Basic Science and Pathogenesis.

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that is characterized by upper and lower motor neuron death that leads to paralysis with the average survival being 3-5 years after diagnosis. The major pathological protein in ALS is TDP-43. TDP-43 becomes hyperphosphorylated and forms inclusions mainly in the cytoplasm. Although only 5-10% of familial cases are caused by mutations in TDP-43; over 90% of all sporadic and familial ALS have TDP-43 pathology. We have developed a cellular over expression model of TDP-43 aggregation.

Methods: Three familial ALS mutations (A315T, M337V, and S379P) were introduced in the TARDBP gene (3X-TDP-43) and cloned into pcDNA 3.1+ then transfected into HEK293 cells. Techniques used: Cell culture, Western blot, immunoprecipitation, immunofluorescence, nuclear/cytoplasmic fractionation, cellular solubility fractionation, and proteomics.

Results: Overexpressing 3X-TDP-43 in HEK cells results in an increase in TDP-43 C-terminal fragments, time dependent change in TDP-43 localization from nucleus to the cytoplasm, and an increase in cytoplasmic phosphorylated TDP-43. We observed reduced solubility of phosphorylated TDP-43 and a time dependent flux of LC3B and an increase in P62 which may indicate potential autophagy dysfunction. Global shotgun proteomics reveal changes in protein expression in importin subunit alpha-1 (KPNA2), heat shock 70 kDa protein 1A (HSPA1A), H2B clustered histone 17 (H2BC17), and protein disulfide-isomerase A3 (PDIA3). TDP-43 also interacts in complex with KPNA2 and PDIA3.

Conclusion: This cellular overexpression model shows a change in solubility and aggregation of phosphorylated TDP-43. Hopefully this model will provide a better understanding of the pathogenesis of TDP-43 proteinopathies.