Investigating TDP-43-Dependent Neuronal Trafficking Disruption in a Drosophila Model of ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is an aggressive motor neuron disease that results in increasingly severe paralysis and eventually death. TDP-43, a nucleic acid-binding protein involved in RNA processing, forms toxic insoluble cytoplasmic puncta in most ALS cases. These aggregates are hypothesized to sequester TDP-43-recognized mRNAs and alter ribosomal access to insolubilized mRNAs, which has been proven for several development-related proteins, the most recent of which is the Drosophila glypican dally-like protein (Dlp). In larvae genetically designed to overexpress TDP-43 in motor neurons, Dlp accumulates into soluble granules within the soma, suggesting functional abnormalities in protein trafficking and/or degradation pathways. Subcellular fractionation of endomembrane trafficking compartments reveals a migration-like phenotype where the endoplasmic reticulum (ER) and late endosomes shift to fractions of differing densities in larva expressing an ALS-related mutant form of TDP-43, consistent with ER fragmentation. Following induction of lysosome-autophagosome fusion with a small molecule inhibitor of PIKfyve, Dlp granulation remained unchanged in VNCs extracted from larva expressing both the wild-type and mutant variants. This may indicate that dysregulated autophagy is not a contributing factor to Dlp mislocalization and accumulation. For future investigations involving genetic interactions, I generated Drosophila stocks that express both Dlp and TDP-43 in the motor neurons, which will allow for more efficient experiments. To conclude, Dlp’s mislocalization and granulation may result from alterations to endomembrane compartment integrity, particularly in the ER. Determining what compartments with which Dlp co-localizes and what mechanisms can induce Dlp clearance will clarify the role of Dlp granules in TDP-43-related neurodegeneration.