Dissecting the Roles of the Nuclear and Mitochondrial Genomes in a Mouse Model of Autoimmune Diabetes.

Mitochondria, the organelles responsible for generating ATP in eukaryotic cells, have been previously implicated as a contributor to diabetes. However, mitochondrial proteins are encoded by both nuclear DNA (nDNA) and mtDNA. In order to better understand the relative contribution of each of these genomes to diabetes, a chimeric mitochondrial-nuclear exchange (MNX) mouse was created via pronuclear transfer carrying nDNA from a strain susceptible to type 1 diabetes (NOD/ShiLtJ) and mtDNA from nondiabetic C57BL/6J mice. Inheritance of the resulting heteroplasmic mtDNA mixture was then tracked across multiple generations, showing that offspring heteroplasmy generally followed that of the mother, with occasional large shifts consistent with an mtDNA bottleneck in the germ line. In addition, survival and incidence of diabetes in MNX mice were tracked and compared with those in unaltered NOD/ShiLtJ control mice. The results indicated improved survival and a delay in diabetes onset in the MNX mice, demonstrating that mtDNA has a critical influence on disease phenotype. Finally, enzyme activity assays showed that the NOD/ShiLtJ mice had significant hyperactivity of complex I of the electron transport chain relative to MNX mice, suggesting that a particular mtDNA variant (m.9461T>C) may be responsible for disease causation in the original NOD/ShiLtJ strain. ARTICLE HIGHLIGHTS: Mitochondria have been previously implicated in diabetes, but the specific genetic factors remain unclear. To better understand the contributions of mitochondrial genes in nuclear DNA (nDNA) versus mtDNA, we created mitochondrial-nuclear exchange (MNX) mice carrying nDNA from a diabetic strain and mtDNA from nondiabetic mice. Long-term tracking of MNX mice showed occasional large shifts in heteroplasmy consistent with an mtDNA bottleneck in the germ line. In addition, the MNX mice showed improved survival and delayed incidence of diabetes relative to the unaltered diabetic mice, which appeared to be linked to the activity of respiratory complex I.

Application of super-resolution microscopy in mitochondria-dynamic diseases.

Limited by spatial and temporal resolution, traditional optical microscopy cannot image the delicate ultra-structure organelles and sub-organelles. The emergence of super-resolution microscopy makes it possible. In this review, we focus on mitochondria. We summarize the process of mitochondrial dynamics, the primary proteins that regulate mitochondrial morphology, the diseases related to mitochondrial dynamics. The purpose is to apply super-resolution microscopy developed during recent years to the mitochondrial research. By providing the right research tools, we will help to promote the application of this technique to the in-depth elucidation of the pathogenesis of diseases related to mitochondrial dynamics, assistdiagnosis and develop the therapeutic treatment.

Mitochondrial Genome Variants as a Cause of Mitochondrial Cardiomyopathy.

Mitochondria are small double-membraned organelles responsible for the generation of energy used in the body in the form of ATP. Mitochondria are unique in that they contain their own circular mitochondrial genome termed mtDNA. mtDNA codes for 37 genes, and together with the nuclear genome (nDNA), dictate mitochondrial structure and function. Not surprisingly, pathogenic variants in the mtDNA or nDNA can result in mitochondrial disease. Mitochondrial disease primarily impacts tissues with high energy demands, including the heart. Mitochondrial cardiomyopathy is characterized by the abnormal structure or function of the myocardium secondary to genetic defects in either the nDNA or mtDNA. Mitochondrial cardiomyopathy can be isolated or part of a syndromic mitochondrial disease. Common manifestations of mitochondrial cardiomyopathy are a phenocopy of hypertrophic cardiomyopathy, dilated cardiomyopathy, and cardiac conduction defects. The underlying pathophysiology of mitochondrial cardiomyopathy is complex and likely involves multiple abnormal processes in the cell, stemming from deficient oxidative phosphorylation and ATP depletion. Possible pathophysiology includes the activation of alternative metabolic pathways, the accumulation of reactive oxygen species, dysfunctional mitochondrial dynamics, abnormal calcium homeostasis, and mitochondrial iron overload. Here, we highlight the clinical assessment of mtDNA-related mitochondrial cardiomyopathy and offer a novel hypothesis of a possible integrated, multivariable pathophysiology of disease.

Premature aging is associated with higher levels of 8-oxoguanine and increased DNA damage in the Polg mutator mouse.

Mitochondrial dysfunction plays an important role in the aging process. However, the mechanism by which this dysfunction causes aging is not fully understood. The accumulation of mutations in the mitochondrial genome (or "mtDNA") has been proposed as a contributor. One compelling piece of evidence in support of this hypothesis comes from the PolgD257A/D257A mutator mouse (Polgmut/mut ). These mice express an error-prone mitochondrial DNA polymerase that results in the accumulation of mtDNA mutations, accelerated aging, and premature death. In this paper, we have used the Polgmut/mut model to investigate whether the age-related biological effects observed in these mice are triggered by oxidative damage to the DNA that compromises the integrity of the genome. Our results show that mutator mouse has significantly higher levels of 8-oxoguanine (8-oxoGua) that are correlated with increased nuclear DNA (nDNA) strand breakage and oxidative nDNA damage, shorter average telomere length, and reduced mtDNA integrity. Based on these results, we propose a model whereby the increased level of reactive oxygen species (ROS) associated with the accumulation of mtDNA mutations in Polgmut/mut mice results in higher levels of 8-oxoGua, which in turn lead to compromised DNA integrity and accelerated aging via increased DNA fragmentation and telomere shortening. These results suggest that mitochondrial play a central role in aging and may guide future research to develop potential therapeutics for mitigating aging process.

Next-Generation Sequencing to Characterize Mitochondrial Genomic DNA Heteroplasmy.

Mitochondria play a very important role in many crucial cellular functions. Each eukaryotic cell contains hundreds of mitochondria with hundreds of mitochondrial genomes. Mutant and wild-type mitochondrial DNA (mtDNA) may co-exist as heteroplasmy and cause human disease. The purpose of the protocols in this article is to simultaneously determine the mtDNA sequence and quantify the heteroplasmy level using parallel sequencing. The protocols include mitochondrial genomic DNA PCR amplification of two full-length products using two distinct sets of PCR primers. The PCR products are mixed at an equimolar ratio, and the samples are then barcoded and sequenced with high-throughput next-generation sequencing technology. This technology is highly sensitive, specific, and accurate in determining mtDNA mutations and the degree/level of heteroplasmy. © 2022 Wiley Periodicals LLC. Basic Protocol 1: PCR amplification of mitochondrial DNA Basic Protocol 2: Analysis of next-generation sequencing of mitochondrial DNA Basic Protocol 3: Mutect2 pipeline for automated sample processing and large-scale data analysis.

PINK1-mediated Drp1S616 phosphorylation modulates synaptic development and plasticity via promoting mitochondrial fission.

Dynamic change of mitochondrial morphology and distribution along neuronal branches are essential for neural circuitry formation and synaptic efficacy. However, the underlying mechanism remains elusive. We show here that Pink1 knockout (KO) mice display defective dendritic spine maturation, reduced axonal synaptic vesicles, abnormal synaptic connection, and attenuated long-term synaptic potentiation (LTP). Drp1 activation via S616 phosphorylation rescues deficits of spine maturation in Pink1 KO neurons. Notably, mice harboring a knockin (KI) phosphor-null Drp1S616A recapitulate spine immaturity and synaptic abnormality identified in Pink1 KO mice. Chemical LTP (cLTP) induces Drp1S616 phosphorylation in a PINK1-dependent manner. Moreover, phosphor-mimetic Drp1S616D restores reduced dendritic spine localization of mitochondria in Pink1 KO neurons. Together, this study provides the first in vivo evidence of functional regulation of Drp1 by phosphorylation and suggests that PINK1-Drp1S616 phosphorylation coupling is essential for convergence between mitochondrial dynamics and neural circuitry formation and refinement.

A Novel Variant of ATP5MC3 Associated with Both Dystonia and Spastic Paraplegia.

BACKGROUND: In a large pedigree with an unusual phenotype of spastic paraplegia or dystonia and autosomal dominant inheritance, linkage analysis previously mapped the disease to chromosome 2q24-2q31. OBJECTIVE: The aim of this study is to identify the genetic cause and molecular basis of an unusual autosomal dominant spastic paraplegia and dystonia. METHODS: Whole exome sequencing following linkage analysis was used to identify the genetic cause in a large family. Cosegregation analysis was also performed. An additional 384 individuals with spastic paraplegia or dystonia were screened for pathogenic sequence variants in the adenosine triphosphate (ATP) synthase membrane subunit C locus 3 gene (ATP5MC3). The identified variant was submitted to the "GeneMatcher" program for recruitment of additional subjects. Mitochondrial functions were analyzed in patient-derived fibroblast cell lines. Transgenic Drosophila carrying mutants were studied for movement behavior and mitochondrial function. RESULTS: Exome analysis revealed a variant (c.318C > G; p.Asn106Lys) (NM_001689.4) in ATP5MC3 in a large family with autosomal dominant spastic paraplegia and dystonia that cosegregated with affected individuals. No variants were identified in an additional 384 individuals with spastic paraplegia or dystonia. GeneMatcher identified an individual with the same genetic change, acquired de novo, who manifested upper-limb dystonia. Patient fibroblast studies showed impaired complex V activity, ATP generation, and oxygen consumption. Drosophila carrying orthologous mutations also exhibited impaired mitochondrial function and displayed reduced mobility. CONCLUSION: A unique form of familial spastic paraplegia and dystonia is associated with a heterozygous ATP5MC3 variant that also reduces mitochondrial complex V activity.

Validation of low-coverage whole-genome sequencing for mitochondrial DNA variants suggests mitochondrial DNA as a genetic cause of preterm birth.

Preterm birth (PTB), or birth that occurs earlier than 37 weeks of gestational age, is a major contributor to infant mortality and neonatal hospitalization. Mutations in the mitochondrial genome (mtDNA) have been linked to various rare mitochondrial disorders and may be a contributing factor in PTB given that maternal genetic factors have been strongly linked to PTB. However, to date, no study has found a conclusive connection between a particular mtDNA variant and PTB. Given the high mtDNA copy number per cell, an automated pipeline was developed for detecting mtDNA variants using low-coverage whole-genome sequencing (lcWGS) data. The pipeline was first validated against samples of known heteroplasmy, and then applied to 929 samples from a PTB cohort from diverse ethnic backgrounds with an average gestational age of 27.18 weeks (range: 21-30). Our new pipeline successfully identified haplogroups and a large number of mtDNA variants in this large PTB cohort, including 8 samples carrying known pathogenic variants and 47 samples carrying rare mtDNA variants. These results confirm that lcWGS can be utilized to reliably identify mtDNA variants. These mtDNA variants may make a contribution toward preterm birth in a small proportion of live births.

Nanoscopic quantification of sub-mitochondrial morphology, mitophagy and mitochondrial dynamics in living cells derived from patients with mitochondrial diseases.

SLC25A46 mutations have been found to lead to mitochondrial hyper-fusion and reduced mitochondrial respiratory function, which results in optic atrophy, cerebellar atrophy, and other clinical symptoms of mitochondrial disease. However, it is generally believed that mitochondrial fusion is attributable to increased mitochondrial oxidative phosphorylation (OXPHOS), which is inconsistent with the decreased OXPHOS of highly-fused mitochondria observed in previous studies. In this paper, we have used the live-cell nanoscope to observe and quantify the structure of mitochondrial cristae, and the behavior of mitochondria and lysosomes in patient-derived SLC25A46 mutant fibroblasts. The results show that the cristae have been markedly damaged in the mutant fibroblasts, but there is no corresponding increase in mitophagy. This study suggests that severely damaged mitochondrial cristae might be the predominant cause of reduced OXPHOS in SLC25A46 mutant fibroblasts. This study demonstrates the utility of nanoscope-based imaging for realizing the sub-mitochondrial morphology, mitophagy and mitochondrial dynamics in living cells, which may be particularly valuable for the quick evaluation of pathogenesis of mitochondrial morphological abnormalities.

Mitochondrial replacement therapy: Genetic counselors' experiences, knowledge, and opinions.

Mitochondrial disorders affect at least 1 in 5,000 individuals worldwide and are often incurable and fatal. Mitochondrial replacement therapy (MRT) is an in vitro fertilization technique used to prevent the transmission of mitochondrial disorders. Currently, MRT is the only approach that provides mothers who carry a pathogenic variant in their mitochondrial DNA (mtDNA), the opportunity to have a biological child without a mitochondrial disease. MRT involves the combination of nuclear DNA from the egg of the carrier mother and the cytoplasm from an oocyte donor, which contains healthy mitochondria. While MRT was approved for use in the UK in 2015, the ban on congressional funding for research on 'heritable genetic modification' has made MRT unavailable within the US borders. This survey-based study aimed to describe genetic counselors' experience, knowledge, and opinions about MRT. Additionally, we also assessed whether genetic counselors' comfort discussing MRT with patients, and feelings about clinical use of MRT in the United States changed after providing information about MRT compared with baseline. Responses were received from 139 genetic counselors in North America. Findings indicate low awareness and knowledge about MRT among participants. However, more participants expressed comfort with discussing MRT with patients and more participants were able to form opinions about statements about MRT after they were provided with information about MRT. This study is the first to assess genetic counselors' opinions toward MRT and suggests the need for more education about novel technologies such as MRT among genetic counselors.

Mitochondrial genome variant m.3250T>C as a possible risk factor for mitochondrial cardiomyopathy.

The MT-TL1 gene codes for the mitochondrial leucine transfer RNA (tRNALeu(UUR) ) necessary for mitochondrial translation. Pathogenic variants in the MT-TL1 gene result in mitochondriopathy in humans. The m.3250T>C variant in the MT-TL1 gene has been previously associated with exercise intolerance and mitochondrial myopathy, yet disease classification for this variant has not been consistently reported. Molecular studies suggest the m.3250T>C variant does not alter tRNALeu(UUR) structure but may have a modest impact on aminoacylation capacity. However, functional studies are limited. Our study aimed to further define the clinical presentation, inheritance pattern, and molecular pathology of the m.3250T>C variant. Families with the m.3250T>C variant were recruited from the Mitochondrial Disease Clinic at Cincinnati Children's Hospital Medical Center and GeneDx laboratory database. Affected individuals most frequently presented with cardiac findings, exercise intolerance, and muscle weakness. Hypertrophic cardiomyopathy was the most frequent cardiac finding. Many asymptomatic individuals had homoplasmic or near homoplasmic levels of the m.3250T>C variant, suggesting the penetrance is incomplete. Patient-derived fibroblasts demonstrated lowered ATP production and increased levels of reactive oxygen species. Our results demonstrate that the m.3250T>C variant exhibits incomplete penetrance and may be a possible cause of cardiomyopathy by impacting cellular respiration in mitochondria.

Systemic Delivery of AAV-Fdxr Mitigates the Phenotypes of Mitochondrial Disorders in Fdxr Mutant Mice.

Gene therapy now provides a novel approach for treating inherited monogenetic disorders, including nuclear gene mutations associated with mitochondrial diseases. In this study, we have utilized a mouse model carrying a p.Arg389Gln mutation of the mitochondrial Ferredoxin Reductase gene (Fdxr) and treated them with neurotropic AAV-PHP.B vector loaded with the mouse Fdxr cDNA sequence. We then used immunofluorescence staining and western blot to test the transduction efficiency of this vector. Toluidine blue staining and electronic microscopy were also utilized to assess the morphology of optic and sciatic nerves, and the mitochondrial respiratory chain activity was determined as well. The AAV vector effectively transduced in the central nervous system and peripheral organs, and AAV-Fdxr treatment reversed almost all the symptoms of the mutants (Fdxr R389Q/R389Q ). This therapy also improved the electronic conductivity of the sciatic nerves, prevented optic atrophy, improved mobility, and restored mitochondrial complex function. Most notably, the sensory neuropathy, neurodegeneration, and chronic neuroinflammation in the brain were alleviated. Overall, we present the first demonstration of a potential definitive treatment that significantly improves optic and sciatic nerve atrophy, sensory neuropathy, and mitochondrial dysfunction in FDXR-related mitochondriopathy. Our study provides substantial support for the translation of AAV-based Fdxr gene therapy into clinical applications.

Integrated analysis of the molecular pathogenesis of FDXR-associated disease.

The mitochondrial flavoprotein ferredoxin reductase (FDXR) is required for biogenesis of iron-sulfur clusters and for steroidogenesis. Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, and an increasing number of disorders are associated with disruptions in the synthesis of Fe-S clusters. Our previous studies have demonstrated that hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans and mice, attributed in part to reduced function of the electron transport chain (ETC) as well as elevated production of reactive oxygen species (ROS). Inflammation and peripheral neuropathy are also hallmarks of this disease. In this paper, we demonstrate that FDXR mutation leads to significant optic transport defects that are likely to underlie optic atrophy, a major clinical presentation in FDXR patients, as well as a neurodegenerative loss of cells in the central nervous system (CNS). Molecular analysis indicates that FDXR mutation also leads to mitochondrial iron overload and an associated depolarization of the mitochondrial membrane, further supporting the hypothesis that FDXR mutations cause neurodegeneration by affecting FDXR's critical role in iron homeostasis.

The role of mitochondrial-related nuclear genes in age-related common disease.

Mitochondria are critical organelles that provide energy as ATP to the cell. Besides 37 genes encoded by mitochondrial genome, it has been estimated that over 1500 nuclear genes are required for mitochondrial structure and function. Thus, mutations of many genes in the nuclear genome cause dysfunction of mitochondria that can lead to many severe conditions. Mitochondrial dysfunction often results in reduced ATP synthesis, higher levels of reactive oxygen species (ROS), imbalanced mitochondrial dynamics, and other detrimental effects. In addition to rare primary mitochondrial disorders, these mitochondrial-related genes are often associated with many common diseases. For example, in neurodegenerative diseases such as Parkinson's, Alzheimer's, and Huntington disease, mitochondrialand energy metabolism abnormalities can greatly affect brain function. Cancer cells are also known to exhibit repressed mitochondrial ATP production in favor of glycolysis, which fuels the aggressive proliferation and metastasis of tumor tissues, leading many to speculate on a possible relationship between compromised mitochondrial function and cancer. The association between mitochondrial dysfunction and diabetes is also unsurprising, given the organelle's crucial role in cellular energy utilization. Here, we will discuss the multiple lines of evidence connecting mitochondrial dysfunction associated with mitochondria-related nuclear genes to many of the well-known disease genes that also underlie common disease.

The special considerations of gene therapy for mitochondrial diseases.

The recent success of gene therapy across multiple clinical trials has inspired a great deal of hope regarding the treatment of previously intractable genetic diseases. This optimism has been extended to the prospect of gene therapy for mitochondrial disorders, which are not only particularly severe but also difficult to treat. However, this hope must be tempered by the reality of the mitochondrial organelle, which possesses specific biological properties that complicate genetic manipulation. In this perspective, we will discuss some of these complicating factors, including the unique pathways used to express and import mitochondrial proteins. We will also present some ways in which these challenges can be overcome by genetic manipulation strategies tailored specifically for mitochondrial diseases.

Systemic administration of AAV-Slc25a46 mitigates mitochondrial neuropathy in Slc25a46-/- mice.

Mitochondrial disorders are the result of nuclear and mitochondrial DNA mutations that affect multiple organs, with the central and peripheral nervous system often affected. Currently, there is no cure for mitochondrial disorders. Currently, gene therapy offers a novel approach for treating monogenetic disorders, including nuclear genes associated with mitochondrial disorders. We utilized a mouse model carrying a knockout of the mitochondrial fusion-fission-related gene solute carrier family 25 member 46 (Slc25a46) and treated them with neurotrophic AAV-PHP.B vector carrying the mouse Slc25a46 coding sequence. Thereafter, we used immunofluorescence staining and western blot to test the transduction efficiency of this vector. Toluidine blue staining and electronic microscopy were utilized to assess the morphology of optic and sciatic nerves following treatment, and the morphology and respiratory chain activity of mitochondria within these tissues were determined as well. The adeno-associated virus (AAV) vector effectively transduced in the cerebrum, cerebellum, heart, liver and sciatic nerves. AAV-Slc25a46 treatment was able to rescue the premature death in the mutant mice (Slc25a46-/-). The treatment-improved electronic conductivity of the peripheral nerves increased mobility and restored mitochondrial complex activities. Most notably, mitochondrial morphology inside the tissues of both the central and peripheral nervous systems was normalized, and the neurodegeneration, chronic neuroinflammation and loss of Purkinje cell dendrites observed within the mutant mice were alleviated. Overall, our study shows that AAV-PHP.B's neurotrophic properties are plausible for treating conditions where the central nervous system is affected, such as many mitochondrial diseases, and that AAV-Slc25a46 could be a novel approach for treating SLC25A46-related mitochondrial disorders.

Mitochondria and Their Role in Human Reproduction.

Genetic defects of the mitochondrial genome can be especially devastating to patients; moreover, attention on human mitochondrial disorders has grown remarkably in recent years. Mitochondrial DNA (mtDNA) is maternally inherited in most eukaryotes, with paternal mtDNA being eliminated from the embryo through a variety of mechanisms. Consequently, mtDNA mutations acquired in a woman's germline can impair fertility and/or lead to severe (and even fatal) diseases in her offspring. These issues are exacerbated as the age of the mother increases. In this review, we discuss the relationship between mitochondrial dysfunction, aging, and fertility, as well as current practices for screening and diagnosing mitochondrial defects in preimplantation embryos. We also discuss recent developments in the use of mitochondrial replacement therapy to prevent the transmission of maternally-inherited mitochondrial diseases.

Mitochondrial DNA Variants and Common Diseases: A Mathematical Model for the Diversity of Age-Related mtDNA Mutations.

The mitochondrion is the only organelle in the human cell, besides the nucleus, with its own DNA (mtDNA). Since the mitochondrion is critical to the energy metabolism of the eukaryotic cell, it should be unsurprising, then, that a primary driver of cellular aging and related diseases is mtDNA instability over the life of an individual. The mutation rate of mammalian mtDNA is significantly higher than the mutation rate observed for nuclear DNA, due to the poor fidelity of DNA polymerase and the ROS-saturated environment present within the mitochondrion. In this review, we will discuss the current literature showing that mitochondrial dysfunction can contribute to age-related common diseases such as cancer, diabetes, and other commonly occurring diseases. We will then turn our attention to the likely role that mtDNA mutation plays in aging and senescence. Finally, we will use this context to develop a mathematical formula for estimating for the accumulation of somatic mtDNA mutations with age. This resulting model shows that almost 90% of non-proliferating cells would be expected to have at least 100 mutations per cell by the age of 70, and almost no cells would have fewer than 10 mutations, suggesting that mtDNA mutations may contribute significantly to many adult onset diseases.