The role of mitochondrial dynamics in mtDNA maintenance.

The dynamic nature of mitochondria, which can fuse, divide and move throughout the cell, allows these critical organelles to adapt their function in response to cellular demands, and is also important for regulating mitochondrial DNA (mtDNA). While it is established that impairments in mitochondrial fusion and fission impact the mitochondrial genome and can lead to mtDNA depletion, abnormal nucleoid organization or accumulation of deletions, it is not entirely clear how or why remodeling mitochondrial network morphology affects mtDNA. Here, we focus on recent advances in our understanding of how mitochondrial dynamics contribute to the regulation of mtDNA and discuss links to human disease.

A new automated tool to quantify nucleoid distribution within mitochondrial networks.

Mitochondrial DNA (mtDNA) maintenance is essential to sustain a functionally healthy population of mitochondria within cells. Proper mtDNA replication and distribution within mitochondrial networks are essential to maintain mitochondrial homeostasis. However, the fundamental basis of mtDNA segregation and distribution within mitochondrial networks is still unclear. To address these questions, we developed an algorithm, Mitomate tracker to unravel the global distribution of nucleoids within mitochondria. Using this tool, we decipher the semi-regular spacing of nucleoids across mitochondrial networks. Furthermore, we show that mitochondrial fission actively regulates mtDNA distribution by controlling the distribution of nucleoids within mitochondrial networks. Specifically, we found that primary cells bearing disease-associated mutations in the fission proteins DRP1 and MYH14 show altered nucleoid distribution, and acute enrichment of enlarged nucleoids near the nucleus. Further analysis suggests that the altered nucleoid distribution observed in the fission mutants is the result of both changes in network structure and nucleoid density. Thus, our study provides novel insights into the role of mitochondria fission in nucleoid distribution and the understanding of diseases caused by fission defects.

A synthetic peptide rescues rat cortical neurons from anesthetic-induced cell death, perturbation of growth and synaptic assembly.

Anesthetics are deemed necessary for all major surgical procedures. However, they have also been found to exert neurotoxic effects when tested on various experimental models, but the underlying mechanisms remain unknown. Earlier studies have implicated mitochondrial fragmentation as a potential target of anesthetic-induced toxicity, although clinical strategies to protect their structure and function remain sparse. Here, we sought to determine if preserving mitochondrial networks with a non-toxic, short-life synthetic peptide-P110, would protect cortical neurons against both inhalational and intravenous anesthetic-induced neurotoxicity. This study provides the first direct and comparative account of three key anesthetics (desflurane, propofol, and ketamine) when used under identical conditions, and demonstrates their impact on neonatal, rat cortical neuronal viability, neurite outgrowth and synaptic assembly. Furthermore, we discovered that inhibiting Fis1-mediated mitochondrial fission reverses anesthetic-induced aberrations in an agent-specific manner. This study underscores the importance of designing mitigation strategies invoking mitochondria-mediated protection from anesthetic-induced toxicity in both animals and humans.

Characterization of a novel variant in the HR1 domain of MFN2 in a patient with ataxia, optic atrophy and sensorineural hearing loss.

Background: Pathogenic variants in MFN2 cause Charcot-Marie-Tooth disease (CMT) type 2A (CMT2A) and are the leading cause of the axonal subtypes of CMT. CMT2A is characterized by predominantly distal motor weakness and muscle atrophy, with highly variable severity and onset age. Notably, some MFN2 variants can also lead to other phenotypes such as optic atrophy, hearing loss and lipodystrophy. Despite the clear link between MFN2 and CMT2A, our mechanistic understanding of how dysfunction of the MFN2 protein causes human disease pathologies remains incomplete. This lack of understanding is due in part to the multiple cellular roles of MFN2. Though initially characterized for its role in mediating mitochondrial fusion, MFN2 also plays important roles in mediating interactions between mitochondria and other organelles, such as the endoplasmic reticulum and lipid droplets. Additionally, MFN2 is also important for mitochondrial transport, mitochondrial autophagy, and has even been implicated in lipid transfer. Though over 100 pathogenic MFN2 variants have been described to date, only a few have been characterized functionally, and even then, often only for one or two functions. Method: Several MFN2-mediated functions were characterized in fibroblast cells from a patient presenting with cerebellar ataxia, deafness, blindness, and diffuse cerebral and cerebellar atrophy, who harbours a novel homozygous MFN2 variant, D414V, which is found in a region of the HR1 domain of MFN2 where few pathogenic variants occur. Results: We found evidence for impairment of several MFN2-mediated functions. Consistent with reduced mitochondrial fusion, patient fibroblasts exhibited more fragmented mitochondrial networks and had reduced mtDNA copy number. Additionally, patient fibroblasts had reduced oxygen consumption, fewer mitochondrial-ER contacts, and altered lipid droplets that displayed an unusual perinuclear distribution. Conclusion: Overall, this work characterizes D414V as a novel variant in MFN2 and expands the phenotypic presentation of MFN2 variants to include cerebellar ataxia.

Aberrant Mitochondrial Morphology and Function in the BTBR Mouse Model of Autism Is Improved by Two Weeks of Ketogenic Diet.

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder that exhibits a common set of behavioral and cognitive impairments. Although the etiology of ASD remains unclear, mitochondrial dysfunction has recently emerged as a possible causative factor underlying ASD. The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that augments mitochondrial function, and has been shown to reduce autistic behaviors in both humans and in rodent models of ASD. The aim of the current study was to examine mitochondrial bioenergetics in the BTBR mouse model of ASD and to determine whether the KD improves mitochondrial function. We also investigated changes in mitochondrial morphology, which can directly influence mitochondrial function. We found that BTBR mice had altered mitochondrial function and exhibited smaller more fragmented mitochondria compared to C57BL/6J controls, and that supplementation with the KD improved both mitochondrial function and morphology. We also identified activating phosphorylation of two fission proteins, pDRP1S616 and pMFFS146, in BTBR mice, consistent with the increased mitochondrial fragmentation that we observed. Intriguingly, we found that the KD decreased pDRP1S616 levels in BTBR mice, likely contributing to the restoration of mitochondrial morphology. Overall, these data suggest that impaired mitochondrial bioenergetics and mitochondrial fragmentation may contribute to the etiology of ASD and that these alterations can be reversed with KD treatment.

Perturbed Mitochondrial Dynamics Is a Novel Feature of Colitis That Can Be Targeted to Lessen Disease.

BACKGROUND & AIMS: Mitochondria exist in a constantly remodelling network, and excessive fragmentation can be pathophysiological. Mitochondrial dysfunction can accompany enteric inflammation, but any contribution of altered mitochondrial dynamics (ie, fission/fusion) to gut inflammation is unknown. We hypothesized that perturbed mitochondrial dynamics would contribute to colitis. METHODS: Quantitative polymerase chain reaction for markers of mitochondrial fission and fusion was applied to tissue from dextran sodium sulfate (DSS)-treated mice. An inhibitor of mitochondrial fission, P110 (prevents dynamin related protein [Drp]-1 binding to mitochondrial fission 1 protein [Fis1]) was tested in the DSS and di-nitrobenzene sulfonic acid (DNBS) models of murine colitis, and the impact of DSS ± P110 on intestinal epithelial and macrophage mitochondria was assessed in vitro. RESULTS: Analysis of colonic tissue from mice with DSS-colitis revealed increased mRNA for molecules associated with mitochondrial fission (ie, Drp1, Fis1) and fusion (optic atrophy factor 1) and increased phospho-Drp1 compared with control. Systemic delivery of P110 in prophylactic or treatment regimens reduced the severity of DSS- or DNBS-colitis and the subsequent hyperalgesia in DNBS-mice. Application of DSS to epithelial cells or macrophages caused mitochondrial fragmentation. DSS-evoked perturbation of epithelial cell energetics and mitochondrial fragmentation, but not cell death, were ameliorated by in vitro co-treatment with P110. CONCLUSIONS: We speculate that the anti-colitic effect of systemic delivery of the anti-fission drug, P110, works at least partially by maintaining enterocyte and macrophage mitochondrial networks. Perturbed mitochondrial dynamics can be a feature of intestinal inflammation, the suppression of which is a potential novel therapeutic direction in inflammatory bowel disease.

Reciprocal Regulation of Mitochondrial Fission and Fusion.

The dynamic processes of mitochondrial fission and fusion are tightly regulated, determine mitochondrial shape, and influence mitochondrial functions. For example, fission and fusion mediate energy output, production of reactive oxygen species (ROS), and mitochondrial quality control. As our understanding of the molecular machinery and mechanisms regulating dynamic changes in the mitochondrial network continues to grow, we are beginning to unravel important signaling pathways that integrate physiological cues to modulate mitochondrial morphology and function. Here, we highlight reciprocal regulation of mitochondrial fusion and fission as an emerging trend in the regulation of mitochondrial function.

Reversible Mitochondrial Fragmentation in iPSC-Derived Cardiomyocytes From Children With DCMA, a Mitochondrial Cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy with ataxia syndrome (DCMA) is an understudied autosomal recessive disease caused by loss-of-function mutations in the poorly characterized gene DNAJC19. Clinically, DCMA is commonly associated with heart failure and early death in affected children through an unknown mechanism. DCMA has been linked to Barth syndrome, a rare but well-studied disorder caused by deficient maturation of cardiolipin (CL), a key mitochondrial membrane phospholipid. METHODS: Peripheral blood mononuclear cells from 2 children with DCMA and severe cardiac dysfunction were reprogrammed into induced pluripotent stem cells (iPSCs). Patient and control iPSCs were differentiated into beating cardiomyocytes (iPSC-CMs) using a metabolic selection strategy. Mitochondrial structure and CL content before and after incubation with the mitochondrially targeted peptide SS-31 were quantified. RESULTS: Patient iPSCs carry the causative DNAJC19 mutation (rs137854888) found in the Hutterite population, and the iPSC-CMs demonstrated highly fragmented and abnormally shaped mitochondria associated with an imbalanced isoform ratio of the mitochondrial protein OPA1, an important regulator of mitochondrial fusion. These abnormalities were reversible by incubation with SS-31 for 24 hours. Differentiation of iPSCs into iPSC-CMs increased the number of CL species observed, but consistent, significant differences in CL content were not seen between patients and control. CONCLUSIONS: We describe a unique and novel cellular model that provides insight into the mitochondrial abnormalities present in DCMA and identifies SS-31 as a potential therapeutic for this devastating disease.

SS-31 Peptide Reverses the Mitochondrial Fragmentation Present in Fibroblasts From Patients With DCMA, a Mitochondrial Cardiomyopathy.

We used patient dermal fibroblasts to characterize the mitochondrial abnormalities associated with the dilated cardiomyopathy with ataxia syndrome (DCMA) and to study the effect of the mitochondrially-targeted peptide SS-31 as a potential novel therapeutic. DCMA is a rare and understudied autosomal recessive disorder thought to be related to Barth syndrome but caused by mutations in DNAJC19, a protein of unknown function localized to the mitochondria. The clinical disease is characterized by 3-methylglutaconic aciduria, dilated cardiomyopathy, abnormal neurological development, and other heterogeneous features. Until recently no effective therapies had been identified and affected patients frequently died in early childhood from intractable heart failure. Skin fibroblasts from four pediatric patients with DCMA were used to establish parameters of mitochondrial dysfunction. Mitochondrial structure, reactive oxygen species (ROS) production, cardiolipin composition, and gene expression were evaluated. Immunocytochemistry with semi-automated quantification of mitochondrial structural metrics and transmission electron microscopy demonstrated mitochondria to be highly fragmented in DCMA fibroblasts compared to healthy control cells. Live-cell imaging demonstrated significantly increased ROS production in patient cells. These abnormalities were reversed by treating DCMA fibroblasts with SS-31, a synthetic peptide that localizes to the inner mitochondrial membrane. Levels of cardiolipin were not significantly different between control and DCMA cells and were unaffected by SS-31 treatment. Our results demonstrate the abnormal mitochondria in fibroblasts from patients with DCMA and suggest that SS-31 may represent a potential therapy for this devastating disease.

Characterization of the C584R variant in the mtDNA depletion syndrome gene FBXL4, reveals a novel role for FBXL4 as a regulator of mitochondrial fusion.

Mutations in FBXL4 (F-Box and Leucine rich repeat protein 4), a nuclear-encoded mitochondrial protein with an unknown function, cause mitochondrial DNA depletion syndrome. We report two siblings, from consanguineous parents, harbouring a previously uncharacterized homozygous variant in FBXL4 (c.1750 T > C; p.Cys584Arg). Both patients presented with encephalomyopathy, lactic acidosis and cardiac hypertrophy, which are reported features of FBXL4 impairment. Remarkably, dichloroacetate (DCA) administration to the younger sibling improved metabolic acidosis and reversed cardiac hypertrophy. Characterization of FBXL4 patient fibroblasts revealed severe bioenergetic defects, mtDNA depletion, fragmentation of mitochondrial networks, and abnormalities in mtDNA nucleoids. These phenotypes, observed with other pathogenic FBXL4 variants, confirm the pathogenicity of the p.Cys584Arg variant. Although treating FBXL4 fibroblasts with DCA improved extracellular acidification, in line with reduced lactate levels in patients, DCA treatment did not improve any of the other mitochondrial functions. Nonetheless, we highlight DCA as a potentially effective drug for the management of elevated lactate and cardiomyopathy in patients with pathogenic FBXL4 variants. Finally, as the exact mechanism through which FBXL4 mutations lead to mtDNA depletion was unknown, we tested the hypothesis that FBXL4 promotes mitochondrial fusion. Using a photo-activatable GFP fusion assay, we found reduced mitochondrial fusion rates in cells harbouring a pathogenic FBXL4 variant. Meanwhile, overexpression of wildtype FBXL4, but not the p.Cys584Arg variant, promoted mitochondrial hyperfusion. Thus, we have uncovered a novel function for FBXL4 in promoting mitochondrial fusion, providing important mechanistic insights into the pathogenic mechanism underlying FBXL4 dysfunction.

The R941L mutation in MYH14 disrupts mitochondrial fission and associates with peripheral neuropathy.

BACKGROUND: Peripheral neuropathies are often caused by disruption of genes responsible for myelination or axonal transport. In particular, impairment in mitochondrial fission and fusion are known causes of peripheral neuropathies. However, the causal mechanisms for peripheral neuropathy gene mutations are not always known. While loss of function mutations in MYH14 typically cause non-syndromic hearing loss, the recently described R941L mutation in MYH14, encoding the non-muscle myosin protein isoform NMIIC, leads to a complex clinical presentation with an unexplained peripheral neuropathy phenotype. METHODS: Confocal microscopy was used to examine mitochondrial dynamics in MYH14 patient fibroblast cells, as well as U2OS and M17 cells overexpressing NMIIC. The consequence of the R941L mutation on myosin activity was modeled in C. elegans. FINDINGS: We describe the third family carrying the R941L mutation in MYH14, and demonstrate that the R941L mutation impairs non-muscle myosin protein function. To better understand the molecular basis of the peripheral neuropathy phenotype associated with the R941L mutation, which has been hindered by the fact that NMIIC is largely uncharacterized, we have established a previously unrecognized biological role for NMIIC in mediating mitochondrial fission in human cells. Notably, the R941L mutation acts in a dominant-negative fashion to inhibit mitochondrial fission, especially in the cell periphery. In addition, we observed alterations to the organization of the mitochondrial genome. INTERPRETATION: As impairments in mitochondrial fission cause peripheral neuropathy, this insight into the function of NMIIC likely explains the peripheral neuropathy phenotype associated with the R941L mutation. FUND: This study was supported by the Alberta Children's Hospital Research Institute, the Canadian Institutes of Health Research and the Care4Rare Canada Consortium.

PISD is a mitochondrial disease gene causing skeletal dysplasia, cataracts, and white matter changes.

Exome sequencing of two sisters with congenital cataracts, short stature, and white matter changes identified compound heterozygous variants in the PISD gene, encoding the phosphatidylserine decarboxylase enzyme that converts phosphatidylserine to phosphatidylethanolamine (PE) in the inner mitochondrial membrane (IMM). Decreased conversion of phosphatidylserine to PE in patient fibroblasts is consistent with impaired phosphatidylserine decarboxylase (PISD) enzyme activity. Meanwhile, as evidence for mitochondrial dysfunction, patient fibroblasts exhibited more fragmented mitochondrial networks, enlarged lysosomes, decreased maximal oxygen consumption rates, and increased sensitivity to 2-deoxyglucose. Moreover, treatment with lyso-PE, which can replenish the mitochondrial pool of PE, and genetic complementation restored mitochondrial and lysosome morphology in patient fibroblasts. Functional characterization of the PISD variants demonstrates that the maternal variant causes an alternative splice product. Meanwhile, the paternal variant impairs autocatalytic self-processing of the PISD protein required for its activity. Finally, evidence for impaired activity of mitochondrial IMM proteases suggests an explanation as to why the phenotypes of these PISD patients resemble recently described "mitochondrial chaperonopathies." Collectively, these findings demonstrate that PISD is a novel mitochondrial disease gene.

Mesenchymal Stem Cells Shift Mitochondrial Dynamics and Enhance Oxidative Phosphorylation in Recipient Cells.

Mesenchymal stem cells (MSCs) are the most commonly used cells in tissue engineering and regenerative medicine. MSCs can promote host tissue repair through several different mechanisms including donor cell engraftment, release of cell signaling factors, and the transfer of healthy organelles to the host. In the present study, we examine the specific impacts of MSCs on mitochondrial morphology and function in host tissues. Employing in vitro cell culture of inherited mitochondrial disease and an in vivo animal experimental model of low-grade inflammation (high fat feeding), we show human-derived MSCs to alter mitochondrial function. MSC co-culture with skin fibroblasts from mitochondrial disease patients rescued aberrant mitochondrial morphology from a fission state to a more fused appearance indicating an effect of MSC co-culture on host cell mitochondrial network formation. In vivo experiments confirmed mitochondrial abundance and mitochondrial oxygen consumption rates were elevated in host tissues following MSC treatment. Furthermore, microarray profiling identified 226 genes with differential expression in the liver of animals treated with MSC, with cellular signaling, and actin cytoskeleton regulation as key upregulated processes. Collectively, our data indicate that MSC therapy rescues impaired mitochondrial morphology, enhances host metabolic capacity, and induces widespread host gene shifting. These results highlight the potential of MSCs to modulate mitochondria in both inherited and pathological disease states.

The Keap1-Nrf2 Stress Response Pathway Promotes Mitochondrial Hyperfusion Through Degradation of the Mitochondrial Fission Protein Drp1.

AIMS: Mitochondrial function is coupled to metabolic and survival pathways through both direct signaling cascades and dynamic changes in mitochondrial morphology. For example, a hyperfused mitochondrial reticulum is activated upon cellular stress and is protective against cell death. As part of a genome-wide small inhibitory ribonucleic acid screen, we identified the central redox regulator, Keap1, as a novel regulator of mitochondrial morphology. Here, we aimed to determine the mechanism through which redox signaling and Keap1 mediate changes in mitochondrial morphology. RESULTS: We found that the Nrf2 transcription factor is required for mitochondrial hyperfusion induced by knockdown of Keap1. Nrf2, which is negatively regulated by Keap1, mediates the cell's response to stress by controlling the expression of several hundred genes, including proteasome expression. We next showed that increased proteasome activity, a result of increased Nrf2 activity, is responsible for the degradation of the mitochondrial fission protein Drp1, which occurs in an ubiquitin-independent manner. INNOVATION: Our study described a novel pathway by which Nrf2 activation, known to occur in response to increased oxidative stress, decreases mitochondrial fission and contributes to a hyperfused mitochondrial network. CONCLUSION: This study has identified the Keap1-Nrf2 nexus and modulation of proteasomal activity as novel avenues to inhibit mitochondrial fission. These findings are important, because inhibiting mitochondrial fission is a promising therapeutic approach to restore the balance between fission and fusion, which is attractive for an increasing number of disorders linked to mitochondrial dysfunction. Antioxid. Redox Signal. 27, 1447-1459.