Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension,lactic acidemia,and failure to thrive

COX5A is a nuclear‐encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early‐onset pulmonary arterial hypertension, lactic acidemia, failure to thrive, and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts.     

In-vitro translation of mitochondrial mRNAs by yeast mitochondrial ribosomes is hampered by the lack of start-codon recognition

Summary In an attempt to reconstitute an homologous in-vitro translation system for yeast mitochondrial mRNAs, we have isolated ribosomes, supernatant factors, and tRNAs from mitochondria of Saccharomyces carlsbergensis. While poly(U) is translated faithfully in this system, no translation of in-vitro synthesised cytochrome c oxidase subunit II (COX2) mRNA could be detected. Formation of formylmethionyl-puromycin on mitochondrial ribosomes is stimulated by ApUpG, but not by COX2 mRNA, although mitochondrial small ribosomal subunits bind to this mRNA in vitro, even without added tRNA and initiation factors. We conclude, therefore, that the inability to faithfully translate mitochondrial mRNAs in vitro may be the result of an inability of mitochondrial ribosomes to recognize the initiation codon.     

雷帕霉素通过诱导细胞自噬在细胞和动物模型中治疗神经退行性疾病的新思路

目的 使用雷帕霉素保护神经退行性疾病损伤模型中神经元的线粒体从而降低神经元的损伤.方法 建立帕金森氏症（PD）细胞和动物的损伤模型,在损伤模型中加入雷帕霉素,经染色后于激光共聚焦显微镜下观察雷帕霉素诱导细胞自噬作用、对线粒体膜极性变化的作用、对细胞内活性氧（ROS）的清除作用以及NF-κB的入核情况.结果 雷帕霉素能诱导细胞自噬在MPP＋诱导的细胞损伤中,并能维持线粒体的正常形态、降低线粒体膜电位的变化、降低细胞ROS水平并减少NF-κB的入核.此外,雷帕霉素在MPTP诱发的斑马鱼和小鼠PD模型中对神经元有显著保护作用.结论 雷帕霉素能通过诱导细胞自噬作用于线粒体从而保护神经元免于神经退行性疾病的损伤,加强对线粒体的保护是一种治疗神经退行性疾病的新思路.     

Neuroprotection of kaempferol by autophagy in models of rotenone-mediated acute toxicity: possible implications for Parkinson's disease

This study aims to elucidate the processes underlying neuroprotection of kaempferol in models of rotenone-induced acute toxicity. We demonstrate that kaempferol, but not quercetin, myricetin or resveratrol, protects SH-SY5Y cells and primary neurons from rotenone toxicity, as a reduction of caspases cleavage and apoptotic nuclei are observed. Reactive oxygen species (ROS) levels and mitochondrial carbonyls decrease significantly. Mitochondrial network, transmembrane potential and oxygen consumption are also deeply preserved. We demonstrate that the main event responsible for the kaempferol-mediated antiapoptotic and antioxidant effects is the enhancement of mitochondrial turnover by autophagy. Indeed, fluorescence and electron microscopy analyses show an increase of the mitochondrial fission rate and mitochondria-containing autophagosomes. Moreover, the autophagosome-bound microtubule-associated protein light chain-3 (LC3-II) increases during kaempferol treatment and chemical/genetic inhibitors of autophagy abolish kaempferol protective effects. Autophagy affords protection also toward other mitochondrial toxins (1-methyl-4-phenyilpiridinium, paraquat) used to reproduce the typical features of Parkinson's disease (PD), but is inefficient against apoptotic stimuli not directly affecting mitochondria (H₂O₂, 6-hydroxydopamine, staurosporine). Striatal glutamatergic response of rat brain slices is also preserved by kaempferol, suggesting a more general protection of kaempferol in Parkinson's disease. Overall, the data provide further evidence for kaempferol to be identified as an autophagic enhancer with potential therapeutic capacity.     

The role of C/EBP-α expression in human liver and liver fibrosis and its relationship with autophagy

Aim: To investigate the expression of CCAAT enhancer binding protein-α (C/EBP-α) in normal human liver and liver fibrosis and its probable association with autophagy. Methods: Double label immunohistochemistry was used to detect the location of C/EBP-α in hepatocytes and hepatic stellate cells (HSCs). The expression of C/EBP-α, Atg5, and Atg6 was also evaluated by immunohistochemistry in paraffin sections of human liver. HSC-T6 cells were treated with rapamycin and 3-methyladenine (3MA) to induce or inhibit autophagy, and the expression of C/EBP-α protein was detected by Western blotting. Results: Double label immunohistochemistry showed that C/EBP-α was predominantly located in hepatocytes and that its expression was significantly decreased in fibrosis compared with normal liver. Atg5 expression was increased in fibrosis but was located primarily in liver septa and peri-vascular areas, which was consistent with the distribution of HSCs. In contrast, Atg6 was not expressed in normal or fibrotic liver. Treatment of HSC-T6 cells in culture with rapamycin or 3MA decreased or increased C/EBP-α expression, respectively, as shown by Western blotting. Conclusion: C/EBP-α was primarily expressed in hepatocytes in normal liver, but its expression decreased significantly in liver fibrosis. Autophagy might play a role in liver fibrosis through its association with C/EBP-α, but this hypothesis warrants further investigation.     

Liver injury in Wilson's disease: An immunohistochemical study

PurposeWilson's disease (WD) is an inherited disorder involving copper accumulation in the liver and brain. An important mechanism responsible for hepatocyte injury in WD is mitochondria destruction, although damage may also be caused by oxidative stress and lipid peroxidation.Patients/methodsThe study included 54 treated patients with WD without liver cirrhosis and 10 healthy controls. All patients had liver biopsy and immunohistochemical analysis of liver samples was performed using targeted staining for markers of mitochondrial injury (thioredoxin-2 [TRX2], cytochrome c oxidases subunit 2 [COX2], and cytochrome c oxidases complex IV subunit 4 isoform 1 [COX4-1]), of oxidative stress (peroxiredoxin-1 [PRDX1] and 8-hydroxyguanosine [8-OHdG]), and of lipid peroxidation (4-hydroxynonenal [4-HNE]).ResultsExpression, measured as mean strengths of intensity (SI) of immunohistochemical reactions per 5 fields of view, was significantly lower in patients with WD compared to controls for COX2 (2.9 vs 8.3), 8-OHdG (0.05 vs 3.8), TRX2 (4.9 vs 10.1), and PRDX1 (4.6 vs 10.1) (all P ?< ?10^?5). COX4-1 expression was undetected in patients with WD but detected in control specimens (8.1) (P ?< ?10^?5). 4-HNE was overexpressed in patients with WD compared to controls (10.1 vs 9.1; P ?< ?0.07).ConclusionsNegligible COX4-1 and low COX2 expression in liver specimens may serve as markers of inner mitochondrial membrane injury in treated patients with WD and early stages of liver fibrosis.     

ULK1, mammalian target of rapamycin, and mitochondria: linking nutrient availability and autophagy

A fundamental function of autophagy conserved from yeast to mammals is mobilization of macromolecules during times of limited nutrient availability, permitting organisms to survive under starvation conditions. In yeast, autophagy is initiated following nitrogen or carbon deprivation, and autophagy mutants die rapidly under these conditions. Similarly, in mammals, autophagy is upregulated in most organs following initiation of starvation, and is critical for survival in the perinatal period following abrupt termination of the placental nutrient supply. The nutrient-sensing kinase, mammalian target of rapamycin, coordinates cellular proliferation and growth with nutrient availability, at least in part by regulating protein synthesis and autophagy-mediated degradation. This review focusses on the regulation of autophagy by Tor, a mammalian target of rapamycin, and Ulk1, a mammalian homolog of Atg1, in response to changes in nutrient availability. Given the importance of mitochondria in maintaining bioenergetic homestasis, and potentially as a source of membrane for autophagosomes during starvation, possible roles for mitochondria in this process are also discussed.     

TRPM2-dependent autophagy inhibition exacerbates oxidative stress-induced CXCL16 secretion by keratinocytes in vitiligo

Vitiligo is a depigmented skin disease due to the destruction of melanocytes. Under oxidative stress, keratinocyte-derived chemokine C-X-C motif ligand 16 (CXCL16) plays a critical role in recruiting CD8⁺ T cells, which kill melanocytes. Autophagy serves as a protective cell survival mechanism and impairment of autophagy has been linked to increased secretion of the proinflammatory cytokines. However, the role of autophagy in the secretion of CXCL16 under oxidative stress has not been investigated. Herein, we initially found that autophagy was suppressed in both keratinocytes of vitiligo lesions and keratinocytes exposed to oxidative stress in vitro. Autophagy inhibition also promoted CXCL16 secretion. Furthermore, upregulated transient receptor potential cation channel subfamily M member 2 (TRPM2) functioned as an upstream oxidative stress sensor to inhibit autophagy. Moreover, TRPM2-mediated Ca²⁺ influx activated calpain to shear autophagy related 5 (Atg5) and Atg12–Atg5 conjugate formation was blocked to inhibit autophagy under oxidative stress. More importantly, Atg5 downregulation enhanced the binding of interferon regulatory factor 3 (IRF3) to the CXCL16 promoter region by activating Tank-binding kinase 1 (TBK1), thus promoting CXCL16 secretion. These findings suggested that TRPM2-restrained autophagy promotes CXCL16 secretion via the Atg5-TBK1-IRF3 signaling pathway under oxidative stress. Inhibition of TRPM2 may serve as a potential target for the treatment of vitiligo. © 2024 The Pathological Society of Great Britain and Ireland.     

Decreased Cytochrome c Oxidase Subunit VIIa in Aged Rat Heart Mitochondria: Immunocytochemistry

Aging decreases oxidative phosphorylation through cytochrome oxidase (COX) in cardiac interfibrillar mitochondria (IFM) in 24‐month old (aged) rats compared to 6‐month old adult Fischer 344 rats, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Immunoelectron microscopy (IEM) reveals in aged rats a 25% reduction in cardiac COX subunit VIIa in cardiac IFM, but not in SSM. In contrast, the content of subunit IV remains unchanged in both SSM and IFM, irrespective of age. These subunits are localized mainly on cristae membranes. In contrast, semi‐quantitative immunoblotting, which detects denatured protein, indicates that the content of COX VIIa is similar in IFM and SSM from both aged and adult hearts. IEM provides a sensitive method for precise localizing and quantifying specific mitochondrial proteins. The lack of immunoreaction of COX VIIa subunit by IEM in aged IFM is not explained by a reduction in protein, but rather by a masking phenomenon or by an in situ change in protein structure affecting COX activity. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Mutations in COA6 cause Cytochrome c Oxidase Deficiency and Neonatal Hypertrophic Cardiomyopathy

COA6/C1ORF31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and isolated complex IV deficiency. For the first time, clinical details about a COA6‐deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady‐state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse‐chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. Although monomeric complex IV is decreased in patient fibroblasts, the CI/CIII₂/CIV_n‐supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6‐deficient patients is provided.     

Mss51p, a putative translational activator of cytochrome c oxidase subunit-1 (COX1) mRNA, is required for synthesis of Cox1p in Saccharomyces cerevisiae

 Mutants of Saccharomyces cerevisiae that lack a functional MSS51 gene are respiratory deficient due to the absence of cytochrome c oxidase subunit 1 (Cox1p). It has been previously suggested, but not formally proven, that Mss51p is required for translational activation of COX1 mRNA, rather than being involved in a subsequent step in the synthesis of Cox1p or its assembly into cytochrome c oxidase. Pulse-chase labelling experiments now show that the absence of detectable levels of Cox1p in mss51-null strains is indeed due to the lack of synthesis of Cox1p, and is not caused by reduced stability of the protein. To gain more insight into the exact function of Mss51p, we determined the subcellular localization of the protein. We were able to show that an epitope-tagged version of Mss51p (Mss51HA) complements the mutation and can be localized in mitochondria, where it is firmly associated with the mitochondrial inner membrane. In addition, we characterized the previously identified mutant allele mss51-3. Sequence analysis revealed the presence of a short open reading frame upstream of MSS51 resulting from the creation of an extra ATG startcodon. Received: 3 December 1999 / 5 January 2000     

Cytochrome c mRNA levels decrease in senescent rat heart

The concentration of mitochondria decrease in the heart as rodents age from maturity to senescence. The reason for this change is not known. One purpose of the present study was to determine if cytochrome c mRNA, representative of proteins of the inner mitochondrial membrane, decreased in the hearts of Fischer 344 rats as they aged from 12 to 24 months. Twenty-two percent less cytochrome c mRNA existed per given quantity of extracted RNA from the heart in 24-month-old rats as compared with the 12-month-old group. No change in the quantities of cardiac α-actin mRNA, Ca²⁺/calmodulin protein kinase II mRNA or 18S rRNA was noted between 12- und 24-month-old hearts. Thus, the decrease in cytochrome c mRNA suggests that decreased in mRNAs for proteins of the inner mitochondrial membrane could play some role in the diminished concentration of mitochondria that exists in the senescent heart.     

Alteration of the sialylation pattern and memory deficits by injection of Aβ(25-35) into the hippocampus of rats

Previous studies in Parkinson's disease (PD) models suggest that early events along the path to neurodegeneration involve activation of the ubiquitin-proteasome system (UPS), endoplasmic reticulum-associated degradation (ERAD), and the unfolded protein response (UPR) pathways, in both the sporadic and familial forms of the disease, and thus ER stress may be a common feature. Furthermore, impairments in protein degradation have been linked to oxidative stress as well as pathways associated with ER stress. We hypothesize that oxidative stress is a primary initiator in a multi-factorial cascade driving dopaminergic (DA) neurons towards death in the early stages of the disease. We now report results from proteomic analysis of a rotenone-induced oxidative stress model of PD in the human neuroblastoma cell line, SH-SY5Y. Cells were exposed to sub-micromolar concentrations of rotenone for 48h prior to whole cell protein extraction and shotgun proteomic analysis. Evidence for activation of the UPR comes from our observation of up-regulated binding immunoglobulin protein (BiP), heat shock proteins, and foldases. We also observed up-regulation of proteins that contribute to the degradation of misfolded or unfolded proteins controlled by the UPS and ERAD pathways. Activation of the UPR may allow neurons to maintain protein homeostasis in the cytosol and ER despite an increase in reactive oxygen species due to oxidative stress, and activation of the UPS and ERAD may further augment clean-up and quality control in the cell.     

Mitochondria and Apoptosis: HQ or High-Security Prison?

Whether we view the mitochondria as the headquarters for the leader of a crack suicide squad or as a prison for the leader of a militant coup, the role of the mitochondria in the apoptotic process is now well established. During apoptosis the integrity of the mitochondria is breeched, the mitochondrial transmembrane potential drops, the electron transport chain is disrupted, and proteins from the mitochondrial intermembrane space (MIS) such as cytochrome c are released into the cytosol, although not necessarily in that order. In the cytosol, cytochrome c forms part of a proteinaceous complex that directly activates caspase-9, one of the apical enzymes responsible for the dismantling of the cell. In this way a mitochondrial factor which is normally locked away from the rest of the cell can directly trigger apoptosis. The need to regulate the release of cytochrome c suggests that the mitochondria may be the decision center for whether a cell lives or dies. Various hypotheses have been formulated to explain how proteins of the MIS are released and how this process is regulated. These include the Bcl-2-regulated opening of a permeability transition pore or an increase in mitochondrial transmembrane potential followed by outer membrane rupture. It remains to be clarified which mitochondria specific events are essential for apoptosis and which are merely consequences of apoptosis.     

Mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa 3 and c in Neurospora crassa

The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa ₃ and c. Using a sib-selection procedure we have isolated the cyt-12 ⁺ allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12 ⁺ allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa ₃ .     

Atg17 recruits Atg9 to organize the pre-autophagosomal structure

Autophagy is a degradation system of cytoplasmic proteins and organelles via formation of double-membrane vesicles called autophagosomes. In the yeast Saccharomyces cerevisiae , autophagosomes are formed via the pre-autophagosomal structure (PAS) in a manner dependent on Atg proteins. Under nutrient-rich condition, Atg9 is recruited to the PAS by binding to Atg11 for the Cvt pathway. However, because Atg9 is recruited to the PAS in atg11 Δ cells in starved condition and autophagy is induced, autophagy-specific mechanism for the Atg9 recruitment to the PAS has been assumed. Here, we demonstrate that, in autophagy-inducing condition, Atg9 is recruited to the PAS in a manner dependent on Atg17. Atg9 physically interacts with Atg17 in the presence of rapamycin. This interaction requires Atg1, a protein kinase essential for autophagy. Consistently, the Atg17-dependent PAS localization of Atg9 requires Atg1. However, its kinase activity is dispensable for this process. It rather regulates the equilibrium of assembly and disassembly of Atg9 at the PAS.     

Mutant protein kinase C gamma that causes spinocerebellar ataxia type 14 (SCA14) is selectively degraded by autophagy

Several causal missense mutations in the protein kinase Cγ (γPKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously showed that mutant γPKC found in SCA14 is susceptible to aggregation and causes apoptosis. Aggregation of misfolded proteins is generally involved in the pathogenesis of many neurodegenerative diseases. Growing evidence indicates that macroautophagy (autophagy) is important for the degradation of misfolded proteins and the prevention of neurodegenerative diseases. In the present study, we examined whether autophagy is involved in the degradation of the mutant γPKC that causes SCA14. Mutant γPKC‐GFP was transiently expressed in SH‐SY5Y cells by using an adenoviral tetracycline‐regulated system. Subsequently, temporal changes in clearance of aggregates and degradation of γPKC‐GFP were evaluated. Rapamycin, an autophagic inducer, accelerated clearance of aggregates and promoted degradation of mutant γPKC‐GFP, but it did not affect degradation of wild‐type γPKC‐GFP. These effects of rapamycin were not observed in embryonic fibroblast cells from Atg5‐deficient mice, which are not able to perform autophagy. Furthermore, lithium, another type of autophagic inducer, also promoted the clearance of mutant γPKC aggregates. These results indicate that autophagy contributes to the degradation of mutant γPKC, suggesting that autophagic inducers could provide therapeutic potential for SCA14.     

The protection role of Atg16l1 in CD11c+dendritic cells in murine colitis

The autophagy-related 16-like 1 gene (Atg16l1) is associated with inflammatory bowel disease (IBD) and has been shown to play an essential role in paneth cell function and intestinal homeostasis. However, the functional consequences of Atg16l1 deficiency in myeloid cells, particularly in dendritic cells (DCs), are not fully characterized. The aim of this study is to investigate the functional consequence of Atg16l1 in CD11c⁺DCs in murine colitis. We generated mice deficient in Atg16l1 in CD11c⁺DCs. Dextran Sulfate Sodium (DSS) and S. typhimurium infection induced colitis was used to assess the role of DCs specific Atg16l1 deficiency in vivo in murine colitis. Bone marrow derived dendritic cells (BMDC) were isolated and autophagy function was assessed with microtubule-associated protein 1 light chain 3β (Map1lc3b or LC3) by western blot. Uptake of Salmonella enteric serovar typhimurium (S. typhimurium) was assessed by flow cytometry and transmission electron microscopy (TEM). The production of reactive oxygen species (ROS) and intracellular S. typhimurium killing in BMDCs were assessed. We showed worsened colonic inflammation in Atg16l1 deficiency mice in DSS induced murine colitis with increased proinflammatory cytokines of IL-1β and TNF-α. Mechanistic studies performed in primary murine BMDCs showed that Atg16l1 deficiency increased ROS production, reduced microbial killing and impaired antigen processing for altered intracellular trafficking. Together, these results indicate impaired CD11c⁺DCs function with Atg16l1 deficiency contributes to the severity of murine colitis.     

Mitochondrial complex IV deficiency,caused by mutated COX6B1, is associated with encephalomyopathy,hydrocephalus and cardiomyopathy

Isolated cytochrome c oxidase (COX) deficiency is a prevalent cause of mitochondrial disease and is mostly caused by nuclear-encoded mutations in assembly factors while rarely by mutations in structural subunits. We hereby report a case of isolated COX deficiency manifesting with encephalomyopathy, hydrocephalus and hypertropic cardiomyopathy due to a missense p.R20C mutation in the COX6B1 gene, which encodes an integral, nuclear-encoded COX subunit. This novel mutation was predicted to be severe in silico. In accord, enzymatic activity was undetectable in muscle and fibroblasts, was severely decreased in lymphocytes and the COX6B1 protein was barely detectable in patient''s muscle mitochondria. Complementation with the wild-type cDNA by a lentiviral construct restored COX activity, and mitochondrial function was improved by 5-aminoimidazole-4-carboxamide ribonucleotide, resveratrol and ascorbate in the patient''s fibroblasts. We suggest that genetic analysis of COX6B1should be included in the investigation of isolated COX deficiency, including patients with cardiac defects. Initial measurement of COX activity in lymphocytes may be useful as it might circumvent the need for invasive muscle biopsy. The evaluation of ascorbate supplementation to patients with mutated COX6B1 is warranted.