Search

Purpose: Families living with mitochondrial diseases (MD) often endure prolonged diagnostic journeys and invasive testing, yet many remain without a molecular diagnosis. The Australian Genomics Mitochondrial Flagship, comprising clinicians, diagnostic, and research scientists, conducted a prospective national study to identify the diagnostic utility of singleton genomic sequencing using blood samples., Methods: A total of 140 children and adults living with suspected MD were recruited using modified Nijmegen criteria (MNC) and randomized to either exome + mitochondrial DNA (mtDNA) sequencing or genome sequencing., Results: Diagnostic yield was 55% (n = 77) with variants in nuclear (n = 37) and mtDNA (n = 18) MD genes, as well as phenocopy genes (n = 22). A nuclear gene etiology was identified in 77% of diagnoses, irrespective of disease onset. Diagnostic rates were higher in pediatric-onset (71%) than adult-onset (31%) cases and comparable in children with non-European (78%) vs European (67%) ancestry. For children, higher MNC scores correlated with increased diagnostic yield and fewer diagnoses in phenocopy genes. Additionally, 3 adult patients had a mtDNA deletion discovered in skeletal muscle that was not initially identified in blood., Conclusion: Genomic sequencing from blood can simplify the diagnostic pathway for individuals living with suspected MD, especially those with childhood onset diseases and high MNC scores., Competing Interests: Conflict of Interest John Christodoulou is an approved pathology provider for Victorian Clinical Genetics Services., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

CLPB is a mitochondrial intermembrane space AAA+ domain-containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease. Utilizing proteomic and biochemical approaches, we show a stress-specific aggregation phenotype in a CLPB-null environment and define the CLPB substrate profile. We illustrate an interplay between intermembrane space proteins including CLPB, HAX1, HTRA2, and the inner membrane quality control proteins (STOML2, PARL, YME1L1; SPY complex), with CLPB deficiency impeding SPY complex function by virtue of protein aggregation in the intermembrane space. We conclude that there is an interdependency of mitochondrial QC components at the intermembrane space/inner membrane interface, and perturbations to this network may underscore CLPB disease pathology., (© 2024 Baker et al.)

TIMM50 is a core subunit of the TIM23 complex, the mitochondrial inner membrane translocase responsible for the import of pre-sequence-containing precursors into the mitochondrial matrix and inner membrane. Here we describe a mitochondrial disease patient who is homozygous for a novel variant in TIMM50 and establish the first proteomic map of mitochondrial disease associated with TIMM50 dysfunction. We demonstrate that TIMM50 pathogenic variants reduce the levels and activity of endogenous TIM23 complex, which significantly impacts the mitochondrial proteome, resulting in a combined oxidative phosphorylation (OXPHOS) defect and changes to mitochondrial ultrastructure. Using proteomic data sets from TIMM50 patient fibroblasts and a TIMM50 HEK293 cell model of disease, we reveal that laterally released substrates imported via the TIM23 SORT complex pathway are most sensitive to loss of TIMM50. Proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23 SORT substrate pool, providing a biochemical mechanism for the specific defects in TIMM50-associated mitochondrial disease patients. These results highlight the power of using proteomics to elucidate molecular mechanisms of disease and uncovering novel features of fundamental biology, with the implication that human TIMM50 may have a more pronounced role in lateral insertion than previously understood.

Background: This paper examines the factors that led to the collapse of hemp grown for cannabidiol (CBD) in Arizona, the United States of America (USA), and particularly in Yuma County, which is a well-established agricultural area in the state., Methods: This research uses a combination of mapping analysis along with a survey of hemp farmers to assess the reasons why the hemp industry collapsed as well as to foster solutions to these problems., Results: In 2019, 5430 acres were sown with hemp seed in Arizona with 3890 acres inspected by the state to determine if they could be harvested. By 2021, there were only 156 acres planted, and only 128 of those acres were inspected by the state for compliance. (Crop mortality accounts for the difference between acres sown and acres inspected.) CONCLUSIONS: A lack of knowledge about the hemp life cycle greatly contributed to the failure of high CBD hemp crops in Arizona. Other problems included noncompliance with tetrahydrocannabinol limits, poor sources for seeds and inconsistent genetics of the hemp varieties sold to farmers, and diseases that hemp plants were susceptible to such as Pythium crown and root rot and beet curly top virus. Addressing these factors will go far in making hemp a profitable and widespread crop in Arizona. Additionally, hemp grown for other traditional uses (e.g., fiber or seed oil) as well as new applications (e.g., microgreens, hempcrete, and phytoremediation) offers other pathways for successful hemp agriculture in this state., (© 2023. The Author(s).)

Environmental managers have been striving to optimize landscape structure to achieve a sustained supply of ecosystem services (ESs). However, we still lack a full understanding of the relationships between landscape structure and ESs due to the absence of thorough investigations on the variability of these relationships in space and time. To fill this critical gap, we assessed landscape structure alongside four important ESs (agricultural production (AP), carbon sequestration (CS), soil conservation (SC), and water retention (WR)) in the Wuhan metropolitan area (WMA), and then analyzed the spatiotemporal impacts of landscape structure on ESs from 2000 to 2020 using Geographically and Temporally Weighted Regression. The results show only AP maintained a stable growth trend over the past two decades, while the other ESs fluctuated considerably with a noticeable decline in SC and WR. The importance of landscape structure in influencing ESs varies by time and place, depending on the local landscape composition and configuration. In general, landscape composition has a stronger and less temporally stable impact on ESs compared to configuration. Furthermore, increases in landscape diversity, as measured through Shannon's diversity index, and the percentage of woodlands were found to contribute to the simultaneous benefits of multiple ESs, but in most cases the effects of landscape structure on different ESs were different or even opposite, suggesting that trade-offs are critical in landscape management. The findings highlight the complex response of ESs to dramatically changing landscapes in the WMA and can guide decision-makers in precise spatial arrangement and temporal adjustments to improve current landscape management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mitochondrial diseases are a broad, genetically heterogeneous class of metabolic disorders characterized by deficits in oxidative phosphorylation (OXPHOS). Primary mitochondrial disease (PMD) defines pathologies resulting from mutation of mitochondrial DNA (mtDNA) or nuclear genes affecting either mtDNA expression or the biogenesis and function of the respiratory chain. Secondary mitochondrial disease (SMD) arises due to mutation of nuclear-encoded genes independent of, or indirectly influencing OXPHOS assembly and operation. Despite instances of novel SMD increasing year-on-year, PMD is much more widely discussed in the literature. Indeed, since the implementation of next generation sequencing (NGS) techniques in 2010, many novel mitochondrial disease genes have been identified, approximately half of which are linked to SMD. This review will consolidate existing knowledge of SMDs and outline discrete categories within which to better understand the diversity of SMD phenotypes. By providing context to the biochemical and molecular pathways perturbed in SMD, we hope to further demonstrate the intricacies of SMD pathologies outside of their indirect contribution to mitochondrial energy generation.

SignificanceMitochondria are double-membraned eukaryotic organelles that house the proteins required for generation of ATP, the energy currency of cells. ATP generation within mitochondria is performed by five multisubunit complexes (complexes I to V), the assembly of which is an intricate process. Mutations in subunits of these complexes, or the suite of proteins that help them assemble, lead to a severe multisystem condition called mitochondrial disease. We show that SFXN4, a protein that causes mitochondrial disease when mutated, assists with the assembly of complex I. This finding explains why mutations in SFXN4 cause mitochondrial disease and is surprising because SFXN4 belongs to a family of amino acid transporter proteins, suggesting that it has undergone a dramatic shift in function through evolution.

Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible, inexpensive therapeutic intervention that can improve mitochondrial bioenergetics and quality of life. By combining multiple omics techniques with biochemical and in silico normalisation, we removed the bias arising from the training-induced increase in mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritised mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the overall increase in mitochondrial content. Our findings suggest enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and do not support the hypothesis that training-induced supercomplex formation enhances mitochondrial bioenergetics. Our study provides an analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding, and calling for careful reinterpretation of previous findings., (© 2021. The Author(s).)

Mitochondrial diseases disrupt cellular energy production and are among the most complex group of inherited genetic disorders. Affecting approximately 1 in 5000 live births, they are both clinically and genetically heterogeneous, and can be highly tissue specific, but most often affect cell types with high energy demands in the brain, heart, and kidneys. There are currently no clinically validated treatment options available, despite several agents showing therapeutic promise. However, modelling these disorders is challenging as many non-human models of mitochondrial disease do not completely recapitulate human phenotypes for known disease genes. Additionally, access to disease-relevant cell or tissue types from patients is often limited. To overcome these difficulties, many groups have turned to human pluripotent stem cells (hPSCs) to model mitochondrial disease for both nuclear-DNA (nDNA) and mitochondrial-DNA (mtDNA) contexts. Leveraging the capacity of hPSCs to differentiate into clinically relevant cell types, these models permit both detailed investigation of cellular pathomechanisms and validation of promising treatment options. Here we catalogue hPSC models of mitochondrial disease that have been generated to date, summarise approaches and key outcomes of phenotypic profiling using these models, and discuss key criteria to guide future investigations using hPSC models of mitochondrial disease.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in AGK cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis. We mapped the proteomic changes in Sengers patient fibroblasts and AGK KO cell lines to understand the effects of AGK dysfunction on mitochondria. This uncovered down-regulation of a number of proteins at the inner mitochondrial membrane, including many SLC25 carrier family proteins, which are predicted substrates of the complex. We also observed down-regulation of SFXN proteins, which contain five transmembrane domains, and show that they represent a novel class of TIM22 complex substrate. Perturbed biogenesis of SFXN proteins in cells lacking AGK reduces the proliferative capabilities of these cells in the absence of exogenous serine, suggesting that dysregulation of one-carbon metabolism is a molecular feature in the biology of Sengers syndrome.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by defects in the skeletal system, such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients have germ line compound biallelic protein-truncating mutations of RECQL4 . As existing murine models employ Recql4 null alleles, we have attempted to more accurately model RTS by generating mice with patient-mimicking truncating Recql4 mutations. Truncating mutations impaired the stability and subcellular localization of RECQL4 and resulted in homozygous embryonic lethality and a haploinsufficient low-bone mass phenotype. Combination of a truncating mutation with a conditional Recql4 null allele demonstrated that the skeletal defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis. We utilized murine Recql4 null cells to assess the impact of human RECQL4 mutations using an in vitro complementation assay. While some mutations created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations in mice lead to an osteoporosis-like phenotype through defects in early osteoblast progenitors and identify RECQL4 gene dosage as a novel regulator of bone mass.

Background: In about half of all patients with a suspected monogenic disease, genomic investigations fail to identify the diagnosis. A contributing factor is the difficulty with repetitive regions of the genome, such as those generated by segmental duplications. The ATAD3 locus is one such region, in which recessive deletions and dominant duplications have recently been reported to cause lethal perinatal mitochondrial diseases characterized by pontocerebellar hypoplasia or cardiomyopathy, respectively., Methods: Whole exome, whole genome and long-read DNA sequencing techniques combined with studies of RNA and quantitative proteomics were used to investigate 17 subjects from 16 unrelated families with suspected mitochondrial disease., Findings: We report six different de novo duplications in the ATAD3 gene locus causing a distinctive presentation including lethal perinatal cardiomyopathy, persistent hyperlactacidemia, and frequently corneal clouding or cataracts and encephalopathy. The recurrent 68 Kb ATAD3 duplications are identifiable from genome and exome sequencing but usually missed by microarrays. The ATAD3 duplications result in the formation of identical chimeric ATAD3A/ATAD3C proteins, altered ATAD3 complexes and a striking reduction in mitochondrial oxidative phosphorylation complex I and its activity in heart tissue., Conclusions: ATAD3 duplications appear to act in a dominant-negative manner and the de novo inheritance infers a low recurrence risk for families, unlike most pediatric mitochondrial diseases. More than 350 genes underlie mitochondrial diseases. In our experience the ATAD3 locus is now one of the five most common causes of nuclear-encoded pediatric mitochondrial disease but the repetitive nature of the locus means ATAD3 diagnoses may be frequently missed by current genomic strategies., Funding: Australian NHMRC, US Department of Defense, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance and Australian Mito Foundation.

As mosquito-borne diseases are a growing human health concern in the United States, the distribution and potential arbovirus risk from container-breeding Aedes mosquitoes is understudied in the southern Great Plains. The aim of the study was to assess landscape and anthropogenic factors associated with encountering adult container-breeding mosquitoes in small cities in southern Oklahoma. Collections were carried out over a 10 week period from June to August 2017 along two geographical transects, each consisting of three cities, equally distant from the Red River/Texas border. Mosquitoes were collected weekly using two trap types along with data for 13 landscape, vegetation, and anthropogenic variables. After five rounds of collection, 6628 female mosquitoes were collected over 2110 trap-nights involving 242 commercial or residential sites in six cities. Of the mosquitoes collected, 80% consisted of container-breeding species: Aedes albopictus (72%), Culex pipiens complex (16%) and Aedes aegypti (8%). Regionally, Aedes aegypti was more likely present in cities closest to the Texas border while Ae. albopictus was spread throughout the region. In general, Ae. aegypti and Ae. albopictus were significantly more present in sites featuring no or low vegetation and residential sites. Variables associated with Ae. albopictus presence and abundance varied between cities and highlighted the urban nature of the species. The study highlighted the distribution of Ae. aegypti geographically and within the urban context, indicated potential habitat preferences of container-breeding mosquito species in small towns, and demonstrated the usefulness of Gravid Aedes traps (GAT) traps for monitoring Aedes populations in urban habitats in small cities.

Measurement of the individual enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) forms a key part of diagnostic investigations in patients with suspected mitochondrial disease, and can provide crucial information on mitochondrial OXPHOS function in a variety of cells and tissues that are applicable to many research investigations. In this chapter, we present methods for analysis of mitochondrial respiratory chain enzymes in cells and tissues based on assays performed in two geographically separate diagnostic referral centers, as part of clinical diagnostic investigations. Techniques for sample preparation from cells and tissues, and spectrophotometric assays for measurement of the activities of OXPHOS complexes I-V, the combined activity of complexes II+III, and the mitochondrial matrix enzyme citrate synthase, are provided. The activities of mitochondrial respiratory chain enzymes are often expressed relative to citrate synthase activity, since these ratios may be more robust in accounting for variability that may arise due to tissue quality, handling and storage, cell growth conditions, or any mitochondrial proliferation that may be present in tissues from patients with mitochondrial disease. Considerations for adaption of these techniques to other cells, tissues, and organisms are presented, as well as comparisons to alternate methods for analysis of respiratory chain function. In this context, a quantitative immunofluorescence protocol is also provided that is suitable for measurement of the amount of multiple respiratory chain complexes in small diagnostic tissue samples. The analysis and interpretation of OXPHOS enzyme activities are then placed in the context of mitochondrial disease tissue pathology and diagnosis., (© 2020 Elsevier Inc. All rights reserved.)

Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease. We show that hTim8a is required for the assembly of Complex IV in neurons, which is mediated through a transient interaction with Complex IV assembly factors, in particular the copper chaperone COX17. Complex IV assembly defects resulting from loss of hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c, which primes cells for death. Alleviation of oxidative stress with Vitamin E treatment rescues cells from apoptotic vulnerability. We hypothesise that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS., Competing Interests: YK, AA, TJ, CP, DD, KF, TS, AF, NC, DT, DT, MM, MR, DS, DS No competing interests declared, (© 2019, Kang et al.)

Small unmanned aircraft systems (sUAS) are rapidly transforming atmospheric research. With the advancement of the development and application of these systems, improving knowledge of best practices for accurate measurement is critical for achieving scientific goals. We present results from an intercomparison of atmospheric measurement data from the Lower Atmospheric Process Studies at Elevation-a Remotely piloted Aircraft Team Experiment (LAPSE-RATE) field campaign. We evaluate a total of 38 individual sUAS with 23 unique sensor and platform configurations using a meteorological tower for reference measurements. We assess precision, bias, and time response of sUAS measurements of temperature, humidity, pressure, wind speed, and wind direction. Most sUAS measurements show broad agreement with the reference, particularly temperature and wind speed, with mean value differences of 1.6 ± 2 . 6 ∘ C and 0.22 ± 0 . 59 m/s for all sUAS, respectively. sUAS platform and sensor configurations were found to contribute significantly to measurement accuracy. Sensor configurations, which included proper aspiration and radiation shielding of sensors, were found to provide the most accurate thermodynamic measurements (temperature and relative humidity), whereas sonic anemometers on multirotor platforms provided the most accurate wind measurements (horizontal speed and direction). We contribute both a characterization and assessment of sUAS for measuring atmospheric parameters, and identify important challenges and opportunities for improving scientific measurements with sUAS.

Inherited disorders of oxidative phosphorylation cause the clinically and genetically heterogeneous diseases known as mitochondrial energy generation disorders, or mitochondrial diseases. Over the last three decades, mutations causing these disorders have been identified in almost 290 genes, but many patients still remain without a molecular diagnosis. Moreover, while our knowledge of the genetic causes is continually expanding, our understanding into how these defects lead to cellular dysfunction and organ pathology is still incomplete. Here, we review recent developments in disease gene discovery, functional characterization, and shared pathogenic parameters influencing disease pathology that offer promising avenues toward the development of effective therapies., (© 2019 by The American Society for Biochemistry and Molecular Biology, Inc.)

BCL-2 proteins are known to engage each other to determine the fate of a cell after a death stimulus. However, their evolutionary conservation and the many other reported binding partners suggest an additional function not directly linked to apoptosis regulation. To identify such a function, we studied mice lacking the BH3-only protein BIM. BIM -/- cells had a higher mitochondrial oxygen consumption rate that was associated with higher mitochondrial complex IV activity. The consequences of increased oxygen consumption in BIM -/- mice were significantly lower body weights, reduced adiposity and lower hepatic lipid content. Consistent with reduced adiposity, BIM -/- mice had lower fasting blood glucose, improved insulin sensitivity and hepatic insulin signalling. Lipid oxidation was increased in BIM -/- mice, suggesting a mechanism for their metabolic phenotype. Our data suggest a role for BIM in regulating mitochondrial bioenergetics and metabolism and support the idea that regulation of metabolism and cell death are connected.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

The mitochondrial inner membrane harbors three protein translocases. Presequence translocase and carrier translocase are essential for importing nuclear-encoded proteins. The oxidase assembly (OXA) translocase is required for exporting mitochondrial-encoded proteins; however, different views exist about its relevance for nuclear-encoded proteins. We report that OXA plays a dual role in the biogenesis of nuclear-encoded mitochondrial proteins. First, a systematic analysis of OXA-deficient mitochondria led to an unexpected expansion of the spectrum of OXA substrates imported via the presequence pathway. Second, biogenesis of numerous metabolite carriers depends on OXA, although they are not imported by the presequence pathway. We show that OXA is crucial for the biogenesis of the Tim18-Sdh3 module of the carrier translocase. The export translocase OXA is thus required for the import of metabolite carriers by promoting assembly of the carrier translocase. We conclude that OXA is of central importance for the biogenesis of the mitochondrial inner membrane., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The vast majority of cellular ATP is produced by the oxidative phosphorylation (OXPHOS) system, which comprises the four complexes of the electron transfer chain plus the ATP synthase. Complex I is the largest of the OXPHOS complexes, and mutation of the genes encoding either the subunits or assembly factors of Complex I can result in Complex I deficiency, which is the most common OXPHOS disorder. Mutations in the Complex I gene NDUFS4 lead to Leigh syndrome, which is the most frequent presentation of Complex I deficiency in children presenting with progressive encephalopathy shortly after birth. Symptoms include motor and intellectual retardation, often accompanied by dystonia, ataxia, and growth retardation, and most patients die by 3 years of age. To understand the origins of this disease, we have generated a series of mouse embryonic stem cell lines from blastocysts that were wild type, heterozygous, and homozygous for the deletion of the Ndufs4 gene. We have demonstrated their pluripotency and potential to differentiate into all cell types of the body. Although the loss of Ndufs4 did not affect the stability of the mitochondrial and nuclear genomes, there were significant differences in patterns of chromosomal gene expression following both spontaneous differentiation and directed neural differentiation into astrocytes. The defect also affected the potential of the cells to generate beating embryoid bodies. These outcomes demonstrate that defects associated with Complex I deficiency affect early gene expression patterns, which escalate during early and later stages of differentiation and are mediated by the defect and not other chromosomal or mitochondrial DNA defects.

Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of COA6 activity using gene editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mitochondrial DNA-encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in COA6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore, we show that COA6 has the capacity to bind copper and can associate with newly translated COX2 and the mitochondrial copper chaperone SCO1. Our data reveal that COA6 is intricately involved in the copper-dependent biogenesis of COX2., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Human mitochondrial complex I is the largest enzyme of the respiratory chain and is composed of 44 different subunits. Complex I subunits are encoded by both nuclear and mitochondrial (mt) DNA and their assembly requires a number of additional proteins. FAD-dependent oxidoreductase domain-containing protein 1 (FOXRED1) was recently identified as a putative assembly factor and FOXRED1 mutations in patients cause complex I deficiency; however, its role in assembly is unknown. Here, we demonstrate that FOXRED1 is involved in mid-late stages of complex I assembly. In a patient with FOXRED1 mutations, the levels of mature complex I were markedly decreased, and a smaller ∼475 kDa subcomplex was detected. In the absence of FOXRED1, mtDNA-encoded complex I subunits are still translated and transiently assembled into a late stage ∼815 kDa intermediate; but instead of transitioning further to the mature complex I, the intermediate breaks down to an ∼475 kDa complex. As the patient cells contained residual assembled complex I, we disrupted the FOXRED1 gene in HEK293T cells through TALEN-mediated gene editing. Cells lacking FOXRED1 had ∼10% complex I levels, reduced complex I activity, and were unable to grow on galactose media. Interestingly, overexpression of FOXRED1 containing the patient mutations was able to rescue complex I assembly. In addition, FOXRED1 was found to co-immunoprecipitate with a number of complex I subunits. Our studies reveal that FOXRED1 is a crucial component in the productive assembly of complex I and that mutations in FOXRED1 leading to partial loss of function cause defects in complex I biogenesis., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Huntington's disease (HD) is a neurodegenerative disorder, involving psychiatric, cognitive and motor symptoms, caused by a CAG-repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. Oxidative stress and excitotoxicity have previously been implicated in the pathogenesis of HD. We hypothesized that N-acetylcysteine (NAC) may reduce both excitotoxicity and oxidative stress through its actions on glutamate reuptake and antioxidant capacity. The R6/1 transgenic mouse model of HD was used to investigate the effects of NAC on HD pathology. It was found that chronic NAC administration delayed the onset and progression of motor deficits in R6/1 mice, while having an antidepressant-like effect on both R6/1 and wild-type mice. A deficit in the astrocytic glutamate transporter protein, GLT-1, was found in R6/1 mice. However, this deficit was not ameliorated by NAC, implying that the therapeutic effect of NAC is not due to rescue of the GLT-1 deficit and associated glutamate-induced excitotoxicity. Assessment of mitochondrial function in the striatum and cortex revealed that R6/1 mice show reduced mitochondrial respiratory capacity specific to the striatum. This deficit was rescued by chronic treatment with NAC. There was a selective increase in markers of oxidative damage in mitochondria, which was rescued by NAC. In conclusion, NAC is able to delay the onset of motor deficits in the R6/1 model of Huntington's disease and it may do so by ameliorating mitochondrial dysfunction. Thus, NAC shows promise as a potential therapeutic agent in HD. Furthermore, our data suggest that NAC may also have broader antidepressant efficacy.

Mitochondrial dysfunction causes a range of early-onset neurological diseases and contributes to neurodegenerative conditions. The mechanisms of neurological damage however are poorly understood, as accessing relevant tissue from patients is difficult, and appropriate models are limited. Hence, we assessed mitochondrial function in neurologically relevant primary cell lines from a CI (complex I) deficient Ndufs4 KO (knockout) mouse (Ndufs4fky/fky) modelling aspects of the mitochondrial disease LS (Leigh syndrome), as well as MEFs (mouse embryonic fibroblasts). Although CI structure and function were compromised in all Ndufs4fky/fky cell types, the mitochondrial membrane potential was selectively impaired in the MEFs, correlating with decreased CI-dependent ATP synthesis. In addition, increased ROS (reactive oxygen species) generation and altered sensitivity to cell death were only observed in Ndufs4fky/fky primary MEFs. In contrast, Ndufs4fky/fky primary isocortical neurons and primary isocortical astrocytes displayed only impaired ATP generation without mitochondrial membrane potential changes. Therefore the neurological dysfunction in the Ndufs4fky/fky mouse may partly originate from a more severe ATP depletion in neurons and astrocytes, even at the expense of maintaining the mitochondrial membrane potential. This may provide protection from cell death, but would ultimately compromise cell functionality in neurons and astrocytes. Furthermore, RET (reverse electron transfer) from complex II to CI appears more prominent in neurons than MEFs or astrocytes, and is attenuated in Ndufs4fky/fky cells.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterised by neurodegeneration and cardiomyopathy that is caused by an insufficiency of the mitochondrial protein, frataxin. Our previous studies described the generation of FRDA induced pluripotent stem cell lines (FA3 and FA4 iPS) that retained genetic characteristics of this disease. Here we extend these studies, showing that neural derivatives of FA iPS cells are able to differentiate into functional neurons, which don't show altered susceptibility to cell death, and have normal mitochondrial function. Furthermore, FA iPS-derived neural progenitors are able to differentiate into functional neurons and integrate in the nervous system when transplanted into the cerebellar regions of host adult rodent brain. These are the first studies to describe both in vitro and in vivo characterization of FA iPS-derived neurons and demonstrate their capacity to survive long term. These findings are highly significant for developing FRDA therapies using patient-derived stem cells.