Research Article10 September 2018Open Access Source DataTransparent process OXA1L mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect Kyle Thompson Kyle Thompson Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Nicole Mai Nicole Mai Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Monika Oláhová Monika Oláhová Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Filippo Scialó Filippo Scialó Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Luke E Formosa Luke E Formosa Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Vic., Australia Search for more papers by this author David A Stroud David A Stroud Department of Biochemistry and Molecular Biology and The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Madeleine Garrett Madeleine Garrett Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Vic., Australia Search for more papers by this author Nichola Z Lax Nichola Z Lax Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Fiona M Robertson Fiona M Robertson Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Cristina Jou Cristina Jou Pathology Department, Hospital Sant Joan de Déu, CIBERER, Barcelona, Spain Search for more papers by this author Andres Nascimento Andres Nascimento Neuromuscular Unit, Neuropaediatrics Department, Hospital Sant Joan de Déu, CIBERER - ISCIII, Barcelona, Spain Search for more papers by this author Carlos Ortez Carlos Ortez Neuromuscular Unit, Neuropaediatrics Department, Hospital Sant Joan de Déu, CIBERER - ISCIII, Barcelona, Spain Search for more papers by this author Cecilia Jimenez-Mallebrera Cecilia Jimenez-Mallebrera Neuromuscular Unit, Neuropaediatrics Department, Hospital Sant Joan de Déu, CIBERER - ISCIII, Barcelona, Spain Search for more papers by this author Steven A Hardy Steven A Hardy Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Langping He Langping He Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Garry K Brown Garry K Brown Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK Search for more papers by this author Paula Marttinen Paula Marttinen Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Robert McFarland Robert McFarland orcid.org/0000-0002-8833-2688 Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Alberto Sanz Alberto Sanz Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Brendan J Battersby Brendan J Battersby orcid.org/0000-0002-8136-2753 Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Penelope E Bonnen Penelope E Bonnen Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Michael T Ryan Michael T Ryan Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Vic., Australia Search for more papers by this author Zofia MA Chrzanowska-Lightowlers Zofia MA Chrzanowska-Lightowlers Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Robert N Lightowlers Robert N Lightowlers Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Robert W Taylor Corresponding Author Robert W Taylor [email protected] orcid.org/0000-0002-7768-8873 Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Kyle Thompson Kyle Thompson Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Nicole Mai Nicole Mai Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Monika Oláhová Monika Oláhová Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Filippo Scialó Filippo Scialó Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Luke E Formosa Luke E Formosa Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Vic., Australia Search for more papers by this author David A Stroud David A Stroud Department of Biochemistry and Molecular Biology and The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Madeleine Garrett Madeleine Garrett Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Vic., Australia Search for more papers by this author Nichola Z Lax Nichola Z Lax Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Fiona M Robertson Fiona M Robertson Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Cristina Jou Cristina Jou Pathology Department, Hospital Sant Joan de Déu, CIBERER, Barcelona, Spain Search for more papers by this author Andres Nascimento Andres Nascimento Neuromuscular Unit, Neuropaediatrics Department, Hospital Sant Joan de Déu, CIBERER - ISCIII, Barcelona, Spain Search for more papers by this author Carlos Ortez Carlos Ortez Neuromuscular Unit, Neuropaediatrics Department, Hospital Sant Joan de Déu, CIBERER - ISCIII, Barcelona, Spain Search for more papers by this author Cecilia Jimenez-Mallebrera Cecilia Jimenez-Mallebrera Neuromuscular Unit, Neuropaediatrics Department, Hospital Sant Joan de Déu, CIBERER - ISCIII, Barcelona, Spain Search for more papers by this author Steven A Hardy Steven A Hardy Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Langping He Langping He Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Garry K Brown Garry K Brown Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK Search for more papers by this author Paula Marttinen Paula Marttinen Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Robert McFarland Robert McFarland orcid.org/0000-0002-8833-2688 Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Alberto Sanz Alberto Sanz Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Brendan J Battersby Brendan J Battersby orcid.org/0000-0002-8136-2753 Institute of Biotechnology, University of Helsinki, Helsinki, Finland Search for more papers by this author Penelope E Bonnen Penelope E Bonnen Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Michael T Ryan Michael T Ryan Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Vic., Australia Search for more papers by this author Zofia MA Chrzanowska-Lightowlers Zofia MA Chrzanowska-Lightowlers Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Robert N Lightowlers Robert N Lightowlers Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Robert W Taylor Corresponding Author Robert W Taylor [email protected] orcid.org/0000-0002-7768-8873 Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Author Information Kyle Thompson1, Nicole Mai1, Monika Oláhová1, Filippo Scialó2, Luke E Formosa3, David A Stroud4, Madeleine Garrett3, Nichola Z Lax1, Fiona M Robertson1, Cristina Jou5, Andres Nascimento6, Carlos Ortez6, Cecilia Jimenez-Mallebrera6, Steven A Hardy1, Langping He1, Garry K Brown7, Paula Marttinen8, Robert McFarland1, Alberto Sanz2, Brendan J Battersby8, Penelope E Bonnen9, Michael T Ryan3, Zofia MA Chrzanowska-Lightowlers1, Robert N Lightowlers1 and Robert W Taylor *,1 1Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK 2Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, Newcastle University, Newcastle upon Tyne, UK 3Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Vic., Australia 4Department of Biochemistry and Molecular Biology and The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Vic., Australia 5Pathology Department, Hospital Sant Joan de Déu, CIBERER, Barcelona, Spain 6Neuromuscular Unit, Neuropaediatrics Department, Hospital Sant Joan de Déu, CIBERER - ISCIII, Barcelona, Spain 7Oxford Medical Genetics Laboratories, Churchill Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK 8Institute of Biotechnology, University of Helsinki, Helsinki, Finland 9Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA *Corresponding author. Tel: +44 191 2083685; E-mail: [email protected] EMBO Mol Med (2018)10:e9060https://doi.org/10.15252/emmm.201809060 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic OXA1L variants (c.500_507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild-type human OXA1L in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or Drosophila melanogaster caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA-encoded subunits of complexes I, IV and V. Our data verify the pathogenicity of these OXA1L variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes. Synopsis This work describes the first confirmed pathogenic variants in OXA1L in a mitochondrial disease patient with tissue-specific combined OXPHOS deficiencies. OXA1L was shown to be important for complex IV assembly in human cell lines with targeted depletion of OXA1L and in patient tissues. Bi-allelic variants in OXA1L (NM_005015.3; c.500_507dup, p.(Ser170Glnfs*18); c.620G>T, p.(Cys207Phe) were identified by whole exome sequencing in a patient with hypotonia, developmental delay and severe encephalopathy who died following cardiorespiratory arrest. Patient fibroblasts showed decreased protein levels of OXA1L and subunits of OXPHOS complexes I, IV and V. Introduction of wild-type OXA1L into patient fibroblasts by retroviral transduction rescued the OXPHOS defects, confirming pathogenicity of the OXA1L variants. Depletion of OXA1L by siRNA in human cell lines and Drosophila caused combined OXPHOS deficiencies including severe effects on complex IV. An OXA1L CRISPR/Cas9 knockout cell line with inducible expression of FLAG-tagged OXA1L was used for OXA1L immunoprecipitation and identified an enrichment of mtDNA-encoded subunits of complexes I, IV and V. Introduction Mitochondrial disorders encompass a wide range of clinical phenotypes and are genetically heterogeneous. Mitochondrial dysfunction can arise from mutations in either the maternally inherited mitochondrial genome (mtDNA) or in nuclear genes that encode mitochondrial proteins. The 13 mtDNA-encoded polypeptides are essential components of the oxidative phosphorylation (OXPHOS) system. Many nuclear genetic defects causing mitochondrial disease are in gene products required for maintaining correct expression of mtDNA. These include proteins involved in replication, mtDNA maintenance, transcription, mtRNA processing, maturation and translation (Lightowlers et al, 2015). In recent years, next-generation sequencing (NGS) technologies, particularly whole exome sequencing (WES), have proven successful in identifying an increasing number of mutations in genes causing primary mitochondrial disorders (Calvo et al, 2012; Taylor et al, 2014; Wortmann et al, 2015; Kohda et al, 2016; Pronicka et al, 2016). There are currently more than 250 nuclear-encoded genes associated with mitochondrial disease (Mayr et al, 2015; Alston et al, 2017; Frazier et al, 2017). However, considering the mitochondrial proteome is estimated to include approximately 1,200 proteins, it is likely that there are many more candidate genes still to be described. One such gene is OXA1L, which has long been proposed as a candidate gene to be screened for mutations in patients presenting with combined respiratory chain deficiencies (Rotig et al, 1997). To date, no mutations have been linked to mitochondrial disease (Coenen et al, 2005). OXA1L is a member of the YidC/Alb3/Oxa1 membrane protein insertase family (Hennon et al, 2015). The yeast orthologue of OXA1L, Oxa1p, was identified as an important factor for the assembly of complex IV (Bonnefoy et al, 1994a) and has also been shown to be important for the assembly of complex V (Altamura et al, 1996). Oxa1p interacts with the mitoribosome (Jia et al, 2003; Kohler et al, 2009) and is required for the co-translational membrane insertion of mitochondrial-encoded Atp6p, Atp9p, Cox1p, Cox2p, Cox3p and Cytb (Hell et al, 1997, 1998, 2001; Jia et al, 2007). Oxa1p also appears to have a direct role in the insertion of nuclear-encoded inner mitochondrial membrane (IMM) proteins (Hell et al, 2001) including Oxa1p itself (Hell et al, 1998) and an indirect effect on many more IMM proteins including several metabolite transporters (Hildenbeutel et al, 2012), since Oxa1p is crucial for the biogenesis of the Tim18-Sdh3 module of the carrier translocase (Stiller et al, 2016). The majority of studies into the function of Oxa1 have been conducted in yeast. Human OXA1L shares 33% identity with the Oxa1p yeast protein and was identified as a homologue by functional complementation and expression of the human gene into an Oxa1p null strain of Saccharomyces cerevisiae. This partially rescued the phenotype of impaired cytochrome c oxidase (COX) assembly, suggesting that OXA1L likely performs a similar role in human cells (Bonnefoy et al, 1994b). Indeed, human OXA1L has since been reported to bind to the mammalian mitoribosome via its C-terminal tail (Haque et al, 2010). In contrast to yeast, shRNA-mediated knockdown of OXA1L in human cells was shown to cause a defect in complex I and complex V assembly, but did not affect complex IV (Stiburek et al, 2007). As other insertases may be present in mitochondria, clarification into the importance of OXA1L is required. Here, we present the clinical, biochemical and molecular characterisation of a patient with a severe, childhood-onset mitochondrial encephalopathy and combined respiratory chain deficiency due to biallelic variants in OXA1L identified by WES. Results from cellular and biochemical approaches suggest that OXA1L plays a major role as the insertase for the biogenesis of respiratory chain complexes. Results Case report The index case, a 5-year-old male, was born to non-consanguineous healthy Chinese parents. Three previous pregnancies had resulted in miscarriages in the first and second trimesters without obvious cause, but this pregnancy had been uneventful, though delivery was complicated by a clavicular fracture. He had a good birthweight of 4.1kg and did not require resuscitation with Apgars recorded as 91 and 105. He showed signs of severe hypotonia from birth with subsequent neurodevelopmental delay, achieving independent sitting at 12 months, but never being able to stand or walk. Language skills were also severely delayed in that he was unable to understand even simple instructions and made no attempt to speak or supplement communication with non-verbal behaviour. He was reliant on parents for all activities of daily living. Obstructive sleep apnoea was confirmed by polysomnography at the age of 3 years, and he had a tonsillectomy prior to commencing non-invasive nocturnal ventilation. On examination at 4 years, he was noted to be obese (32 kg) and exhibited generalised weakness, hypotonia and areflexia in his lower limbs. Iron deficiency anaemia was identified though the cause was unclear. Brain MRI revealed dysgenesis of the corpus callosum but was otherwise normal (Fig EV1). Electrophysiological testing showed normal motor nerve velocities, but low amplitude CMAPs and a neurogenic pattern on electromyography. At 5 years, he presented with a brief febrile illness associated with a mild metabolic acidosis (venous lactate 2.48 mmol/l, normal range 0.7–2.1 mmol/l) and leucocytosis. Further metabolic workup revealed increased serum alanine (520 μmol/l; normal range < 416), but ammonia, CDG and biotinidase activity were normal, as was PDHc activity in patient fibroblasts. Acylcarnitines and urinary organic acids were not determined. His condition deteriorated rapidly with generalised seizures and encephalopathy prior to a cardiorespiratory arrest from which he could not be resuscitated. An older female sibling, also considered to have neurodevelopmental delay, died in China aged 12 months. This death was also preceded by a febrile illness, but the cause remains unclear. A younger male sibling was born following pre-natal testing for the genetic mutation identified in the index case (Fig 1A). Click here to expand this figure. Figure EV1. Patient Neuroimaging A. Sagittal T1-weighted FSE showing thinning of the posterior part of the corpus callosum with agenesis of the splenium. Prominent subcutaneous fat deposition can also be seen. B. Axial T1 FSPGR reveals that the third ventricle is bigger and the ventricular atrium has a parallel orientation, due to agenesis of the splenium. C. MRS of the patient's brain shows a normal spectroscopy pattern. Download figure Download PowerPoint Figure 1. Molecular genetics and biochemical studies of OXA1L variants A. Family pedigree detailing recessive inheritance pattern of OXA1L variants, index case denoted with red asterisk. B. Haematoxylin & Eosin (i) and modified Gomori trichrome (ii) staining demonstrate expected variability in muscle fibre size, with isolated internal nuclei. There is no evidence of regenerative fibres or necrosis, nor subsarcolemmal mitochondrial aggregates typical of "ragged-red" changes. The absence of mitochondrial proliferation is confirmed in both the NADH-tetrazolium reductase (iii) and SDH (iv) reactions. The individual COX reaction (v) reveals a uniform loss of enzyme reactivity across the muscle cryosection, accentuated in the sequential COX-SDH reaction (vi) in which COX-deficient, SDH-positive fibres are prominent uniformly. Scale bar shown is 50 μm. C. Activity of mitochondrial respiratory complexes in control (red) and patient (blue) skeletal muscle samples. Mean enzyme activities of control muscle (n = 25) are set to 100%, and error bars represent standard deviation. Asterisk represents values outside of the control range. D, E. Confirmatory Sanger sequencing to show each parent was heterozygous for a single OXA1L variant. Asterisk indicates position of the labelled variant. F. Analysis of mRNA from the patient by RT–PCR showed that the c.620G>T variant affects splicing and can lead to skipping of exon 4 (p.(Cys207_Glu2545del)) or the p.Cys207Phe amino acid substitution. G. Evolutionary conservation of OXA1L human Cys207 residue (blue box). Download figure Download PowerPoint Diagnostic mitochondrial investigations of patient muscle Postmortem tissue from the patient was made available for diagnostic evaluation of suspected mitochondrial disease. Histopathological assessment, including routine histology and oxidative enzyme histochemistry, showed no obvious morphological abnormalities although the individual COX staining was weak, which was confirmed by a generalised decrease in COX activity following sequential COX-SDH histochemistry (Fig 1B). In agreement with these observations, the assessment of mitochondrial respiratory chain complex activities in skeletal muscle showed decreased activity of complex IV, with activities of complexes I-III within the normal range (Fig 1C). Molecular genetic investigations A prominent complex IV deficiency in patient skeletal muscle demonstrated a mitochondrial aetiology. Assessment of mtDNA in patient samples showed no mutations, and mtDNA copy number was normal. Candidate genes including SURF1, SCO1, SCO2, COX10, COX14, COX15, COX20, COA3, COA5 and APOPT1 were screened but no pathogenic variants were identified. Further analysis using WES revealed biallelic variants in OXA1L (NM_005015.3) c.500_507dup, p.(Ser170Glnfs*18) (ClinVar: SCV000803663) and c.620G>T, p.(Cys207Phe) (ClinVar: SCV000803664). The c.500_507dup variant is not listed in the gnomAD database (http://gnomad.broadinstitute.org), whereas the c.620G>T variant is present in one individual of East Asian ethnicity (1 in 246,200, allele frequency 0.00041%). Sanger sequencing confirmed the presence of the variants and that the variants segregated with disease in the family (Fig 1D and E). The c.620G>T OXA1L variant is predicted to result in a p.(Cys207Phe) amino acid substitution, but is also predicted to affect splicing due to this variant being at the first nucleotide of the exon. Analysis of cDNA confirmed a splicing defect with evidence of skipping of exon 4 (Fig 1F) demonstrating the c.620G>T variant causes an amino acid substitution and exon skipping (p.[Cys207Phe, Cys207_Glu254del]), with the amino acid substitution affecting the moderately well-conserved Cys207 residue (Fig 1G). Since one variant causes a frameshift and the other affects splicing, American College of Medical Genetics and Genomics (ACMGG) guidelines consider these to be loss-of-function alleles. MutationTaster (www.mutationtaster.org) predicts the c.620G>T variant to be disease-causing, and the variant has a scaled CADD score of 24.9 (http://cadd.gs.washington.edu). Neuropathology The pons exhibited bilateral cavitation with microvascular proliferation, which severely affected the pedunculopontine (PPN) nucleus (Fig EV2Ai, ii and iii) and was accompanied by astrogliosis (Fig EV2Aiv) and microglial activation (Fig EV2Av) with preserved neuronal cell density. PPN neurons and the surrounding neuropil showed profound loss in the levels of complex I subunits NDUFB8 and NDUFS3 expression (Fig EV2Avi), while nuclear-encoded complex II subunit SDHA (Fig EV2Aviii), mitochondrially encoded complex IV subunit COXI (Fig EV2Ax) and complex V subunit ATP5B (Fig EV2Axii) levels were maintained. Substantia nigra neurons showed a similar pattern of NDUFS3-specific loss (Fig EV2Bi), while SDHA (Fig EV2Bii), COXI (Fig EV2Biii) and ATP5B (Fig EV2Biv) were preserved. The spinal cord demonstrates prominent demyelination of the fasciculus gracile tract (Fig EV2Ci and ii) with relative preservation of myelin in the fasciculus cuneatus (Fig EV2Ciii). The motor neurons were well populated with remaining neurons exhibiting reduced levels of NDUFB8 (Fig EV2Civ) and NDUFS3 (Fig EV2Cv), while SDHA (Fig EV2Cvi), COXI (Fig EV2Cvii) and ATP5B (Fig EV2Cviii) were apparently normal within neurons. Macroscopically, the frontal lobe was atrophic, and the corpus callosum was affected by hypoplasia, particularly in the splenium (Fig EV3A). The basal ganglia were also affected by microcavitation affecting the thalamus (Fig EV3B), and striatum with microvascular proliferation, activated microglia and reactive gliosis (Fig EV3B). The cortex revealed a normal laminar architecture, with discrete cell loss affecting the frontal cortex. Slight downregulation in the levels of NDUFS3 and NDUFB8 relative to SDHA, COXI and ATP5B was observed in neurons within the frontal (Fig EV3C), parietal (Fig EV3D) and occipital cortex (Fig EV3E). The architecture of the cerebellum was preserved with normal Purkinje cell density, minimal neuronal cell loss from the dentate nucleus and intact expression of NDUFS3, NDUFB8, SDHA, COXI and ATP5B. Click here to expand this figure. Figure EV2. Neuropathology: Bilateral cavitation of the pons, demyelination of the fasciculus gracile tract and downregulation of complex I subunits NDUFS3 and NDUFB8 A. The pons demonstrate bilateral cavitation (i and ii), specifically affecting the pedunculopontine nucleus, accompanied by microvascular proliferation (iii), reactive astrogliosis (iv) and microglial activation (v). There is downregulation of NDUFS3 (vi; arrowheads) expression within neurons and the surrounding neuropil (control staining: vii), while SDHA (viii; control shown in ix), COXI (x; control shown in xi) and ATP5B (xii; control shown in xiii) expression is maintained within normal levels. B. The neurons of the substantia nigra also reveal a loss of NDUFS3 (i; arrowheads, control shown on right) expression, while SDHA (ii left panel; control shown on right), COXI (iii left panel; control shown on right) and ATP5B (iv left panel; control shown on right) expression levels are comparable to control. C. The fasciculus gracile tracts reveal demyelination (i and ii), while the adjacent fasciculus cuneatus is myelinated (iii). Neuronal population density of the motor neurons is maintained, while there is a loss of NDUFB8 (iv) and NDUFS3 expression (v) and intact SDHA (vi), COXI (vii) and ATP5B (viii) expression. Data information: Scale bar = 100 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Neuropathology: Frontal lobe atrophy, corpus callosum hypoplasia and thalamic cavitation. The laminar distribution of neurons in cortical regions is intact, and mitochondrial OXPHOS expression is maintained throughout the frontal, parietal and occipital cortices A. Macroscopically, the frontal cortex was atrophic, while the corpus callosum was hypoplastic. B. There was microcavitation of the thalamus with microvascular proliferation, activated microglia and reactive gliosis, which was revealed with immunohistochemical staining for glial fibrillary acidic protein (PGFA). Scale bars: top panels = 500 μm, bottom left = 200 μm, bottom right = 100 μm. C–F. Mitochondrial respiratory chain protein expression is maintained in patient neurons within frontal (C), parietal (D) and occipital (E) cortices comparable with levels observed in control neurons (F), and there is only slight downregulation of NDUFS3 (i) expression, while expression in patient tissues of SDHA (ii), COXI (iii) and ATP5B (iv) is high. Scale bar = 100 μm. Download figure Download PowerPoint OXPHOS steady-state levels and complex assembly in fibroblasts and skeletal muscle Western blot analysis of patient fibroblasts and skeletal muscle was carried out to assess the steady-state levels of OXA1L and OXPHOS components. OXA1L protein levels were decreased both in patient fibroblasts (Fig 2A) and in skeletal muscle (Fig 2B) compared to controls demonstrating a functional consequence of the identified OXA1L variants. Interestingly, there was no observable difference in the size of the OXA1L protein in the patient sample, suggesting that the protein missing exon 4 is either not produced or rapidly degraded. This would infer that the residual levels of OXA1L in the patient are likely to be the variant carrying the p.(Cys207Phe) amino acid substitution only. Western blot assessment of sub
Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.
Key points Ageing is associated with an upregulation of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) in human skeletal muscle with the increased abundance of Mfn2 being exclusive to type II muscle fibres. These changes occur despite a similar content of mitochondria, as measured by COXIV, NDUFA9 and complexes in their native states (Blue Native PAGE). Following 12 weeks of high‐intensity training (HIT), older adults exhibit a robust increase in mitochondria content, while there is a decline in Mfn2 in type II fibres. We propose that the upregulation of Mfn2 and MiD49 with age may be a protective mechanism to protect against mitochondrial dysfunction, in particularly in type II skeletal muscle fibres, and that exercise may have a unique protective effect negating the need for an increased turnover of mitochondria. Abstract Mitochondrial dynamics proteins are critical for mitochondrial turnover and maintenance of mitochondrial health. High‐intensity interval training (HIT) is a potent training modality shown to upregulate mitochondrial content in young adults but little is known about the effects of HIT on mitochondrial dynamics proteins in older adults. This study investigated the abundance of protein markers for mitochondrial dynamics and mitochondrial content in older adults compared to young adults. It also investigated the adaptability of mitochondria to 12 weeks of HIT in older adults. Both older and younger adults showed a higher abundance of mitochondrial respiratory chain subunits COXIV and NDUFA9 in type I compared with type II fibres, with no difference between the older adults and young groups. In whole muscle homogenates, older adults had higher mitofusin‐2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) contents compared to the young group. Also, older adults had higher levels of Mfn2 in type II fibres compared with young adults. Following HIT in older adults, MiD49 and Mfn2 levels were not different in whole muscle and Mfn2 content decreased in type II fibres. Increases in citrate synthase activity (55%) and mitochondrial respiratory chain subunits COXIV (37%) and NDUFA9 (48%) and mitochondrial respiratory chain complexes (∼70–100%) were observed in homogenates and/or single fibres. These findings reveal (i) a similar amount of mitochondria in muscle from young and healthy older adults and (ii) a robust increase of mitochondrial content following 12 weeks of HIT exercise in older adults.
Cytochrome c oxidase or mitochondrial respiratory chain complex IV catalyses the transfer of electrons from cytochrome c in the intermembrane space, to molecular oxygen in the matrix and therefore contributes to the proton gradient that drives mitochondrial ATP synthesis.Complex IV dysfunction is a significant cause of human mitochondrial disease.Complex IV requires the incorporation of three copper ions, heme a and heme a3 cofactors for the assembly and activity of the complex.Complex IV assembly factors are required for subunit maturation, co-factor incorporation and stabilization of intermediate assemblies of complex IV in humans.Lossof-function mutations in several genes encoding complex IV assembly factors have been shown to result in diminished complex IV activity and severe pathologic conditions in affected infants [1].Our study focuses on two mitochondrial complex IV assembly factors, Coa6 and Coa7, that are located in the intermembrane space of mitochondria and contain intramolecular disulfide bonds.Coa6 binds copper with femtomolar affinity and has been proposed to play a role in the biogenesis of the CuA site of complex IV [2,3].The W59C pathogenic mutation in Coa6 does not affect copper binding or import of the protein into mitochondria but affects the maturation and stability of the protein [3].The precise role of Coa7 in the biogenesis of complex IV is not completely understood.However, patients with Coa7 pathogenic mutations suffer from mitochondrial diseases owing to complex IV deficiency.This presentation will describe the crystal structures of the Coa7 and Coa6 (wild-type and the W59C mutant) proteins and implications for their roles in complex IV assembly and function.
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