An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants.

Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.

A fructose receptor functions as a nutrient sensor in the Drosophila brain.

Internal nutrient sensors play important roles in feeding behavior, yet their molecular structure and mechanism of action are poorly understood. Using Ca(2+) imaging and behavioral assays, we show that the gustatory receptor 43a (Gr43a) functions as a narrowly tuned fructose receptor in taste neurons. Remarkably, Gr43a also functions as a fructose receptor in the brain. Interestingly, hemolymph fructose levels are tightly linked to feeding status: after nutritious carbohydrate consumption, fructose levels rise several fold and reach a concentration sufficient to activate Gr43a in the brain. By using different feeding paradigms and artificial activation of Gr43a-expressing brain neurons, we show that Gr43a is both necessary and sufficient to sense hemolymph fructose and promote feeding in hungry flies but suppress feeding in satiated flies. Thus, our studies indicate that the Gr43a-expressing brain neurons function as a nutrient sensor for hemolymph fructose and assign opposing valence to feeding experiences in a satiation-dependent manner.

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.

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.

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.

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.

Biparental Inheritance of Mitochondrial DNA in Humans.

Although there has been considerable debate about whether paternal mitochondrial DNA (mtDNA) transmission may coexist with maternal transmission of mtDNA, it is generally believed that mitochondria and mtDNA are exclusively maternally inherited in humans. Here, we identified three unrelated multigeneration families with a high level of mtDNA heteroplasmy (ranging from 24 to 76%) in a total of 17 individuals. Heteroplasmy of mtDNA was independently examined by high-depth whole mtDNA sequencing analysis in our research laboratory and in two Clinical Laboratory Improvement Amendments and College of American Pathologists-accredited laboratories using multiple approaches. A comprehensive exploration of mtDNA segregation in these families shows biparental mtDNA transmission with an autosomal dominantlike inheritance mode. Our results suggest that, although the central dogma of maternal inheritance of mtDNA remains valid, there are some exceptional cases where paternal mtDNA could be passed to the offspring. Elucidating the molecular mechanism for this unusual mode of inheritance will provide new insights into how mtDNA is passed on from parent to offspring and may even lead to the development of new avenues for the therapeutic treatment for pathogenic mtDNA transmission.

Functional characterization of odorant receptors in the ponerine ant, Harpegnathos saltator.

Animals use a variety of sensory modalities-including visual, acoustic, and chemical-to sense their environment and interact with both conspecifics and other species. Such communication is especially critical in eusocial insects such as honey bees and ants, where cooperation is critical for survival and reproductive success. Various classes of chemoreceptors have been hypothesized to play essential roles in the origin and evolution of eusociality in ants, through their functional roles in pheromone detection that characterizes reproductive status and colony membership. To better understand the molecular mechanisms by which chemoreceptors regulate social behaviors, we investigated the roles of a critical class of chemoreceptors, the odorant receptors (ORs), from the ponerine ant Harpegnathos saltator in detecting cuticular hydrocarbon pheromones. In light of the massive OR expansion in ants (∼400 genes per species), a representative survey based on phylogenetic and transcriptomic criteria was carried out across discrete odorant receptor subfamilies. Responses to several classes of semiochemicals are described, including cuticular hydrocarbons and mandibular gland components that act as H. saltator pheromones, and a range of more traditional general odorants. When viewed through the prism of caste-specific OR enrichment and distinctive OR subfamily odorant response profiles, our findings suggest that whereas individual HsOrs appear to be narrowly tuned, there is no apparent segregation of tuning responses within any discrete HsOr subfamily. Instead, the HsOR gene family as a whole responds to a broad array of compounds, including both cuticular hydrocarbons and general odorants that are likely to mediate distinct behaviors.

Biallelic mutations in FDXR cause neurodegeneration associated with inflammation.

Mitochondrial dysfunction lies behind many neurodegenerative disorders, owing largely to the intense energy requirements of most neurons. Such mitochondrial dysfunction may work through a variety of mechanisms, from direct disruption of the electron transport chain to abnormal mitochondrial biogenesis. Recently, we have identified biallelic mutations in the mitochondrial flavoprotein "ferredoxin reductase" (FDXR) gene as a novel cause of mitochondriopathy, peripheral neuropathy, and optic atrophy. In this report, we expand upon those results by describing two new cases of disease-causing FDXR variants in patients with variable severity of phenotypes, including evidence of an inflammatory response in brain autopsy. To investigate the underlying pathogenesis, we examined neurodegeneration in a mouse model. We found that Fdxr mutant mouse brain tissues share pathological changes similar to those seen in patient autopsy material, including increased astrocytes. Furthermore, we show that these abnormalities are associated with increased levels of markers for both neurodegeneration and gliosis, with the latter implying inflammation as a major factor in the pathology of Fdxr mutations. These data provide further insight into the pathogenic mechanism of FDXR-mediated central neuropathy, and suggest an avenue for mechanistic studies that will ultimately inform treatment.

Phylogenetic and transcriptomic analysis of chemosensory receptors in a pair of divergent ant species reveals sex-specific signatures of odor coding.

Ants are a highly successful family of insects that thrive in a variety of habitats across the world. Perhaps their best-known features are complex social organization and strict division of labor, separating reproduction from the day-to-day maintenance and care of the colony, as well as strict discrimination against foreign individuals. Since these social characteristics in ants are thought to be mediated by semiochemicals, a thorough analysis of these signals, and the receptors that detect them, is critical in revealing mechanisms that lead to stereotypic behaviors. To address these questions, we have defined and characterized the major chemoreceptor families in a pair of behaviorally and evolutionarily distinct ant species, Camponotus floridanus and Harpegnathos saltator. Through comprehensive re-annotation, we show that these ant species harbor some of the largest yet known repertoires of odorant receptors (Ors) among insects, as well as a more modest number of gustatory receptors (Grs) and variant ionotropic glutamate receptors (Irs). Our phylogenetic analyses further demonstrate remarkably rapid gains and losses of ant Ors, while Grs and Irs have also experienced birth-and-death evolution to different degrees. In addition, comparisons of antennal transcriptomes between sexes identify many chemoreceptors that are differentially expressed between males and females and between species. We have also revealed an agonist for a worker-enriched OR from C. floridanus, representing the first case of a heterologously characterized ant tuning Or. Collectively, our analysis reveals a large number of ant chemoreceptors exhibiting patterns of differential expression and evolution consistent with sex/species-specific functions. These differentially expressed genes are likely associated with sex-based differences, as well as the radically different social lifestyles observed between C. floridanus and H. saltator, and thus are targets for further functional characterization. Our findings represent an important advance toward understanding the molecular basis of social interactions and the differential chemical ecologies among ant species.

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.

FDXR variants cause adrenal insufficiency and atypical sexual development.

Genetic defects affecting steroid biosynthesis cause cortisol deficiency and differences of sex development; among them recessive mutations in the steroidogenic enzymes CYP11A1 and CYP11B, whose function is supported by reducing equivalents donated by ferredoxin reductase (FDXR) and ferredoxin. So far, mutations in the mitochondrial flavoprotein FDXR have been associated with a progressive neuropathic mitochondriopathy named FDXR-Related Mitochondriopathy (FRM), but cortisol insufficiency has not been documented. However, FRM patients often experience worsening or demise following stress associated with infections. We investigated two female FRM patients carrying the novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life; they presented with cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. In addition, steroidogenic FDXR-variant cell lines reprogrammed from three FRM patients' fibroblasts displayed deficient mineralocorticoid and glucocorticoid production. Finally, Fdxr-mutant mice allelic to the severe p.R386W human variant, showed reduced progesterone and corticosterone production. Therefore, our comprehensive studies show that human FDXR variants may cause compensated, but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance.

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.

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.

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.