PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation

Abstract

Mitophagy is a highly specialized process to remove dysfunctional or superfluous mitochondria through the macroautophagy/autophagy pathway, aimed at protecting cells from the damage of disordered mitochondrial metabolism and apoptosis induction. PINK1, a neuroprotective protein mutated in autosomal recessive Parkinson disease, has been implicated in the activation of mitophagy by selectively accumulating on depolarized mitochondria, and promoting PARK2/Parkin translocation to them. While these steps have been characterized in depth, less is known about the process and site of autophagosome formation upon mitophagic stimuli. A previous study reported that, in starvation-induced autophagy, the proautophagic protein BECN1/Beclin1 (which we previously showed to interact with PINK1) relocalizes at specific regions of contact between the endoplasmic reticulum (ER) and mitochondria called mitochondria-associated membranes (MAM), from which the autophagosome originates. Here we show that, following mitophagic stimuli, autophagosomes also form at MAM; moreover, endogenous PINK1 and BECN1 were both found to relocalize at MAM, where they promoted the enhancement of ER-mitochondria contact sites and the formation of omegasomes, that represent autophagosome precursors. PARK2 was also enhanced at MAM following mitophagy induction. However, PINK1 silencing impaired BECN1 enrichment at MAM independently of PARK2, suggesting a novel role for PINK1 in regulating mitophagy. MAM have been recently implicated in many key cellular events. In this light, the observed prevalent localization of PINK1 at MAM may well explain other neuroprotective activities of this protein, such as modulation of mitochondrial calcium levels, mitochondrial dynamics, and apoptosis.

Reduction of PINK1 or DJ‐1 impair mitochondrial motility in neurites and alter ER‐mitochondria contacts

“…PINK1 has a mitochondrial targeting peptide, while the mature protein has been shown to localize both to mitochondria and cytosol . On the outer mitochondrial membrane, PINK1 kinase activity recruits Parkin from the cytosol and triggers the autophagy of damaged mitochondria . PINK1 can also phosphorylate motor‐adaptor protein Miro1 and thereby modulate mitochondrial motility in a Parkin‐dependent manner .…”

From dysfunctional endoplasmic reticulum-mitochondria coupling to neurodegeneration

“…Parkin was shown to be enriched in the MAM fraction of neurons exposed to glutamate excitotoxicity (Van Laar et al, 2015) and cell lines, upon induction of mitophagy (Gelmetti et al 2017), and to ubiquitylate several proteins of the ER-mitochondria interface, including Mfn2, VDACs and Miro (Pickrell and Youle, 2015;Sarraf et al, 2013). PINK1 was also found in the MAM fraction during mitophagy (Gelmetti et al 2017).…”

“…Approximately 10% of the cases are due to monogenic mutations . Physical and/or functional association with ER-mitochondria contacts was demonstrated in the case of the PD-related proteins α-synuclein (Cali et al, 2012;Guardia-Laguarta et al, 2014), DJ-1 , PINK1 (Celardo et al, 2016;Gelmetti et al, 2017) and Parkin Celardo et al, 2016;Gautier et al, 2016;Gelmetti et al, 2017;Van Laar et al, 2015;Zheng et al, 2017).…”

“…wt or diseaseassociated variants Zampese et al, 2011); downregulation has opposite effects • ↑ juxtaposition, ↑ biosynthesis of phospholipids/cholesteryl esters, or ↑ Ca 2+ transfers in KO MEFs and fibroblasts from mutation carriers (Area-Gomez et al, 2012;Filadi et al, 2016) • ↑ juxtaposition, ↑ biosynthesis of phospholipids/cholesteryl esters in fibroblasts from mutation carriers (Area-Gomez et al, 2012) • ↑ juxtaposition, ↑ Ca 2+ transfers in mouse hippocampal neurons exposed to Aβ (Hedskog et al, 2013) • ↑ juxtaposition, ↓ decreased Aβ production upon Mfn2 ablation in cell lines (Leal et al, 2016) ↑ phospholipid/cholesteryl ester production in exposed fibroblasts and mouse hippocampal neurons (Tambini et al, (Van Laar et al, 2015); cell lines (Gelmetti et al, 2017) • ↑ juxtaposition, ↑ Ca 2+ transfers when overexpr. in cell lines; ↓ Ca 2+ transfers, mt fragmentation when down-regulated; ↓ Ca 2+ transfers, no effect on mt morphology in cells with cytosolic α-synuclein aggregates (Cali et al, 2012) • ↓ juxtaposiZon, ↓ phospholipid biosynthesis, mt fragmentaZon in cells overexpr.…”

“…PTEN induced putative kinase 1 (PINK1) cell lines ( (Gelmetti et al, 2017); our unpublished data) fibroblasts from mutation carriers; ↑ ER-to-mitochondria Ca 2+ transfers in KO MEFs and fibroblasts/iPSC-derived neurons from mutation carriers (Celardo et al, 2016;Gautier et al, 2016) • ↑juxtaposition in brain tissue from pink1 flies and fibroblasts from mutation carriers (Celardo et al, 2016) • ↑ juxtaposiZon due to presence in the MAM upon induction of mitophagy in cell lines (Gelmetti et al, 2017) Amyotrophic lateral sclerosis (ALS) (Hayashi and Su, 2007) in cell lines, together with OM membrane protein PTPIP51; ↑ for disease-associated variant VAPBP56S not present not present ↓ juxtaposiZon, perturbed Ca 2+ signaling, cell death when downregulated in mouse motor neurons (Bernard-Marissal et al, 2015) • ↓ juxtaposiZon, ↓ Ca 2+ transfers, ↑ autophagy upon down-regulation of VAPB or PTPIP51 in cell lines • VAPBP56S expr. leads to ↑ interacZon with PTPIP51, ↑ mt Ca 2+ uptake, mt clustering in cell lines (De Vos et al, 2012;Gomez-Suaga et al, 2017) • No effect of down-regulation • ↓ juxtaposiZon, ↓ Ca 2+ transfers, disruption of VAPB-PTPIP51 interaction, when overexpr.…”