TARDBP pathogenic mutations increase cytoplasmic translocation of TDP-43 and cause reduction of endoplasmic reticulum Ca2Â + signaling in motor neurons

- Cellular models expressing the full-length genomic DNA TARDBP gene with M337V or A382T mutations were generated.
- The M337V mutation induces cytoplasmic mislocalization of TDP-43 which is further potentiated by oxidative stress.
- ER stress induces cytoplasmic mislocalization of wild-type TDP-43 (wt-TDP).
- M337V and A382T mutations associate with elevated Bcl-2 levels.
- The M337V and A382T mutations induce Bcl-2 mediated reduced ER Ca2 + signaling.

The transactive response DNA binding protein (TDP-43) is a major component of the characteristic neuronal cytoplasmic inclusions seen in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, are found in sporadic and familial ALS cases. To study the molecular mechanisms of cellular toxicity due to TDP-43 mutations we generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying one of two ALS-associated mutations, A382T or M337V, which were used to generate site-specific bacterial artificial chromosome (BAC) human stable cell lines and BAC transgenic mice. In cell lines and primary motor neurons in culture, TDP-M337V mislocalized to the cytoplasm more frequently than wild-type TDP (wt-TDP) and TDP-A382T, an effect potentiated by oxidative stress. Expression of mutant TDP-M337V correlated with increased apoptosis detected by cleaved caspase-3 staining. Cells expressing mislocalized TDP-M337V spontaneously developed cytoplasmic aggregates, while for TDP-A382T aggregates were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin. Lowering Ca2 + concentration in the ER of wt-TDP cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca2 + dysregulation as a potential mediator of pathology through alterations in Bcl-2 protein levels. Ca2 + signaling from the ER was impaired in immortalized cells and primary neurons carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca2 + stores content and delayed Ca2 + release compared with cells carrying wt-TDP. The deficits in Ca2 + release in human cells correlated with the upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored the amplitude of Ca2 + oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 and Bcl-2 mediated ER Ca2 + signaling dysregulation.