Department of Biomedical Sciences, University of Padova, 35131 Padova, Italy.
CNR-Neuroscience Institute, 35131 Padova, Italy.
Int J Mol Sci. 2021 Nov 1;22(21):11853. doi: 10.3390/ijms222111853.
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 GSK3β, previously associated with MERCs disruption.
线粒体-内质网接触（MERCs）由许多连接蛋白严格调节，这些连接蛋白作为两种细胞器之间的分子和功能连接，对于维持各种细胞功能至关重要。这种接触在许多神经退行性疾病的早期阶段经常受到损害，包括肌萎缩侧索硬化症（ALS）。TDP-43 是一种主要参与 RNA 代谢的核蛋白，与 ALS 发病机制和其他神经退行性疾病反复相关。尽管 TDP-43 的神经病理学机制尚不清楚，但该蛋白在细胞质包含体中的积累可能是蛋白功能丧失效应的基础。因此，我们使用 GFP 基探针进行 MERCs 定量和 aequorin 基探针进行局部 Ca 测量，结合靶向蛋白和 mRNA 分析，研究了 siRNA 介导的 TDP-43 沉默对 HeLa 细胞中 MERCs 和相关细胞参数的影响。我们的结果表明，TDP-43 的下调降低了 MERCs 的密度，从而显著减少了 ER Ca 释放后线粒体的 Ca 摄取。全面的 mRNA 和蛋白质分析并未突出显示参与 MERCs 组装或 Ca 介导的 ER-线粒体交叉对话的蛋白质表达或活性的改变，也没有通过共聚焦显微镜观察到线粒体密度和形态的改变。然而，进一步的机制检查表明，观察到的细胞变化与 GSK3β 的表达/活性增加相关，GSK3β 先前与 MERCs 的破坏有关。
