Abstract
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 Ca2+ measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca2+ uptake after ER Ca2+ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca2+-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 GSK3β, previously associated with MERCs disruption.
Highlights
It is widely accepted that organelles within cells are strictly connected with each other through the transmission of soluble signalling molecules and the formation of dynamic platforms of inter-organellar connections called membrane contact sites (MCSs)
The loss of (TAR) DNA binding protein 43 (TDP-43) functionality caused by its mislocalisation and accumulation into cytoplasmic inclusions may contribute to processes governing amyotrophic lateral sclerosis (ALS) pathogenesis
For the first time to our knowledge, here, we demonstrated that TDP-43 down-regulation induces both an increased expression and a higher activation of glycogen synthase kinase 3β (GSK3β), allowing us to speculate that this pathway may significantly contribute to the reported reduction in endoplasmic reticulum (ER)–mitochondria tethering after TDP-43 silencing
Summary
It is widely accepted that organelles within cells are strictly connected with each other through the transmission of soluble signalling molecules and the formation of dynamic platforms of inter-organellar connections called membrane contact sites (MCSs). MCSs maintain adjacent membranes in close proximity thanks to the presence of several molecules/proteins acting as molecular bridges and crucially contribute to the functional cross-talk between organelles [1,2]. Through MERCs, mitochondria and the ER lumen directly and continuously communicate with each other regulating key physiological processes [5], such as mitochondria biogenesis [8] and dynamics [9,10], autophagosome formation [11] and lipid synthesis and transfer [12,13,14]. Ca2+ exchange between ER and mitochondria needs to be tightly regulated because it is essential for key mitochondrial functions [16,17,19], e.g., the regulation of key enzymes of the tricarboxylic acid cycle and cellular Ca2+ buffering [20,21], while excessive mitochondrial Ca2+ accumulation can lead to cell death by triggering the opening of the mitochondrial permeability transition pore [22]
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