Membrane depolarization activates the mitochondrial protease OMA1 by stimulating self‐cleavage

Mitochondrial inner membrane fusion depends on the dynamin‐related GTPase OPA1 and the function of OPA1 is regulated by proteolytic cleavage. The mitochondrial proteases Yme1L and OMA1 cleave OPA1 at S2 and S1 sites, respectively. Here, we show that OMA1 is cleaved to a short form (S‐OMA1) by itself upon mitochondrial membrane depolarization; S‐OMA1 is degraded quickly but could be stabilized by CCCP treatment or Prohibitin knockdown in cells. In addition, OMA1 processing is positively correlated with OPA1 cleavage at the S1 site and the regulation of mitochondrial morphology. Thus, our results reveal the molecular mechanism for OMA1 activation toward OPA1 processing.     

Mitochondrial proteolytic stress induced by loss of mortalin function is rescued by Parkin and PINK1

The mitochondrial chaperone mortalin was implicated in Parkinson''s disease (PD) because of its reduced levels in the brains of PD patients and disease-associated rare genetic variants that failed to rescue impaired mitochondrial integrity in cellular knockdown models. To uncover the molecular mechanisms underlying mortalin-related neurodegeneration, we dissected the cellular surveillance mechanisms related to mitochondrial quality control, defined the effects of reduced mortalin function at the molecular and cellular levels and investigated the functional interaction of mortalin with Parkin and PINK1, two PD-related proteins involved in mitochondrial homeostasis. We found that reduced mortalin function leads to: (1) activation of the mitochondrial unfolded protein response (UPR(mt)), (2) increased susceptibility towards intramitochondrial proteolytic stress, (3) increased autophagic degradation of fragmented mitochondria and (4) reduced mitochondrial mass in human cells in vitro and ex vivo. These alterations caused increased vulnerability toward apoptotic cell death. Proteotoxic perturbations induced by either partial loss of mortalin or chemical induction were rescued by complementation with native mortalin, but not disease-associated mortalin variants, and were independent of the integrity of autophagic pathways. However, Parkin and PINK1 rescued loss of mortalin phenotypes via increased lysosomal-mediated mitochondrial clearance and required intact autophagic machinery. Our results on loss of mortalin function reveal a direct link between impaired mitochondrial proteostasis, UPR(mt) and PD and show that effective removal of dysfunctional mitochondria via either genetic (PINK1 and Parkin overexpression) or pharmacological intervention (rapamycin) may compensate mitochondrial phenotypes.     

Lanosterol induces mitochondrial uncoupling and protects dopaminergic neurons from cell death in a model for Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder marked by the selective degeneration of dopaminergic neurons in the nigrostriatal pathway. Several lines of evidence indicate that mitochondrial dysfunction contributes to its etiology. Other studies have suggested that alterations in sterol homeostasis correlate with increased risk for PD. Whether these observations are functionally related is, however, unknown. In this study, we used a toxin-induced mouse model of PD and measured levels of nine sterol intermediates. We found that lanosterol is significantly (～50%) and specifically reduced in the nigrostriatal regions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, indicative of altered lanosterol metabolism during PD pathogenesis. Remarkably, exogenous addition of lanosterol rescued dopaminergic neurons from 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in culture. Furthermore, we observed a marked redistribution of lanosterol synthase from the endoplasmic reticulum to mitochondria in dopaminergic neurons exposed to MPP+, suggesting that lanosterol might exert its survival effect by regulating mitochondrial function. Consistent with this model, we find that lanosterol induces mild depolarization of mitochondria and promotes autophagy. Collectively, our results highlight a novel sterol-based neuroprotective mechanism with direct relevance to PD.     

Stress‐induced OMA1 activation and autocatalytic turnover regulate OPA1‐dependent mitochondrial dynamics

The dynamic network of mitochondria fragments under stress allowing the segregation of damaged mitochondria and, in case of persistent damage, their selective removal by mitophagy. Mitochondrial fragmentation upon depolarisation of mitochondria is brought about by the degradation of central components of the mitochondrial fusion machinery. The OMA1 peptidase mediates the degradation of long isoforms of the dynamin‐like GTPase OPA1 in the inner membrane. Here, we demonstrate that OMA1‐mediated degradation of OPA1 is a general cellular stress response. OMA1 is constitutively active but displays strongly enhanced activity in response to various stress insults. We identify an amino terminal stress‐sensor domain of OMA1, which is only present in homologues of higher eukaryotes and which modulates OMA1 proteolysis and activation. OMA1 activation is associated with its autocatalyic degradation, which initiates from both termini of OMA1 and results in complete OMA1 turnover. Autocatalytic proteolysis of OMA1 ensures the reversibility of the response and allows OPA1‐mediated mitochondrial fusion to resume upon alleviation of stress. This differentiated stress response maintains the functional integrity of mitochondria and contributes to cell survival.     

Structural and mechanistic basis of Parl activity and regulation

The mitochondrial rhomboid protease Parl governs apoptosis, morphology, metabolism and might be implicated in Parkinson's disease, but the structural basis of its activity and complex regulation remain unknown. We report the discovery of γ-cleavage, a proteolytic event on the loop connecting the first transmembrane helix (TMH) of Parl to the 6-TMH catalytic rhomboid domain of the protease. This cleavage disrupts the '1+6' structure that defines every mitochondrial rhomboid and generates a new form of Parl, PROD (Parl-rhomboid-domain). Structure-function analysis of Parl suggests that γ-cleavage could be implicated in eliminating Parl proteolytic activity, and structural modeling of PROD reveals structural conservation with the bacterial rhomboid GlpG. However, unlike bacterial rhomboids, which employ a diad-based mechanism of catalysis, Parl appears to use a conserved mitochondrial rhomboid-specific Asp residue on TMH-5 in a triad-based mechanism of catalysis. This work provides unexpected insights into the structural determinants regulating Parl stability and activity in vivo, and reveals a complex cascade of proteolytic events controlling the function of the protease in the mitochondrion.     

Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30–35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer''s disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.     

Loss of Drp1 function alters OPA1 processing and changes mitochondrial membrane organization

RNAi mediated loss of Drp1 function changes mitochondrial morphology in cultured HeLa and HUVEC cells by shifting the balance of mitochondrial fission and fusion towards unopposed fusion. Over time, inhibition of Drp1 expression results in the formation of a highly branched mitochondrial network along with “bulge”-like structures. These changes in mitochondrial morphology are accompanied by a reduction in levels of Mitofusin 1 (Mfn1) and 2 (Mfn2) and a modified proteolytic processing of OPA1 isoforms, resulting in the inhibition of cell proliferation. In addition, our data imply that bulge formation is driven by Mfn1 action along with particular proteolytic short-OPA1 (s-OPA1) variants: Loss of Mfn2 in the absence of Drp1 results in an increase of Mfn1 levels along with processed s-OPA1-isoforms, thereby enhancing continuous “fusion” and bulge formation. Moreover, bulge formation might reflect s-OPA1 mitochondrial membrane remodeling activity, resulting in the compartmentalization of cytochrome c deposits. The proteins Yme1L and PHB2 appeared not associated with the observed enhanced OPA1 proteolysis upon RNAi of Drp1, suggesting the existence of other OPA1 processing controlling proteins. Taken together, Drp1 appears to affect the activity of the mitochondrial fusion machinery by unbalancing the protein levels of mitofusins and OPA1.     

PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.     

Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

Mutations in PINK1 and Parkin are associated with early-onset Parkinson''s disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser⁶⁵) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser⁶⁵ phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser⁶⁵ by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser⁶⁵ phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin''s ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser⁶⁵ is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson''s disease.     

High-dose of vitamin C supplementation reduces amyloid plaque burden and ameliorates pathological changes in the brain of 5XFAD mice

Blood–brain barrier (BBB) breakdown and mitochondrial dysfunction have been implicated in the pathogenesis of Alzheimer''s disease (AD), a neurodegenerative disease characterized by cognitive deficits and neuronal loss. Besides vitamin C being as one of the important antioxidants, recently, it has also been reported as a modulator of BBB integrity and mitochondria morphology. Plasma levels of vitamin C are decreased in AD patients, which can affect disease progression. However, investigation using animal models on the role of vitamin C in the AD pathogenesis has been hampered because rodents produce with no dependence on external supply. Therefore, to identify the pathogenic importance of vitamin C in an AD mouse model, we cross-bred 5 familial Alzheimer''s disease mutation (5XFAD) mice (AD mouse model) with ι-gulono-γ-lactone oxidase (Gulo) knockout (KO) mice, which are unable to synthesize their own vitamin C, and produced Gulo KO mice with 5XFAD mice background (KO-Tg). These mice were maintained on either low (0.66 g/l) or high (3.3 g/l) supplementation of vitamin C. We found that the higher supplementation of vitamin C had reduced amyloid plaque burden in the cortex and hippocampus in KO-Tg mice, resulting in amelioration of BBB disruption and mitochondrial alteration. These results suggest that intake of a larger amount of vitamin C could be protective against AD-like pathologies.     

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson''s disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 ¹. Because ageing is the most important risk factor, cases of PD will increase during the next decades ². Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD ^3-11.After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.     

Alzheimer's disease‐causing proline substitutions lead to presenilin 1 aggregation and malfunction

Do different neurodegenerative maladies emanate from the failure of a mutual protein folding mechanism? We have addressed this question by comparing mutational patterns that are linked to the manifestation of distinct neurodegenerative disorders and identified similar neurodegeneration‐linked proline substitutions in the prion protein and in presenilin 1 that underlie the development of a prion disorder and of familial Alzheimer's disease (fAD), respectively. These substitutions were found to prevent the endoplasmic reticulum (ER)‐resident chaperone, cyclophilin B, from assisting presenilin 1 to fold properly, leading to its aggregation, deposition in the ER, reduction of γ‐secretase activity, and impaired mitochondrial distribution and function. Similarly, reduced quantities of the processed, active presenilin 1 were observed in brains of cyclophilin B knockout mice. These discoveries imply that reduced cyclophilin activity contributes to the development of distinct neurodegenerative disorders, propose a novel mechanism for the development of certain fAD cases, and support the emerging theme that this disorder can stem from aberrant presenilin 1 function. This study also points at ER chaperones as targets for the development of counter‐neurodegeneration therapies.