Regulation of endoplasmic reticulum-mitochondria tethering and Ca²⁺ fluxes by TDP-43 via GSK3?

Mitochondria-ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca uptake after ER Ca release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca-mediated ER-mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3?, previously associated with MERCs disruption.