Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells

Abstract

Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy), mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER) and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca2+ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1), as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT) and the IP3-receptors (IP3Rs) as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca2+ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca2+ uniporter (MCU) primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca2+ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca2+ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis.

Miro proteins prime mitochondria for Parkin translocation and mitophagy

“…It has been suggested that cytosolic Ca 2+ levels are one of the factors contributing to mitophagy (East & Campanella, ; Rimessi et al , ). For example, MacVicar et al () demonstrated that treatment with the Ca 2+ chelator BAPTA‐AM blocked CCCP‐induced mitophagy and suggested that transient and local Ca 2+ spikes might be needed for mitophagy. Cherra et al () demonstrated that excess cytosolic Ca 2+ is associated with profound mitophagy and that restoring Ca 2+ homeostasis through Ca 2+ chelation or inhibition of voltage‐gated Ca 2+ channels was sufficient to prevent the mutant LRRK2 phenotype related to Parkinson disease.…”

Involvement of PINK1/parkin-mediated mitophagy in ZnO nanoparticle-induced toxicity in BV-2 cells

“…The results demonstrated that the downregulation of the PINK1 gene did not significantly change the cell growth rate, which is consistent with the results of previous research. 36 Different responses between wild-type cells and PINK1 gene knockdown cells to ZnO NP exposure were observed, and the results suggested that the downregulation of PINK1 caused a significant reduction in the survival rate after ZnO NP exposure compared with that of control cells. These results indicated that the PINK1 gene may not be an essential factor in the survival of BV-2 cells but that it possibly exerts a function in the defense of ZnO NP stimulation.…”

Peeking into Sigma-1 Receptor Functions Through the Retina

“…S1R was also reported to interact with G-protein coupled receptors (see review) [12]. Recently, evidence showed that S1R is involved in autophagy [31, 32]. In accordance, S1R also participates in ER stress responses; e.g., S1R interacts with and stabilizes ER stress sensor IRE1 [29, 33, 34].…”

“…Nevertheless, in an earlier study using a human RPE cell line (ARPE-19) and adult human primary RPE cells, Bucolo et al were able to reduce H 2 O 2 -induced DNA damage and cell loss by pre-treatment with PRE084, an effect abolished by S1R antagonists [71]. Most recently, using targeted siRNA screening in a human RPE1 cell line, MacVicar et al identified S1R as a potential regulator of autophagosome homeostasis involving mitochondrial dynamics [31]. Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents.…”

“…While autophagy is found to be an active process in the RPE in vivo [72], evidence from AMD donors indicates a decline of autophagic flux in the RPE [73]. Although not yet specifically investigated in RPE cells in vivo, S1R has been reported to influence autophagy in vitro [31, 32, 74]. A possible protective role of the S1R via autophagic regulations in RPE cells needs to be further determined experimentally.…”