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.

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.

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.

AbstractAmyotrophic 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 a combination of RNA immunoprecipitations and tagged ribosome affinity purifications in Drosophila models of TDP-43 proteinopathy, we identified several TDP-43 dependent translational alterations including the glypican Dally like protein (Dlp), a wingless (Wg/Wnt) signaling component. Here we show that dlp mRNA is enriched in TDP-43 protein complexes and depleted from ribosomes in the context of TDP-43 proteinopathy. We also show that dlp mRNA is insolubilized and Dlp protein is significantly depleted from neuromuscular synapses while steady state transcript levels remain unchanged, consistent with mRNA sequestration and translation inhibition. Furthermore, we find that Dlp accumulates in cytoplasmic puncta in the Drosophila ventral cord, supporting the possibility of added axonal transport deficits, a well-established ALS phenotype. Notably, overexpression of dlp in Drosophila motor neurons is sufficient to mitigate TDP-43 dependent neurodegenerative phenotypes indicating that dlp is a physiologically relevant target of TDP-43. Finally, we show that similar to Dlp in the Drosophila ventral cord, the human ortholog GPC6 forms puncta-like structures in ALS patient spinal cords, further supporting a role for Dlp/GPC6 in TDP-43 induced neurodegeneration.