Molecular dissection of TDP-43 in ALS and FTLD

Abstract

Proteomic and immunochemical analyses have shown that hyperphosphorylated TDP-43 is a major component of ubiquitin-positive inclusions from brain of frontotemporal lobar degeneration (FTLD) patients. In 2008, TDP43 gene mutations were discovered in familial and sporadic amyotrophic lateral sclerosis (ALS), indicating that TDP43 protein abnormality is associated with neurodegeneration. We raised antibodies against 36 synthetic phosphopeptides and demonstrated that abnormal phosphorylation takes place in the C-terminal region of TDP-43. One antibody, pS409/410, stained the inclusions in both FTLD and ALS brains, with no nuclear staining. Immunoblotting revealed the presence of hyperphosphorylated 45 kDa band, smearing substances and 18-26 kDa fragments in deposits, and the band patterns were different between FTLD and ALS. Overexpression of TDP43 C-terminal fragments as GFP-fusions resulted in formation of inclusions positive for antibodies to phosphorylated TDP43 and ubiquitin. We further investigated the protease-resistant TDP43 and found that it is also different between ALS and FTLD, supporting the idea that the different band patterns reflect different conformations of abnormal TDP-43. Interestingly, the C-terminal band pattern is indistinguishable among brain regions and spinal cord in each individual patient. These results suggest that abnormal TDP43 produced in a cell may be transferred to different regions and propagated during disease progression.