DELE1 promotes translation-associated homeostasis, growth, and survival in mitochondrial myopathy.

Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1's protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.

CHCHD10 mutations induce tissue-specific mitochondrial DNA deletions with a distinct signature.

Mutations affecting the mitochondrial intermembrane space protein CHCHD10 cause human disease, but it is not known why different amino acid substitutions cause markedly different clinical phenotypes, including amyotrophic lateral sclerosis-frontotemporal dementia, spinal muscular atrophy Jokela-type, isolated autosomal dominant mitochondrial myopathy and cardiomyopathy. CHCHD10 mutations have been associated with deletions of mitochondrial DNA (mtDNA deletions), raising the possibility that these explain the clinical variability. Here, we sequenced mtDNA obtained from hearts, skeletal muscle, livers and spinal cords of WT and Chchd10 G58R or S59L knockin mice to characterise the mtDNA deletion signatures of the two mutant lines. We found that the deletion levels were higher in G58R and S59L mice than in WT mice in some tissues depending on the Chchd10 genotype, and the deletion burden increased with age. Furthermore, we observed that the spinal cord was less prone to the development of mtDNA deletions than the other tissues examined. Finally, in addition to accelerating the rate of naturally occurring deletions, Chchd10 mutations also led to the accumulation of a novel set of deletions characterised by shorter direct repeats flanking the deletion breakpoints. Our results indicate that Chchd10 mutations in mice induce tissue-specific deletions which may also contribute to the clinical phenotype associated with these mutations in humans.

A mitochondrial iron-responsive pathway regulated by DELE1.

The heme-regulated kinase HRI is activated under heme/iron deficient conditions; however, the underlying molecular mechanism is incompletely understood. Here, we show that iron-deficiency-induced HRI activation requires the mitochondrial protein DELE1. Notably, mitochondrial import of DELE1 and its subsequent protein stability are regulated by iron availability. Under steady-state conditions, DELE1 is degraded by the mitochondrial matrix-resident protease LONP1 soon after mitochondrial import. Upon iron chelation, DELE1 import is arrested, thereby stabilizing DELE1 on the mitochondrial surface to activate the HRI-mediated integrated stress response (ISR). Ablation of this DELE1-HRI-ISR pathway in an erythroid cell model enhances cell death under iron-limited conditions, suggesting a cell-protective role for this pathway in iron-demanding cell lineages. Our findings highlight mitochondrial import regulation of DELE1 as the core component of a previously unrecognized mitochondrial iron responsive pathway that elicits stress signaling following perturbation of iron homeostasis.

CHCHD2 and CHCHD10-related neurodegeneration: molecular pathogenesis and the path to precision therapy.

In the last decade, dominant mutations in the mitochondrial protein CHCHD10 (p.R15L and p.S59L) and its paralog CHCHD2 (p.T61I) were shown to cause familial amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD), respectively, with phenotypes that often resemble the idiopathic forms of the diseases. Different mutations in CHCHD10 cause additional neuromuscular disorders, including the lower motor neuron disease Spinal Muscular Atrophy Jokela type (SMAJ) (p.G66V) and autosomal dominant isolated mitochondrial myopathy (IMMD) (p.G58R). Modeling these disorders is revealing how mitochondrial dysfunction may drive ALS and PD pathogenesis by a gain of function mechanism, driven by protein misfolding of CHCHD2 and CHCHD10 into toxic species. It is also laying the groundwork for precision therapy of CHCHD2/CHCHD10-related neurodegeneration. In this review, we address the normal function of CHCHD2 and CHCHD10, the mechanisms of their disease pathogenesis, the strong genotype-phenotype correlations that have emerged for CHCHD10, and potential therapeutic strategies for these disorders.

OMA1 mediates local and global stress responses against protein misfolding in CHCHD10 mitochondrial myopathy.

Mitochondrial stress triggers a response in the cell's mitochondria and nucleus, but how these stress responses are coordinated in vivo is poorly understood. Here, we characterize a family with myopathy caused by a dominant p.G58R mutation in the mitochondrial protein CHCHD10. To understand the disease etiology, we developed a knockin (KI) mouse model and found that mutant CHCHD10 aggregated in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane. Unexpectedly, the survival of CHCHD10-KI mice depended on a protective stress response mediated by the mitochondrial metalloendopeptidase OMA1. The OMA1 stress response acted both locally within mitochondria, causing mitochondrial fragmentation, and signaled outside the mitochondria, activating the integrated stress response through cleavage of DAP3-binding cell death enhancer 1 (DELE1). We additionally identified an isoform switch in the terminal complex of the electron transport chain as a component of this response. Our results demonstrate that OMA1 was critical for neonatal survival conditionally in the setting of inner mitochondrial membrane stress, coordinating local and global stress responses to reshape the mitochondrial network and proteome.

Heterozygous PRKN mutations are common but do not increase the risk of Parkinson's disease.

PRKN mutations are the most common recessive cause of Parkinson's disease and are a promising target for gene and cell replacement therapies. Identification of biallelic PRKN patients at the population scale, however, remains a challenge, as roughly half are copy number variants and many single nucleotide polymorphisms are of unclear significance. Additionally, the true prevalence and disease risk associated with heterozygous PRKN mutations is unclear, as a comprehensive assessment of PRKN mutations has not been performed at a population scale. To address these challenges, we evaluated PRKN mutations in two cohorts with near complete genotyping of both single nucleotide polymorphisms and copy number variants: the NIH-PD + AMP-PD cohort, the largest Parkinson's disease case-control cohort with whole genome sequencing data from 4094 participants, and the UK Biobank, the largest cohort study with whole exome sequencing and genotyping array data from 200 606 participants. Using the NIH-PD participants, who were genotyped using whole genome sequencing, genotyping array, and multi-plex ligation-dependent probe amplification, we validated genotyping array for the detection of copy number variants. Additionally, in the NIH-PD cohort, functional assays of patient fibroblasts resolved variants of unclear significance in biallelic carriers and suggested that cryptic loss of function variants in monoallelic carriers are not a substantial confounder for association studies. In the UK Biobank, we identified 2692 PRKN copy number variants from genotyping array data from nearly half a million participants (the largest collection to date). Deletions or duplications involving exon 2 accounted for roughly half of all copy number variants and the vast majority (88%) involved exons 2, 3, or 4. In the UK Biobank, we found a pathogenic PRKN mutation in 1.8% of participants and two mutations in ∼1/7800 participants. Those with one PRKN pathogenic variant were as likely as non-carriers to have Parkinson's disease [odds ratio = 0.91 (0.58-1.38), P-value 0.76] or a parent with Parkinson's disease [odds ratio = 1.12 (0.94-1.31), P-value = 0.19]. Similarly, those in the NIH-PD + AMP + PD cohort with one PRKN pathogenic variant were as likely as non-carriers to have Parkinson's disease [odds ratio = 1.29 (0.74-2.38), P-value = 0.43]. Together our results demonstrate that heterozygous pathogenic PRKN mutations are common in the population but do not increase the risk of Parkinson's disease.

Managing risky assets - mitophagy in vivo.

Mitochondria, which resemble their α-proteobacteria ancestors, are a major cellular asset, producing energy 'on the cheap' through oxidative phosphorylation. They are also a liability. Increased oxidative phosphorylation means increased oxidative stress, and damaged mitochondria incite inflammation through release of their bacteria-like macromolecules. Mitophagy (the selective macroautophagy of mitochondria) controls mitochondria quality and number to manage these risky assets. Parkin, BNIP3 and NIX were identified as being part of the first mitophagy pathways identified in mammals over a decade ago, with additional pathways, including that mediated by FUNDC1 reported more recently. Loss of Parkin or PINK1 function causes Parkinson's disease, highlighting the importance of mitophagy as a quality control mechanism in the brain. Additionally, mitophagy is induced in idiopathic Parkinson's disease and Alzheimer's disease, protects the heart and other organs against energy stress and lipotoxicity, regulates metabolism by controlling mitochondrial number in brown and beige fat, and clears mitochondria during terminal differentiation of glycolytic cells, such as red blood cells and neurons. Despite its importance in disease, mitophagy is likely dispensable under physiological conditions. This Review explores the in vivo roles of mitophagy in mammalian systems, focusing on the best studied examples - mitophagy in neurodegeneration, cardiomyopathy, metabolism, and red blood cell development - to draw out common themes.

Discovery of bactericides as an acute mitochondrial membrane damage inducer.

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

α-Synuclein Deposition in Sympathetic Nerve Fibers in Genetic Forms of Parkinson's Disease.

BACKGROUND: Cytoplasmic inclusions of α-synuclein (α-syn) in brainstem neurons are characteristic of idiopathic Parkinson's disease (PD). PD also entails α-syn buildup in sympathetic nerves. Among genetic forms of PD, the relative extents of sympathetic intraneuronal accumulation of α-syn have not been reported. OBJECTIVE: This cross-sectional observational study compared magnitudes of intraneuronal deposition of α-syn in common and rare genetic forms of PD. METHODS: α-Syn deposition was quantified by the α-syn-tyrosine hydroxylase colocalization index in C2 cervical skin biopsies from 65 subjects. These included 30 subjects with pathogenic mutations in SNCA (n = 3), PRKN [biallelic (n = 7) and monoallelic (n = 3)], LRRK2 (n = 7), GBA (n = 7), or PARK7/DJ1 [biallelic (n = 1) and monoallelic (n = 2)]. Twenty-five of the mutation carriers had PD and five did not. Data were also analyzed from 19 patients with idiopathic PD and 16 control participants. RESULTS: α-Syn deposition varied as a function of genotype (F = 16.7, P < 0.0001). It was above the control range in 100% of subjects with SNCA mutations, 100% with LRRK2 mutations, 95% with idiopathic PD, 83% with GBA mutations, and 0% with biallelic PRKN mutations. α-Syn deposition in the biallelic PRKN group was significantly higher than in the control group. In addition, patients with biallelic PRKN mutations had higher α-syn deposition than their unaffected siblings. CONCLUSIONS: Individuals with SNCA, DJ-1, LRRK2, or GBA mutations have substantial intraneuronal α-syn deposition in sympathetic noradrenergic nerves in skin biopsies, whereas those with biallelic PRKN mutations do not. Biallelic PRKN patients may have mildly increased α-syn deposition compared with control subjects. © 2021 International Parkinson and Movement Disorder Society.

Mt-Keima detects PINK1-PRKN mitophagy in vivo with greater sensitivity than mito-QC.

PINK1 and PRKN, which cause Parkinson disease when mutated, form a quality control mitophagy pathway that is well-characterized in cultured cells. The extent to which the PINK1-PRKN pathway contributes to mitophagy in vivo, however, is controversial. This is due in large part to conflicting results from studies using one of two mitophagy reporters: mt-Keima or mito-QC. Studies using mt-Keima have generally detected PINK1-PRKN mitophagy in vivo, whereas those using mito-QC generally have not. Here, we directly compared the performance of mito-QC and mt-Keima in cell culture and in mice subjected to a PINK1-PRKN activating stress. We found that mito-QC was less sensitive than mt-Keima for mitophagy, and that this difference was more pronounced for PINK1-PRKN mitophagy. These findings suggest that mito-QC's poor sensitivity may account for conflicting reports of PINK1-PRKN mitophagy in vivo and caution against using mito-QC as a reporter for PINK1-PRKN mitophagy.Abbreviations: DFP: deferiprone; EE: exhaustive exercise; FBS: fetal bovine serum; OAQ: oligomycin, antimycin, and Q-VD-OPH; OMM: outer mitochondrial membrane; PBS: phosphate-buffered saline; PD: Parkinson disease; UPS: ubiquitin-proteasome system.

Mitochondrial Dysfunction and Mitophagy in Parkinson's Disease: From Mechanism to Therapy.

Mitochondrial dysfunction has been associated with neurodegeneration in Parkinson's disease (PD) for over 30 years. Despite this, the role of mitochondrial dysfunction as an initiator, propagator, or bystander remains undetermined. The discovery of the role of the PD familial genes PTEN-induced putative kinase 1 (PINK1) and parkin (PRKN) in mediating mitochondrial degradation (mitophagy) reaffirmed the importance of this process in PD aetiology. Recently, progress has been made in understanding the upstream and downstream regulators of canonical PINK1/parkin-mediated mitophagy, alongside noncanonical PINK1/parkin mitophagy, in response to mitochondrial damage. Progress has also been made in understanding the role of PD-associated genes, such as SNCA, LRRK2, and CHCHD2, in mitochondrial dysfunction and their overlap with sporadic PD (sPD), opening opportunities for therapeutically targeting mitochondria in PD.

Metabolic Analysis at the Nanoscale with Multi-Isotope Imaging Mass Spectrometry (MIMS).

Incorporation of a stable-isotope metabolic tracer into cells or tissue can be followed at submicron resolution by multi-isotope imaging mass spectrometry (MIMS), a form of imaging secondary ion microscopy optimized for accurate isotope ratio measurement from microvolumes of sample (as small as ∼30 nm across). In a metabolic MIMS experiment, a cell or animal is metabolically labeled with a tracer containing a stable isotope. Relative accumulation of the heavy isotope in the fixed sample is then measured as an increase over its natural abundance by MIMS. MIMS has been used to measure protein turnover in single organelles, track cellular division in vivo, visualize sphingolipid rafts on the plasma membrane, and measure dopamine incorporation into dense-core vesicles, among other biological applications. In this article, we introduce metabolic analysis using NanoSIMS by focusing on two specific applications: quantifying protein turnover in single organelles of cultured cells and tracking cell replication in mouse tissues in vivo. These examples illustrate the versatility of metabolic analysis with MIMS. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Metabolic labeling for MIMS Basic Protocol 2: Embedding of samples for correlative transmission electron microscopy and MIMS with a genetically encoded reporter Alternate Protocol: Embedding of samples for correlative light microscopy and MIMS Support Protocol: Preparation of silicon wafers as sample supports for MIMS Basic Protocol 3: Analysis of MIMS data.

Loss of CHCHD2 and CHCHD10 activates OMA1 peptidase to disrupt mitochondrial cristae phenocopying patient mutations.

Dominant mutations in the mitochondrial paralogs coiled-helix-coiled-helix (CHCHD) domain 2 (C2) and CHCHD10 (C10) were recently identified as causing Parkinson's disease and amyotrophic lateral sclerosis/frontotemporal dementia/myopathy, respectively. The mechanism by which they disrupt mitochondrial cristae, however, has been uncertain. Using the first C2/C10 double knockout (DKO) mice, we report that C10 pathogenesis and the normal function of C2/C10 are intimately linked. Similar to patients with C10 mutations, we found that C2/C10 DKO mice have disrupted mitochondrial cristae, because of cleavage of the mitochondrial-shaping protein long form of OPA1 (L-OPA1) by the stress-induced peptidase OMA1. OMA1 was found to be activated similarly in affected tissues of mutant C10 knock-in (KI) mice, demonstrating that L-OPA1 cleavage is a novel mechanism for cristae abnormalities because of both C10 mutation and C2/C10 loss. Using OMA1 activation as a functional assay, we found that C2 and C10 are partially functionally redundant, and some but not all disease-causing mutations have retained activity. Finally, C2/C10 DKO mice partially phenocopied mutant C10 KI mice with the development of cardiomyopathy and activation of the integrated mitochondrial integrated stress response in affected tissues, tying mutant C10 pathogenesis to C2/C10 function.

Detection of mitophagy in mammalian cells, mice, and yeast.

Selective elimination of superfluous or dysfunctional mitochondria is a fundamental process conserved among both uni- and multicellular eukaryotes, contributing to mitochondrial quality and quantity control. This process depends on autophagy, a cellular self-eating membrane trafficking system, and is thus called mitophagy. In this chapter, we describe methods to detect mitophagy in mammalian cells, mice, and yeast.

Coupling APEX labeling to imaging mass spectrometry of single organelles reveals heterogeneity in lysosomal protein turnover.

Quantification of stable isotope tracers after metabolic labeling provides a snapshot of the dynamic state of living cells and tissue. A form of imaging mass spectrometry quantifies isotope ratios with a lateral resolution <50 nm, using a methodology that we refer to as multi-isotope imaging mass spectrometry (MIMS). Despite lateral resolution exceeding diffraction-limited light microscopy, lack of contrast has largely limited use of MIMS to large or specialized subcellular structures, such as the nucleus and stereocilia. In this study, we repurpose the engineered peroxidase APEX2 as the first genetically encoded marker for MIMS. Coupling APEX2 labeling of lysosomes and metabolic labeling of protein, we identify that individual lysosomes exhibit substantial heterogeneity in protein age, which is lost in iPSC-derived neurons lacking the lysosomal protein progranulin. This study expands the practical use of MIMS for cell biology by enabling measurements of metabolic function from stable isotope labeling within individual organelles in situ.