Despite their importance as signaling hubs, the function of mitochondria-ER contact sites in mitochondrial quality control pathways remains unexplored. Here we describe a mechanism by which Mfn2, a mitochondria-ER tether, gates the autophagic turnover of mitochondria by PINK1 and parkin. Mitochondria-ER appositions are destroyed during mitophagy, and reducing mitochondria-ER contacts increases the rate of mitochondrial degradation. Mechanistically, parkin/PINK1 catalyze a rapid burst of Mfn2 phosphoubiquitination to trigger p97-dependent disassembly of Mfn2 complexes from the outer mitochondrial membrane, dissociating mitochondria from the ER. We additionally demonstrate that a major portion of the facilitatory effect of p97 on mitophagy is epistatic to Mfn2 and promotes the availability of other parkin substrates such as VDAC1. Finally, we reconstitute the action of these factors on Mfn2 and VDAC1 ubiquitination in a cell-free assay. We show that mitochondria-ER tethering suppresses mitophagy and describe a parkin-/PINK1-dependent mechanism that regulates the destruction of mitochondria-ER contact sites.

The best-known hallmarks of Parkinson’s disease (PD) are the motor deficits that result from the degeneration of dopaminergic neurons in the substantia nigra. Dopaminergic neurons are thought to be particularly susceptible to mitochondrial dysfunction. As such, for their survival, they rely on the elaborate quality control mechanisms that have evolved in mammalian cells to monitor mitochondrial function and eliminate dysfunctional mitochondria. Mitophagy is a specialized type of autophagy that mediates the selective removal of damaged mitochondria from cells, with the net effect of dampening the toxicity arising from these dysfunctional organelles. Despite an increasing understanding of the molecular mechanisms that regulate the removal of damaged mitochondria, the detailed molecular link to PD pathophysiology is still not entirely clear. Herein, we review the fundamental molecular pathways involved in PINK1/Parkin-mediated and receptor-mediated mitophagy, the evidence for the dysfunction of these pathways in PD, and recently-developed state-of-the art assays for measuring mitophagy in vitro and in vivo.

In Parkinson’s disease and other synucleinopathies, α-synuclein (α-Syn) misfolds and forms Ser ¹²⁹ -phosphorylated aggregates (pSyn ¹²⁹ ). The factors controlling this process are largely unknown. Here, we used arrayed CRISPR-mediated gene activation and ablation to discover new pSyn ¹²⁹ modulators. Using quadruple-guide RNAs (qgRNAs) and Cas9, or an inactive Cas9 version fused to a synthetic transactivator, we ablated 2304 and activated 2428 human genes related to mitochondrial, trafficking and motility function in HEK293 cells. After exposure of cells to α-Syn fibrils, pSyn ¹²⁹ signals were recorded by high-throughput fluorescence microscopy and aggregates were identified by image analysis. We found that pSyn ¹²⁹ was increased by activating the mitochondrial protein OXR1, which decreased ATP levels and altered the mitochondrial membrane potential. Instead, pSyn ¹²⁹ was reduced by ablation of the endoplasmic reticulum (ER)-associated protein EMC4, which enhanced ER-driven autophagic flux and lysosomal clearance. OXR1 activation preferentially modulated cellular reactions to fibrils derived from multiple system atrophy (MSA) patients, whereas EMC4 ablation broadly reduced pSyn ¹²⁹ across diverse α-Syn polymorphs. These findings were confirmed in human iPSC-derived cortical and dopaminergic neurons, where OXR1 preferentially promoted somatic aggregation and EMC4 reduced both somatic and neuritic aggregates. These results uncover previously unrecognized roles for OXR1 and EMC4 in α-Syn aggregation, thereby broadening our mechanistic understanding of synucleinopathies.

A central hallmark of Parkinson′s disease (PD) is the spread of α-Synuclein (α-Syn) aggregates, which is thought to contribute to its progressive nature. To better understand the mechanism of cellular internalization of α-Syn fibrils, we conducted a genome wide CRISPR activation (CRISPRa) screen to identify genetic modifiers of fibril uptake. We report here BRCA associated protein 1 (BAP1) as a regulator of fibril uptake into cells and validate its association with the genetic risk of PD. BAP1 regulates fibril entry into cells by acting as a master transcriptional regulator of the cell surface glycoproteome. BAP1 downregulates Heparan Sulfate Proteoglycan (HSPG) and upregulates O-linked glycoprotein expression, the net effect of which is to reduce fibril internalization. The effect of enhancing BAP1 expression is cell-type specific, as it reduces αSyn uptake in iPSC-derived dopaminergic neurons, but not in microglia. Using human midbrain organoids with integrated microglia, we find this effect is mediated through ASXL3 another PD risk gene and a non-obligatory component of the BAP1 Polycomb Repressive-Deubiquitinase Complex (PR-DUB). These findings are consistent with higher BAP1/ASXL3 co-expression in regions of the brain less vulnerable to PD pathology. Together, the work uncovers a novel role for BAP1/ASXL3 in regulating αSyn fibril internalization by remodelling the cell surface glycoproteome.

The generation of mitochondrial-derived vesicles (MDVs) is implicated in a plethora of vital cell functions, from mitochondrial quality control to peroxisomal biogenesis. The discovery of distinct subtypes of MDVs has revealed the selective inclusion of mitochondrial cargo in response to varying stimuli. However, the true scope and variety of MDVs is currently unclear, and unbiased approaches have yet to be used to understand their biology. Furthermore, as mitochondrial dysfunction has been implicated in many neurodegenerative diseases, it is essential to understand MDV pathways in the nervous system. To address this, we sought to identify the cargo in brain MDVs. We used an in vitro budding assay and proteomic approach to identify proteins selectively enriched in MDVs. 72 proteins were identified as MDV enriched, of which 31% were OXPHOS proteins. Interestingly, the OXPHOS proteins localized to specific modules of the respiratory complexes, hinting at the inclusion of sub-assemblies in MDVs. Small TIM chaperones were also highly enriched in MDVs, linking mitochondrial chaperone-mediated protein transport to MDV formation. As the two Parkinson’s disease genes PINK1 and Parkin have been previously implicated in MDV biogenesis in response to oxidative stress, we compared the MDV proteomes from the brains of wild-type mice with those of PINK1 ^-/- and Parkin ^-/- mice. No significant difference was found, suggesting that PINK1- and Parkin-dependent MDVs make up a small proportion of all MDVs in the brain. Our findings demonstrate a previously uncovered landscape of MDV complexity and provide a foundation from which to discover further novel MDV functions.

Un tiers de la population européenne est touché par au moins un trouble du cerveau. En effet, la maladie d’Alzheimer, et les gliomes, représentent respectivement le syndrome de démence et les tumeurs cérébrales les plus fréquentes chez l’homme. Plusieurs études épidémiologiques ont montré l’existence d’une corrélation inverse entre le risque de développer une maladie neurodégénérative et un cancer cérébral. Ceci suggère la présence de dénominateurs moléculaires communs entre ces pathologies. Dans les deux cas, un dysfonctionnement mitochondrial est rapporté, représentant une caractéristique partagée par ces deux troubles neurologiques. La protéine kinase mitochondriale PINK1 responsable, lorsqu’elle est mutée, d’une forme précoce et familiale de Parkinson, est particulièrement impliquée dans les processus de maintien de l’homéostasie mitochondriale. Par conséquent, les mécanismes moléculaires régulant PINK1 ainsi que leurs impacts au cours des désordres mitochondriaux répertoriés dans la maladie d’Alzheimer et les tumeurs cérébrales, ont suscité un intérêt central, lors de ma thèse. Au cours de ce travail, nous avons examiné certaines des fonctions mitochondriales de PinK1, associées au maintien de l’homéostasie mitochondriale dans un contexte « Alzheimerisé ». Nous mettons en évidence le rôle de la γ-secretase dans la physiologie mitochondriale en contrôlant la régulation transcriptionnelle de PINK1 par l’AICD, le fragment généré conjointement avec le peptide amyloïde toxique Aβ, à partir du précurseur βAPP. Nous montrons de surcroît, l’initiation de cette régulation par la parkine.