Evaluating TDP-43 Targets in Amyotrophic Lateral Sclerosis Using Drosophila and Patient Spinal Cords Alexander Blythe1, Erik Lehmkuhl1, Suvitha Loganathan1, Eric Alsop4, Dianne Barrameda1, Bhavani B. Siddegowda1, Archi Joardar1, Kendall Jensen4, Daniela C. Zarnescu1,2,3 1Department of Molecular and Cellular Biology, 2Department of Neuroscience, 3Department of Neurology, 4Translational Genomics Research Institute Introduction and Background DLP DLP HRP w1118 D42>TDP-43WT D42>TDP-43G298S Figure 3. Images of dissected larval neuromuscular junctions (NMJ) treated with immunofluorescent staining. Horseradish peroxidase (HRP) is used as a control to emphasize changes in DLP levels. An additional diagram showing the different nervous tissue types in developing Drosophila larvae is shown. Sample Type Spinal Region TDP-43 Pathology Disease Mutation Control Thoracic No NA Control Lumbar No NA Control Cervical No NA Disease Thoracic Unknown C9orf72 Disease Lumbar Yes TDP-43 Disease Cervical Yes C9orf72 Disease Lumbar Yes OPTN, SETX Disease Lumbar Yes DCTN1 Disease Lumbar Yes NEK1 Figure 2. Larval turning times were used to assay locomotor function in 3rd instar larvae. DLP overexpression (OE) resulted in a rescue of locomotor function while DLP knockdown (RNAi) exacerbated TDP-43-induced locomotor dysfunction. N=33- 40. Asterisks (*) indicate p-values less than 0.05. Significance tests were performed using the Mann U Whitney Test. Effect of DLP mRNA Levels on Larval Motor Function DLP Visualized at the Larval Neuromuscular Junction DLP Levels in VNCs and NMJs During TDP-43 Proteinopathy DLP Levels in VNCs and NMJs During TDP-43 Proteinopathy Figure 5. Quantification of DLP levels in the ventral nerve cords (VNCs) of 3rd instar larvae. Western blots were run on homogenized VNCs from control larvae and larvae expressing TDP-43WT and TDP- 43G298S in the motor neurons. 10 VNCs were used in each sample. DLP intensity was normalized to Ponceau stain. Figure 4. Quantification of DLP levels in 4 terminal boutons at the NMJ of 3rd instar larvae relative to bouton size. N=3. Comparison of w1118 to D42>TDP-43G298S yields a p-value of 0.044. Asterisks (*) denote p-values less than 0.05. DLP Human Ortholog (GPC4) Levels in Spinal Tissue In 97% of ALS cases and about 45% of frontotemporal dementia cases, insoluble cytoplasmic aggregates containing highly phosphorylated TDP-43 have been identified as pathological hallmarks1. Considering these complexes and their noted neurotoxicity, a current hypothesis for TDP-43’s potential role in modifying neurodegenerative phenotypes during ALS is the ribostasis hypothesis, which posits that TDP-43 binds to and sequesters mRNAs into cytoplasmic insoluble complexes in motor neurons. These aggregates result in translational repression of affected mRNAs and severely dysregulate protein homeostasis, leading to motor neuron stress and eventually death2. Figure 1. Ribostasis hypothesis: Cytoplasmic TDP-43-induced sequestration of mRNAs into insoluble complexes results in translational repression of affected mRNAs, thereby lowering protein levels and disrupting proteostasis in motor neurons. A Drosophila model of ALS has been developed by overexpressing either wild- type or mutant TDP-43 in motor neurons, using a motor neuron-specific D42 driver with different versions of the TDP-43 bound to UAS promoters. These models have demonstrated ALS phenotypes including cytoplasmic TDP-43 aggregates, reduced lifespan, sleep fragmentation, and locomotor dysfunction3. Following tagged ribosome affinity purifications (TRAP) and RNA immunoprecipitations (RIP), potential mRNA candidates with both low ribosome association and co-precipitation with TDP-43 have been identified4. One such candidate is dally-like protein (DLP), a glypican present in the extracellular matrix of developing motor neurons. By determining if changed DLP levels exacerbate or rescue ALS phenotypes in developing larvae and observing if DLP protein levels are alter