Nuclear-encoded Mitochondrial Genes Research Articles

Polygenic Risk Scores Validated in Patient-Derived Cells Stratify for Mitochondrial Subtypes of Parkinson's Disease.

The aim of our study is to better understand the genetic architecture and pathological mechanisms underlying neurodegeneration in idiopathic Parkinson's disease (iPD). We hypothesized that a fraction of iPD patients may harbor a combination of common variants in nuclear-encoded mitochondrial genes ultimately resulting in neurodegeneration. We used mitochondria-specific polygenic risk scores (mitoPRSs) and created pathway-specific mitoPRSs using genotype data from different iPD case-control datasets worldwide, including the Luxembourg Parkinson's Study (412 iPD patients and 576 healthy controls) and COURAGE-PD cohorts (7,270 iPD cases and 6,819 healthy controls). Cellular models from individuals stratified according to the most significant mitoPRS were subsequently used to characterize different aspects of mitochondrial function. Common variants in genes regulating Oxidative Phosphorylation (OXPHOS-PRS) were significantly associated with a higher PD risk in independent cohorts (Luxembourg Parkinson's Study odds ratio, OR = 1.31[1.14-1.50], p-value = 5.4e-04; COURAGE-PD OR = 1.23[1.18-1.27], p-value = 1.5e-29). Functional analyses in fibroblasts and induced pluripotent stem cells-derived neuronal progenitors revealed significant differences in mitochondrial respiration between iPD patients with high or low OXPHOS-PRS (p-values < 0.05). Clinically, iPD patients with high OXPHOS-PRS have a significantly earlier age at disease onset compared to low-risk patients (false discovery rate [FDR]-adj p-value = 0.015), similar to prototypic monogenic forms of PD. Finally, iPD patients with high OXPHOS-PRS responded more effectively to treatment with mitochondrially active ursodeoxycholic acid. OXPHOS-PRS may provide a precision medicine tool to stratify iPD patients into a pathogenic subgroup genetically defined by specific mitochondrial impairment, making these individuals eligible for future intelligent clinical trial designs. ANN NEUROL 2024.

Mitochondrial and Nuclear DNA Variants in Amyotrophic Lateral Sclerosis: Enrichment in the Mitochondrial Control Region and Sirtuin Pathway Genes in Spinal Cord Tissue.

Amyotrophic Lateral Sclerosis (ALS) is a progressive disease with prevalent mitochondrial dysfunctions affecting both upper and lower motor neurons in the motor cortex, brainstem, and spinal cord. Despite mitochondria having their own genome (mtDNA), in humans, most mitochondrial genes are encoded by the nuclear genome (nDNA). Our study aimed to simultaneously screen for nDNA and mtDNA genomes to assess for specific variant enrichment in ALS compared to control tissues. Here, we analysed whole exome (WES) and whole genome (WGS) sequencing data from spinal cord tissues, respectively, of 6 and 12 human donors. A total of 31,257 and 301,241 variants in nuclear-encoded mitochondrial genes were identified from WES and WGS, respectively, while mtDNA reads accounted for 73 and 332 variants. Despite technical differences, both datasets consistently revealed a specific enrichment of variants in the mitochondrial Control Region (CR) and in several of these genes directly associated with mitochondrial dynamics or with Sirtuin pathway genes within ALS tissues. Overall, our data support the hypothesis of a variant burden in specific genes, highlighting potential actionable targets for therapeutic interventions in ALS.

Correction: Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish.

Correction: Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish.

Differential Expression of Nuclear-Encoded Mitochondrial Protein Genes of ATP Synthase Across Different Tissues of Female Buffalo.

The physiological well-being of buffaloes, encompassing phenotypic traits, reproductive health, and productivity, depends on their energy status. Mitochondria, the architects of energy production, orchestrate a nuanced interplay between nuclear and mitochondrial domains. Oxidative phosphorylation complexes and associated proteins wield significant influence over metabolic functions, energy synthesis, and organelle dynamics, often linked to tissue-specific pathologies. The unexplored role of ATP synthase in buffalo tissues prompted a hypothesis: in-depth exploration of nuclear-derived mitochondrial genes, notably ATP synthase, reveals distinctive tissue-specific diversity. RNA extraction and sequencing of buffalo tissues (kidney, heart, brain, and ovary) enabled precise quantification of nuclear-derived mitochondrial protein gene expression. The analysis unveiled 24 ATP synthase transcript variants, each with unique tissue-specific patterns. Kidney, brain, and heart exhibited elevated gene expression comparedtoovaries, with 10, 8, and 19 up-regulated genes, respectively. The kidney showed 3 and 12 down-regulated genes compared to the brain and heart. The heart-brain comparison highlighted ten highly expressed genes in ATP synthase functions. Gene ontology and pathway analyses revealed enriched functions linked to ATP synthesis and oxidative phosphorylation, offering a comprehensive understanding of energy production in buffalo tissues. This analysis enhances understanding of tissue-specific gene expression, emphasizing the influence of energy demands. Revealing intricate links between mitochondrial gene expression and tissue specialization in buffaloes, it provides nuanced insights into tissue-specific expression of nuclear-encoded mitochondrial protein genes, notably ATP synthase, advancing the comprehension of buffalo tissue biology.

Mitochondrial DNA D-loop variants correlate with a primary open-angle glaucoma subgroup

IntroductionPrimary open-angle glaucoma (POAG) is a characteristic optic neuropathy, caused by degeneration of the optic nerve-forming neurons, the retinal ganglion cells (RGCs). High intraocular pressure (IOP) and aging have been identified as major risk factors; yet the POAG pathophysiology is not fully understood. Since RGCs have high energy requirements, mitochondrial dysfunction may put the survivability of RGCs at risk. We explored in buffy coat DNA whether mtDNA variants and their distribution throughout the mtDNA could be risk factors for POAG.MethodsThe mtDNA was sequenced from age- and sex-matched study groups, being high tension glaucoma (HTG, n=71), normal tension glaucoma patients (NTG, n=33), ocular hypertensive subjects (OH, n=7), and cataract controls (without glaucoma; n=30), all without remarkable comorbidities.ResultsNo association was found between the number of mtDNA variants in genes encoding proteins, tRNAs, rRNAs, and in non-coding regions in the different study groups. Next, variants that controls shared with the other groups were discarded. A significantly higher number of exclusive variants was observed in the D-loop region for the HTG group (~1.23 variants/subject), in contrast to controls (~0.35 variants/subject). In the D-loop, specifically in the 7S DNA sub-region within the Hypervariable region 1 (HV1), we found that 42% of the HTG and 27% of the NTG subjects presented variants, while this was only 14% for the controls and OH subjects. As we have previously reported a reduction in mtDNA copy number in HTG, we analysed if specific D-loop variants could explain this. While the majority of glaucoma patients with the exclusive D-loop variants m.72T&amp;gt;C, m.16163 A&amp;gt;G, m.16186C&amp;gt;T, m.16298T&amp;gt;C, and m.16390G&amp;gt;A presented a mtDNA copy number below controls median, no significant association between these variants and low copy number was found and their possible negative role in mtDNA replication remains uncertain. Approximately 38% of the HTG patients with reduced copy number did not carry any exclusive D-loop or other mtDNA variants, which indicates that variants in nuclear-encoded mitochondrial genes, environmental factors, or aging might be involved in those cases.ConclusionIn conclusion, we found that variants in the D-loop region may be a risk factor in a subgroup of POAG, possibly by affecting mtDNA replication.

Altered Mitochondrial DNA Methylation Patterns in Thrombosis

DNA methylation being one of the chief controllers of gene expression has not only been the reason behind the initiation of a plethora of diseases like Cancer, Alzheimer’s disease, Parkinson’s disease, etc. Still, it is an active contributor to the pathophysiology of several cardiovascular diseases like coronary artery disease (CAD), Atherosclerosis, Stroke, Cardiomyopathy, etc. The role of nuclear DNA methylation in VTE has been studied earlier, but the comparison of methylation in both nuclear genes and mitochondrial genes in high altitude VTE (HA-VTE) and sea level VTE (SL-VTE) patients has not been studied in depth. Through this present study, DNA methylation patterns of mitochondrial encoded as well as nuclear-encoded mitochondrial genes of five high-altitude VTE patients and five sea-level VTE patients have been obtained. On comparing HA-VTE vs. SL-VTE methylation, one hundred and twenty hypermethylated genes and one hundred and thirty-eight hypomethylated genes were observed. Post gene enrichment and ontology study, the TCA cycle and NADH dehydrogenase were found to be the highly enriched pathways in both the study groups. Protein-protein interaction network using STRING pointed out the enriched pathway of L-2-hydroxyglutaric acid when both the gene sets were enriched. These results show the crucial role of mitochondrial DNA methylation in the pathophysiology of thrombosis and show great potential to study the role of mitochondria in thrombosis.

Mitochondrial DNA Haplogroups and Variants Predispose to Chagas Disease Cardiomyopathy

Cardiomyopathies are major causes of heart failure. Chagas disease (CD) is caused by the parasite Trypanosoma cruzi, and it is endemic in Central and South America. Thirty percent of cases evolve into chronic chagas cardiomyopathy (CCC), which has worse prognosis as compared with other cardiomyopathies. In vivo bioenergetic analysis and ex vivo proteomic analysis of myocardial tissues highlighted worse mitochondrial dysfunction in CCC, and previous studies identified nuclear-encoded mitochondrial gene variants segregating with CCC. Here, we assessed the role of the mitochondrial genome through mtDNA copy number variations and mtDNA haplotyping and sequencing from heart or blood tissues of severe, moderate CCC and asymptomatic/indeterminate Chagas disease as well as healthy controls as an attempt to help decipher mitochondrial-intrinsic genetic involvement in Chagas disease development. We have found that the mtDNA copy number was significantly lower in CCC than in heart tissue from healthy individuals, while blood mtDNA content was similar among asymptomatic Chagas disease, moderate, and severe CCC patients. An MtDNA haplogrouping study has indicated that African haplogroups were over represented in the Chagas subject groups in comparison with healthy Brazilian individuals. The European lineage is associated with protection against cardiomyopathy and the macro haplogroup H is associated with increased risk towards CCC. Using mitochondria DNA sequencing, 84 mtDNA-encoded protein sequence pathogenic variants were associated with CCC. Among them, two variants were associated to left ventricular non-compaction and two to hypertrophic cardiomyopathy. The finding that mitochondrial protein-coding SNPs and mitochondrial haplogroups associate with risk of evolving to CCC is consistent with a key role of mitochondrial DNA in the development of chronic chagas disease cardiomyopathy.

Different impact of vitamin D on mitochondrial activity and morphology in normal and malignant keratinocytes, the role of genomic pathway

Deregulation of mitochondria activity is one of the hallmarks of cancerogenesis and an important target for cancer therapy. Therefore, we compared the impact of an active form of vitamin D3 (1,25(OH)2D3) on mitochondrial morphology and bioenergetics in human squamous cell carcinoma (A431) and immortalized HaCaT keratinocytes. It was shown that mitochondria of cancerous A431 cells differ from that observed in HaCaT keratinocytes in terms of network, morphology, bioenergetics, glycolysis, and mitochondrial DNA copy number, while treatment of A431 with 1,25(OH)2D3 partially eliminates these differences. Furthermore, mitochondrial membrane potential, basal respiration, and mitochondrial reactive oxygen species production were decreased in A431 cells treated with 1,25(OH)2D3. Additionally, the expression and protein level of mitophagy marker PINK1 was significantly increased in A431 1,25(OH)2D3 treated cells, but not observed in treated HaCaT cells. Knockout of VDR (vitamin D receptor) or RXRA (binding partner retinoid X receptor) partially altered mitochondrial morphology and function as well as mitochondrial response to 1,25(OH)2D3. Transcriptomic analysis on A431 cells treated with 1,25(OH)2D3 revealed modulation of expression of several mitochondrial-related genes involved in mitochondrial depolarization, mitochondrial protein translation (i.e. LYRM9, MARS2), and fusion-fission (OPA1, FIS1, MFN1 and 2), however, none of the genes coded by mitochondrial DNA was affected. Interestingly, in silico analyses of nuclear-encoded mitochondrial genes revealed that they are rather activated by the secondary genomic response to 1,25(OH)2D3. Taken together, 1,25(OH)2D3 remodels mitochondrial architecture and bioenergetics through VDR-dependent and only partially RXRA-dependent activation of the genomic pathway, thus outlining a new perspective for anticancer properties of vitamin D3 in relation to mitochondria in squamous cell carcinoma.

Mitochondria-Mediated (MAVS) Innate Immune Signaling Drives Inflammation-Induced Bone Marrow Failure

Bone marrow failure syndromes (BMFS) are life threatening diseases, characterized by impaired fitness of hematopoietic stem cells (HSC) and ineffective hematopoiesis causing the absence of one or more hematopoietic lineages in the peripheral blood. Substantial clinical data indicate that hyperactivity of inflammatory cytokines, including TNFα, IL-6, and transforming growth factor-β (TGFβ), directly contribute to BMF. Inflammatory challenges result in irreversible HSC functional decline, including decreased repopulation potential and myeloid-bias differentiation. We recently showed that the combination of inflammatory challenges, including polyinosinc:polycytidilic acid (pIC - a double stranded RNA [dsRNA]) plus high TGFβ causes chronic pancytopenia, anemia, expanded hematopoietic stem and progenitor cell pools, and enhanced bone marrow dysplasia, mimicking a myelodysplastic syndrome (MDS) that developed 3-4 months after pIC challenge (Javier et.al. Haematologica 2022). However, the mechanisms behind this association and/or causality are still ill-defined. Here, we show that mitochondrial antiviral signaling (MAVS) protein can mediate the negative effect of pIC and TGFβ on HSC functions and BMF/ MDS development. Differential gene expression analyses in HSCs suggested that pIC plus TGFβ altered expression of nuclear-encoded mitochondrial genes. Mitochondria serve as platform for the activation of MAVS and downstream innate immune signaling in response to dsRNA. We thus examined the role of mitochondria and MAVS in pIC plus TGFβ-induced BMF/MDS. We show that overexpression TGFβ and pIC together cause alterations in mitochondrial proteins and membrane potential as well as enhanced caspase1 activity, but not the necroptotic signals p-RIP1 and p-RIP3, in HSCs. pIC exposure caused an increase in MAVS expression, and a redistribution into large aggregates that localize onto mitochondria in HSCs, indicative of MAVS activation (two days after pIC exposure in vivo). The MAVS aggregates resolved into smaller and dispersed clusters by 3 months after pIC exposure in control mice. In contrast, in HSCs from TGFβ overexpressing mice, MAVS aggregates persisted 3 months after pIC exposure. To determine the impact of augmented MAVS in dsRNA+TGFβ-driven BMF, we used MAVS deficient mice crossed with TGFβ overexpressing mice. We found that absence of MAVS protein restored normal white blood cell count, including neutrophils and lymphocytes, restored normal red blood cell but not platelet counts in TGFβ overexpressing mice after pIC challenge, compared to controls. Remarkably, MAVS deficiency partially rescued the dysplastic features of bone marrow myeloid cells in TGFβ overexpressing mice after pIC challenge. MAVS-deficiency also reduced the increased caspase1 activity and lowers mitochondria membrane potential in HSCs of these mice. In serial competitive transplant studies, MAVS-deficiency conferred HSC repopulation advantage. These findings suggest that pIC+TGFβ overexpression mediated BMF/MDS phenotype depends on chronic activation of MAVS. Because MAVS aggregation depends on mitochondrial fusion, we used genetic loss of the mitochondrial fission regulator Drp1. Drp1-deficiency caused severe mitochondrial aggregation in HSCs; this was associated with sustained MAVS aggregation. In response to pIC, Drp1-deficient mice exhibited increased caspase-1 activity, increased mitochondrial membrane potential and increase in HSC numbers 6 months after pIC challenges. Drp1 loss resulted in anemia and mild pancytopenia. Interestingly, Drp1-deficient bone marrow myeloid progenitors exhibited severe dysplastic features after pIC challenged. These findings suggest that chronic mitochondrial and MAVS aggregation are sufficient to drive BMF/MDS phenotype. Finally, we investigated the levels of epigenetic markers (acetyl H3K27 and methyl H3 Lys4) in HSCs after pIC stress. H4K4me3 levels were decreased in HSCs long-term after pIC challenges [6 months], this was further decreased in Drp1-deficient HSCs. In conclusion, these findings indicate that non-genetic factors contribute to the onset of BMF/MDS. Mitochondria and MAVS protein together mediate an innate immune mechanism that drives BMF/MDS. This study opens the possibility to target mitochondria-related proteins as a therapeutic strategy to restore ineffective hematopoiesis due to chronic inflammation.

Human forebrain organoid-based multi-omics analyses of PCCB as a schizophrenia associated gene linked to GABAergic pathways

Identifying genes whose expression is associated with schizophrenia (SCZ) risk by transcriptome-wide association studies (TWAS) facilitates downstream experimental studies. Here, we integrated multiple published datasets of TWAS, gene coexpression, and differential gene expression analysis to prioritize SCZ candidate genes for functional study. Convergent evidence prioritized Propionyl-CoA Carboxylase Subunit Beta (PCCB), a nuclear-encoded mitochondrial gene, as an SCZ risk gene. However, the PCCB’s contribution to SCZ risk has not been investigated before. Using dual luciferase reporter assay, we identified that SCZ-associated SNPs rs6791142 and rs35874192, two eQTL SNPs for PCCB, showed differential allelic effects on transcriptional activities. PCCB knockdown in human forebrain organoids (hFOs) followed by RNA sequencing analysis revealed dysregulation of genes enriched with multiple neuronal functions including gamma-aminobutyric acid (GABA)-ergic synapse. The metabolomic and mitochondrial function analyses confirmed the decreased GABA levels resulted from inhibited tricarboxylic acid cycle in PCCB knockdown hFOs. Multielectrode array recording analysis showed that PCCB knockdown in hFOs resulted into SCZ-related phenotypes including hyper-neuroactivities and decreased synchronization of neural network. In summary, this study utilized hFOs-based multi-omics analyses and revealed that PCCB downregulation may contribute to SCZ risk through regulating GABAergic pathways, highlighting the mitochondrial function in SCZ.

NR2F1 shapes mitochondria in the mouse brain, providing new insights into Bosch-Boonstra-Schaaf optic atrophy syndrome.

The nuclear receptor NR2F1 acts as a strong transcriptional regulator in embryonic and postnatal neural cells. In humans, mutations in the NR2F1 gene cause Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), a rare neurodevelopmental disorder characterized by multiple clinical features including vision impairment, intellectual disability and autistic traits. In this study, we identified, by genome-wide and in silico analyses, a set of nuclear-encoded mitochondrial genes as potential genomic targets under direct NR2F1 transcriptional control in neurons. By combining mouse genetic, neuroanatomical and imaging approaches, we demonstrated that conditional NR2F1 loss of function within the adult mouse hippocampal neurogenic niche results in a reduced mitochondrial mass associated with mitochondrial fragmentation and downregulation of key mitochondrial proteins in newborn neurons, the genesis, survival and functional integration of which are impaired. Importantly, we also found dysregulation of several nuclear-encoded mitochondrial genes and downregulation of key mitochondrial proteins in the brain of Nr2f1-heterozygous mice, a validated BBSOAS model. Our data point to an active role for NR2F1 in the mitochondrial gene expression regulatory network in neurons and support the involvement of mitochondrial dysfunction in BBSOAS pathogenesis.

Genetic perturbation of mitochondrial function reveals functional role for specific mitonuclear genes, metabolites, and pathways that regulate lifespan

Altered mitochondrial function is tightly linked to lifespan regulation, but underlying mechanisms remain unclear. Here, we report the chronological and replicative lifespan variation across 167 yeast knock-out strains, each lacking a single nuclear-coded mitochondrial gene, including 144 genes with human homologs, many associated with diseases. We dissected the signatures of observed lifespan differences by analyzing profiles of each strain’s proteome, lipidome, and metabolome under fermentative and respiratory culture conditions, which correspond to the metabolic states of replicative and chronologically aging cells, respectively. Examination of the relationships among extended longevity phenotypes, protein, and metabolite levels revealed that although many of these nuclear-encoded mitochondrial genes carry out different functions, their inhibition attenuates a common mechanism that controls cytosolic ribosomal protein abundance, actin dynamics, and proteasome function to regulate lifespan. The principles of lifespan control learned through this work may be applicable to the regulation of lifespan in more complex organisms, since many aspects of mitochondrial function are highly conserved among eukaryotes.

Identification of two novel RRM2B variants associated with autosomal recessive progressive external ophthalmoplegia in a family with pseudodominant inheritance pattern.

RRM2B encodes the p53-inducible small subunit (p53R2) of ribonucleotide reductase, a key protein for mitochondrial DNA (mtDNA) synthesis. Pathogenic variants in this gene result in familial mitochondrial disease in adults and children, secondary to a maintenance disorder of mtDNA. This study describes two patients, mother and son, with early-onset chronic progressive external ophthalmoplegia (PEO). Skeletal muscle biopsy from the latter was examined: cytochrome c oxidase (COX)-negative fibres were shown, and molecular studies revealed multiple mtDNA deletions. A next-generation sequencing gene panel for nuclear-encoded mitochondrial maintenance genes identified two unreported heterozygous missense variants (c.514 G > A and c.682 G > A) in the clinically affected son. The clinically affected mother harboured the first variant in homozygous state, and the clinically unaffected father harboured the remaining variant in heterozygous state. In silico analyses predicted both variants as deleterious. Cell culture studies revealed that patients' skin fibroblasts, but not fibroblasts from healthy controls, responded to nucleoside supplementation with enhanced mtDNA repopulation, thus suggesting an in vitro functional difference in patients' cells. Our results support the pathogenicity of two novel RRM2B variants found in two patients with autosomal recessive PEO with multiple mtDNA deletions inherited with a pseudodominant pattern.

Differential gene expression and mitonuclear incompatibilities in fast- and slow-developing interpopulation Tigriopus californicus hybrids.

Mitochondrial functions are intimately reliant on proteins and RNAs encoded in both the nuclear and mitochondrial genomes, leading to inter-genomic coevolution within taxa. Hybridization can break apart coevolved mitonuclear genotypes, resulting in decreased mitochondrial performance and reduced fitness. This hybrid breakdown is an important component of outbreeding depression and early-stage reproductive isolation. However, the mechanisms contributing to mitonuclear interactions remain poorly resolved. Here, we scored variation in developmental rate (a proxy for fitness) among reciprocal F2 interpopulation hybrids of the intertidal copepod Tigriopus californicus and used RNA sequencing to assess differences in gene expression between fast- and slow-developing hybrids. In total, differences in expression associated with developmental rate were detected for 2925 genes, whereas only 135 genes were differentially expressed as a result of differences in mitochondrial genotype. Upregulated expression in fast developers was enriched for genes involved in chitin-based cuticle development, oxidation-reduction processes, hydrogen peroxide catabolic processes and mitochondrial respiratory chain complex I. In contrast, upregulation in slow developers was enriched for DNA replication, cell division, DNA damage and DNA repair. Eighty-four nuclear-encoded mitochondrial genes were differentially expressed between fast- and slow-developing copepods, including 12 subunits of the electron transport system (ETS) which all had higher expression in fast developers than in slow developers. Nine of these genes were subunits of ETS complex I. Our results emphasize the major roles that mitonuclear interactions within the ETS, particularly in complex I, play in hybrid breakdown, and resolve strong candidate genes for involvement in mitonuclear interactions.

CROSSTALK BETWEEN UBIQUITINATION AND ADP-RIBOSYLATION IN THE MAINTENANCE OF MITOCHONDRIAL HOMEOSTASIS

Abstract Oxidative stress and increased ROS production, as observed in aging and obesity, commonly lead to the accumulation of mitochondrial dysfunctions. This is met by the activation of a robust mitochondria-to-nucleus stress response promoting the rewiring of nuclear gene expression to limit cellular and tissue damage and promote organelle adaptation. Here we will review previous work uncovering the transcriptional cofactor G-Protein Pathway Suppressor 2 (GPS2) as a mediator of mitochondria retrograde signaling and a key nuclear regulator of nuclear-encoded mitochondrial genes, including mitochondrial chaperones/proteases, mitokines, and other protective enzymes such as the ADP-ribosyltransferase NEURL4. We will also discuss unpublished work showing that shuttling of GPS2 between organelles plays a role in coordinating the transcriptional and translational regulation of antioxidant factors and pro-apoptotic genes by promoting the ubiquitination of mitochondria-associated translation factors.

OLA1 Phosphorylation Governs the Mitochondrial Bioenergetic Function of Pulmonary Vascular Cells.

Mitochondrial function and metabolic homeostasis are integral to cardiovascular function and influence how vascular cells respond to stress. However, little is known regarding how mitochondrial redox control mechanisms and metabolic regulation interact in the developing lungs. Here we show that human OLA1 (Obg-like ATPase-1) couples redox signals to the metabolic response pathway by activating metabolic gene transcription in the nucleus. OLA1 phosphorylation at Ser232/Tyr236 triggers its translocation from the cytoplasm and mitochondria into the nucleus. Subsequent phosphorylation of OLA1 at Thr325 effectively changes its biochemical function from ATPase to GTPase, promoting the expression of genes involved in the mitochondrial bioenergetic function. This process is regulated by ERK1/2 (extracellular-regulated kinases 1 and 2), which were restrained by PP1A (protein phosphatase 1A) when stress abated. Knockdown of ERK1 or OLA1 mutated to a phosphoresistant T325A mutant blocked its nuclear translocation, compromised the expression of nuclear-encoded mitochondrial genes, and consequently led to cellular energy depletion. Moreover, the lungs of OLA1 knockout mice have fewer mitochondria, lower cellular ATP concentrations, and higher lactate concentrations. The ensuing mitochondrial metabolic dysfunction resulted in abnormal behaviors of pulmonary vascular cells and significant vascular remodeling. Our findings demonstrate that OLA1 is an important component of the mitochondrial retrograde communication pathways that couple stress signals with metabolic genes in the nucleus. Thus, phosphorylation-dependent nuclear OLA1 localization that governs cellular energy metabolism is critical to cardiovascular function.

Expression Changes in Mitochondrial Genes Affecting Mitochondrial Morphology, Transmembrane Potential, Fragmentation, Amyloidosis, and Neuronal Cell Death Found in Brains of Alzheimer's Disease Patients.

Alzheimer's disease (AD) is a neurological disease that has both a genetic and non-genetic origin. Mitochondrial dysfunction is a critical component in the pathogenesis of AD as deficits in oxidative capacity and energy production have been reported. Nuclear-encoded mitochondrial genes were studied in order to understand the effects of mitochondrial expression changes on mitochondrial function in AD brains. These expression data were to be incorporated into a testable mathematical model for AD used to further assess the genes of interest as therapeutic targets for AD. RT2-PCR arrays were used to assess expression of 84 genes involved in mitochondrial biogenesis in AD brains. A subset of mitochondrial genes of interest was identified after extensive Ingenuity Pathway Analysis (IPA) (Qiagen). Further filtering of this subset of genes of interest was achieved by individual qPCR analyses. Expression values from this group of genes were included in a mathematical model being developed to identify potential therapeutic targets. Nine genes involved in trafficking proteins to mitochondria, morphology of mitochondria, maintenance of mitochondrial transmembrane potential, fragmentation of mitochondria and mitochondrial dysfunction, amyloidosis, and neuronal cell death were identified as significant to the changes seen. These genes include TP53, SOD2, CDKN2A, MFN2, DNM1L, OPA1, FIS1, BNIP3, and GAPDH. Altered mitochondrial gene expression indicates that a subset of nuclear-encoded mitochondrial genes compromise multiple aspects of mitochondrial function in AD brains. A new mathematical modeling system may provide further insights into potential therapeutic targets.

Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish.

Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a -/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to use localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.

Direct evidence of CRISPR-Cas9-mediated mitochondrial genome editing

Pathogenic mitochondrial DNA (mtDNA) mutations can cause a variety of human diseases. The recent development of genome-editing technologies to manipulate mtDNA, such as mitochondria-targeted DNA nucleases and base editors, offer a promising way for curing mitochondrial diseases caused by mtDNA mutations. The CRISPR-Cas9 system is a widely used tool for genome editing; however, its application in mtDNA editing is still under debate. In this study, we developed a mito-Cas9 system by adding the mitochondria-targeted sequences and 3′ untranslated region of nuclear-encoded mitochondrial genes upstream and downstream of the Cas9 gene, respectively. We confirmed that the mito-Cas9 system was transported into mitochondria and enabled knockin of exogenous single-stranded DNA oligonucleotides (ssODNs) into mtDNA based on proteinase and DNase protection assays. Successful knockin of exogenous ssODNs into mtDNA was further validated using polymerase chain reaction-free third-generation sequencing technology. We also demonstrated that RS-1, an agonist of RAD51, significantly increased knockin efficiency of the mito-Cas9 system. Collectively, we provide direct evidence that mtDNA can be edited using the CRISPR-Cas9 system. The mito-Cas9 system could be optimized as a promising approach for the treatment of mitochondrial diseases caused by pathogenic mtDNA mutations, especially those with homoplasmic mtDNA mutations.

Adaptive mechanisms in no flow vs. low flow ischemia in equine jejunum epithelium: Different paths to the same destination.

Intestinal ischemia reperfusion injury (IRI) is a frequent complication of equine colic. Several mechanisms may be involved in adaptation of the intestinal epithelium to IRI and might infer therapeutic potential, including hypoxia-inducible factor (HIF) 1α, AMP-activated protein kinase (AMPK), nuclear factor-erythroid 2-related factor 2 (NRF2), and induction of autophagy. However, the mechanisms supporting adaptation and thus cellular survival are not completely understood yet. We investigated the activation of specific adaptation mechanisms in both no and low flow ischemia and reperfusion simulated in equine jejunum epithelium in vivo. We found an activation of HIF1α in no and low flow ischemia as indicated by increased levels of HIF1α target genes and phosphorylation of AMPKα tended to increase during ischemia. Furthermore, the protein expression of the autophagy marker LC3B in combination with decreased expression of nuclear-encoded mitochondrial genes indicates an increased rate of mitophagy in equine intestinal IRI, possibly preventing damage by mitochondria-derived reactive oxygen species (ROS). Interestingly, ROS levels were increased only shortly after the onset of low flow ischemia, which may be explained by an increased antioxidative defense, although NFR2 was not activated in this setup. In conclusion, we could demonstrate that a variety of adaptation mechanisms manipulating different aspects of cellular homeostasis are activated in IRI irrespective of the ischemia model, and that mitophagy might be an important factor for epithelial survival following small intestinal ischemia in horses that should be investigated further.