is an RNA binding protein that has emerged as a common denominator in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and related disorders due to the identification of causative mutations altering its function and its accumulation in cytoplasmic aggregates observed in a significant fraction of ALS/FTD cases, regardless of etiology. TDP-43 is involved in multiple aspects of RNA processing including splicing, transport and translation. Given that one of the early events in disease pathogenesis is mislocalization from the nucleus to the cytoplasm, several studies have focused on elucidating the pathogenic role of TDP-43 in cytoplasmic translation. Here we review recent findings describing TDP-43 translational targets and potential mechanisms of translation dysregulation in TDP-43 proteinopathies across multiple experimental models including cultured cells, flies, mice and patient derived neurons.

TDP-43 Proteinopathy Causes Broad Metabolic Alterations including TCA Cycle Intermediates and Dopamine Levels in Drosophila Models of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal, complex neurodegenerative disorder that causes selective degeneration of motor neurons. ALS patients exhibit symptoms consistent with altered cellular energetics such as hypermetabolism, weight loss, dyslipidemia, insulin resistance, and altered glucose tolerance. Although evidence supports metabolic changes in ALS patients, metabolic alterations at a cellular level remain poorly understood. Here, we used a Drosophila model of ALS based on TDP-43 expression in motor neurons that recapitulates hallmark features of motor neuron disease including TDP-43 aggregation, locomotor dysfunction, and reduced lifespan. To gain insights into metabolic changes caused by TDP-43, we performed global metabolomic profiling in larvae expressing TDP-43 (WT or ALS associated mutant variant, G298S) and identified significant alterations in several metabolic pathways. Here, we report alterations in multiple metabolic pathways and highlight upregulation of Tricarboxylic acid (TCA) cycle metabolites and defects in neurotransmitter levels. We also show that modulating TCA cycle flux either genetically or by dietary intervention mitigates TDP-43-dependent locomotor defects. In addition, dopamine levels are significantly reduced in the context of TDP-43G298S, and we find that treatment with pramipexole, a dopamine agonist, improves locomotor function in vivo in Drosophila models of TDP-43 proteinopathy.

Heads-up: new roles for the fragile X mental retardation protein in neural stem and progenitor cells.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function for Fragile X Mental Retardation Protein (FMRP), a selective RNA-binding protein with a demonstrated role in the localized translation of target mRNAs at synapses. Several recent studies provide compelling evidence for a new role of FMRP in the development of the nervous system, during neurogenesis. Using a multi-faceted approach and a variety of model systems ranging from cultured neurospheres and progenitor cells to in vivo Drosophila and mouse models these reports indicate that FMRP is required for neural stem and progenitor cell proliferation, differentiation, survival, as well as regulation of gene expression. Here we compare and contrast these recent reports and discuss the implications of FMRP's new role in embryonic and adult neurogenesis, including the development of novel therapeutic approaches to FXS and related neurological disorders such as autism.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy.

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700µÎ´6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700µÎ´6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700µÎ´6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

TDP-43 proteinopathy alters the ribosome association of multiple mRNAs including the glypican Dally-like protein (Dlp)/GPC6.

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. Using tagged ribosome affinity purifications in Drosophila models of TDP-43 proteinopathy, we identified TDP-43 dependent translational alterations in motor neurons impacting the spliceosome, pentose phosphate and oxidative phosphorylation pathways. A subset of the mRNAs with altered ribosome association are also enriched in TDP-43 complexes suggesting that they may be direct targets. Among these, dlp mRNA, which encodes the glypican Dally like protein (Dlp)/GPC6, a wingless (Wg/Wnt) signaling regulator is insolubilized both in flies and patient tissues with TDP-43 pathology. While Dlp/GPC6 forms puncta in the Drosophila neuropil and ALS spinal cords, it is reduced at the neuromuscular synapse in flies suggesting compartment specific effects of TDP-43 proteinopathy. These findings together with genetic interaction data show that Dlp/GPC6 is a novel, physiologically relevant target of TDP-43 proteinopathy.