The molecular mechanisms of intracellular TDP-43 aggregates

TAR DNA binding protein of 43 kDa (TDP-43) was identified as a major component of the ubiquitin-positive inclusions found in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). To investigate the molecular mechanisms underlying the formation of intracellular aggregates of TDP-43, we have established cellular models of intracellular TDP-43 aggregates similar to the pathological inclusions found in the brain FTLD and ALS patients. Deletion of the sequences near the nuclear localization signal resulted in cytoplasmic localization of TDP-43, whereas deletion mutants that lack the region around the RNA recognition motif localized in the nuclei, forming unique dot-like structures. Proteasome inhibition caused these structures to assemble into round aggregates that are reactive with anti-phosphorylated TDP-43 and anti-ubiquitin antibodies. Green fluorescent protein (GFP)-tagged N-terminal or C-terminal fragments of TDP-43 also promoted the formation of the abnormal intracellular inclusions. Co-expression of DsRed-tagged full-length TDP-43 with GFP-tagged C-terminal fragments of TDP-43 causes the formation of cytoplasmic inclusions that are positive for both GFP and DsRed. Cells with GFP and DsRed positive inclusions were nagative for normal nuclear staining for endogenous TDP-43. These results suggest that GFP-tagged C-terminal fragments of TDP-43 are bound not only to transfected DsRed-full-length TDP-43 but also to endogenous TDP-43. Endogenous TDP-43 may also be included in the cytoplasmic aggregates of TDP-43 C-terminal fragments, which results in the failure of its nuclear localization and therefore inhibits its function. We propose that the generation and aggregation of phosphorylated C-terminal fragments of TDP-43 play a primary role in the formation of inclusions, and the resultant loss of normal TDP-43 localization leads to neuronal degeneration in TDP-43 proteinopathy.