No abstract

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.

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 Mitofusin 2 protein (MFN2), encoded by the MFN2 gene, was first described for its role in mediating mitochondrial fusion. However, MFN2 is now recognized to play additional roles in mitochondrial autophagy (mitophagy), mitochondrial motility, lipid transfer, and as a tether to other organelles including the endoplasmic reticulum (ER) and lipid droplets. The tethering role of MFN2 is an important mediator of mitochondrial-ER contact sites (MERCs), which themselves have many important functions that regulate mitochondria, including calcium homeostasis and lipid metabolism. Exemplifying the importance of MFN2, pathogenic variants in MFN2 are established to cause the peripheral neuropathy Charcot-Marie-Tooth Disease Subtype 2A (CMT2A). However, the mechanistic basis for disease is not clear. Moreover, additional pathogenic phenotypes such as lipomatosis, distal myopathy, optic atrophy, and hearing loss, can also sometimes be present in patients with CMT2A. Given these variable patient phenotypes, and the many cellular roles played by MFN2, the mechanistic underpinnings of the cellular impairments by which MFN2 dysfunction leads to disease are likely to be complex. Here, we will review what is known about the various functions of MFN2 that are impaired by pathogenic variants causing CMT2A, with a specific emphasis on the ties between MFN2 variants and MERCs.

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 (PS) to phosphatidylethanolamine (PE) in the inner mitochondrial membrane (IMM). Decreased conversion of PS to PE, and depletion of total cellular PE levels in patient fibroblasts are consistent with impaired 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 2deoxyglucose. Moreover, treatment with lyso-PE, which can replenish the mitochondrial pool of PE, restored mitochondrial and lysosome morphology in patient fibroblasts. Functional characterization of the PISD mutations 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 explains why the phenotypes of these PISD patients resemble recently described "mitochondrial chaperonopathies". Collectively, these findings demonstrate that PISD is a novel mitochondrial disease gene.

No abstract

Background:The 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 two 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 and 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.

Intestinal homeostasis is highly dependent on optimal epithelial barrier function and permeability. Intestinal epithelial cells (IEC) regulate these properties acting as cellular gatekeepers by selectively absorbing nutrients and controlling the passage of luminal bacteria. These functions are energy demanding processes that are presumably met through mitochondrial-based processes. Routine methods for examining IEC mitochondrial function remain sparse, hence, our objective is to present standardized methods for quantifying mitochondrial energetics in an immortalized IEC line. Employing the murine IEC4.1 cell line, we present adapted methods and protocols to examine mitochondrial function using two well-known platforms: the Seahorse Extracellular Flux Analyzer and Oxygraph-2 k. To demonstrate the applicability of these protocols and instruments, IEC were treated with and without the murine colitogenic agent, dextran sulfate sodium (DSS, 2% w/v). Profound impairments with DSS treatment were found with both platforms, however, the Oxygraph-2 k allowed greater resolution of affected pathways including short-chain fatty acid metabolism. Mitochondrial functional analysis is a novel tool to explore the relationship between IEC energetics and functional consequences within the contexts of health and disease. The outlined methods offer an introductory starting point for such assessment and provide the investigator with insights into platform-specific capabilities.