Abstract 2222 Aggregation of TDP-43 in cellular overexpression model of ALS

Amyotrophic Lateral Sclerosis (ALS), or Lou Gehrig's disease, is an incurable neurodegenerative disease that affects neurons in the brain and spinal cord, causing loss of muscle control which eventually leads to death. Phosphorylated transactive response DNA-binding protein-43 (TDP-43) is the major pathological protein in both sporadic and familial ALS, forming cytoplasmic aggregates in over 95% of cases. Of the 10-15% of ALS cases that are familial, mutations in TDP-43 represent about 5% of those with a family history. We have developed an in vitro overexpression model by introducing three familial ALS mutations (A315T, M337V, and S379P) in the TDP-43 (TARDBP) gene which we define as 3X-TDP-43. TDP-43 is predominantly localized to the nucleus and under pathological conditions localized and aggregates and fragments in the cytoplasm. When 3X-TDP-43 is overexpressed in HEK cells, we see increases in fragmentation localization to both the nucleus and cytoplasm. In addition, there are deficits in autophagy flux and an increase colocalization of TDP-43 to stress granules. In addition there is a time-dependent change in localization of TDP-43 from the nucleus to the cytoplasm. This cell culture model also shows a reduction in solubility of phosphorylated TDP-43 which is similar to what is seen in ALS. Changes in metabolism are associated with progression of ALS and in our overexpression model the glycolytic enzyme, phosphoglycerate kinase one (PGK1) shows a significant decrease in cells expressing 3X-TDP-43. Through shotgun proteomic analysis there are global changes in the importin subunit alpha-1 (KPNA2), heat shock 70 kDa protein 1A (HSPA1A), H2B clustered histone 17 (H2BC17), and protein disulfide-isomerase A3 (PDIA3) expression levels. Coimmunoprecipitation reveals that these proteins complex with TDP-43. These results suggest that this 3X-TDP-43 overexpression model may provide new insights into pathophysiology and possibly provide an avenue for drug screening in vitro for those suffering from ALS and related TDP-43 proteinopathies. This work was funded by the Alzheimer's Association New Investigator Research Grant: NIRG-12-241456, the National Institute on Aging: 1K01AG042500, Delaware IDeA Network of Biomedical Research Excellence (INBRE) Pilot Award: NIH-NIGMS: 5P20GM103446, and NIH-NIGMS Centers of Biomedical Research Excellence (COBRE): 5P20GM103653.