Decreased Brain Protein Levels of Cytochrome Oxidase Subunits in Alzheimer's Disease and in Hereditary Spinocerebella Ataxia Disorders

Abstract : Controversy exists as to the clinical importance, cause, and disease specificity of the cytochrome oxidase (CO) activity reduction observed in some patients with Alzheimer's disease (AD). Although it is assumed that the enzyme is present in normal amount in AD, no direct measurements of specific CO protein subunits have been conducted. We measured protein levels of CO subunits encoded by mitochondrial (COX I, COX II) and nuclear (COX IV, COX VIc) DNA in autopsied brain of patients with AD whom we previously reported had decreased cerebral cortical CO activity. To assess disease specificity, groups of patients with spinocerebellar ataxia type I and Friedreich's ataxia were also included. As compared with the controls, mean protein concentrations of all four CO subunits were significantly decreased (-19 to -47%) in temporal and parietal cortices in the AD group but were not significantly reduced (-12 to -17%) in occipital cortex. The magnitude of the reduction in protein levels of the CO subunits encoded by mitochondrial DNA (-42 to -47%) generally exceeded that encoded by nuclear DNA (-19 to -43%). In the spinocerebellar ataxia disorders, COX I and COX II levels were significantly decreased in cerebellar cortex (-22 to -32%) but were normal or close to normal in cerebral cortex, an area relatively unaffected by neurodegeneration. We conclude that protein levels of mitochondrial- and nuclear-encoded CO subunits are moderately reduced in degenerating but not in relatively spared brain areas in AD and that the decrease is not specific to this disorder. The simplest explanation for our findings is that CO is decreased in human brain disorders as a secondary event in brain areas having reduced neuronal activity or neuronal/synaptic elements consequent to the primary neurodegenerative process.     

hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.     

Evolutionary Rate Acceleration of Cytochrome c Oxidase Subunit I in Simian Primates

We present an analysis of the evolutionary rates of the cytochrome c oxidase subunit I genes of primates and other mammals. Five primate genes were sequenced, and this information was combined with published data from other species. The sequences from simian primates show approximately twofold increases in their nonsynonymous substitution rate compared to those from other primates and other mammals. The species range and the overall magnitude of this rate increase are similar to those previously identified for the cytochrome c oxidase subunit II and cytochrome b genes. Received: 22 July 1999 / Accepted: 21 February 2000     

Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex

Cytochrome c oxidase or complex IV, catalyzes the final step in mitochondrial electron transfer chain, and is regarded as one of the major regulation sites for oxidative phosphorylation. This enzyme is controlled by both nuclear and mitochondrial genomes. Among its 13 subunits, three are encoded by mitochondrial DNA and ten by nuclear DNA. In this work, an RNA interference approach was taken which led to the generation of mouse A9 cell derivatives with suppressed expression of nuclear-encoded subunit IV (COX IV) of this complex. The amounts of this subunit are decrease by 86% to 94% of normal level. A detail biosynthetic and functional analysis of several cell lines with suppressed COX IV expression revealed a loss of assembly of cytochrome c oxidase complex and, correspondingly, a reduction in cytochrome c oxidase-dependent respiration and total respiration. Furthermore, dysfunctional cytochrome c oxidase in the cells leads to a compromised mitochondrial membrane potential, a decreased ATP level, and failure to grow in galactose medium. Interestingly, suppression of COX IV expression also sensitizes the cells to apoptosis. These observations provide the evidence of the essential role of the COX IV subunit for a functional cytochrome c oxidase complex and also demonstrate a tight control of cytochrome c oxidase over oxidative phosphorylation. Finally, our results further shed some insights into the pathogenic mechanism of the diseases caused by dysfunctional cytochrome c oxidase complex.     

Age-Dependent Impairment of Mitochondrial Function in Primate Brain

Abstract: It has been hypothesized that some of the functional impairments associated with aging are the result of increasing oxidative damage to mitochondrial DNA that produces defects in oxidative phosphorylation. To test this hypothesis, we examined the enzymes that catalyze oxidative phosphorylation in crude mitochondrial preparations from frontoparietal cortex of 20 rhesus monkeys (5-34 years old). Samples were assayed for complex I, complex II-III, complex IV, complex V, and citrate synthase activities. When enzyme activities were corrected for citrate synthase activities (to account for variable degrees of mitochondrial enrichment), linear regression analysis demonstrated a significant negative correlation of the activities of complex I (p < 0.002) and complex IV (p < 0.03) with age but no significant change in complex II-III or complex V activities. Relative to animals 6.9 ± 0.9 years old (n = 7), the citrate synthase-corrected activity of complex I was reduced by 17% in animals 22.5 ± 0.9 years old (n = 6) (p < 0.05) and by 22% in animals 30.7 ± 0.9 years old (n = 7) (p < 0.01). Similar age-related reductions in the activities of complexes I and IV were obtained when enzyme activities were corrected for complex II-III activity. These findings show an age-associated progressive impairment of mitochondrial complex I and complex IV activities in cerebral cortices of primates.     

Acetylsalicylic acid and acetaminophen protect against MPP+-induced mitochondrial damage and superoxide anion generation

The effects of 1-methyl-4-phenylpyridinium (MPP+) has been extensively researched due to its selective toxicity to dopaminergic neurons. Mitochondrial dysfunction which is common in the etiology of Parkinson's disease (PD), has been widely implicated in MPP+-induced toxicity. MPP+-induced mitochondrial dysfunction is believed to result in the generation of free radicals. This study was therefore performed to assess the effect of MPP+ on mitochondrial function and the ability of MPP+ to generate superoxide free radicals. Furthermore, we assessed the ability of the non-narcotic analgesics, acetaminophen and acetylsalicylic acid to prevent any diliterious effects of the potent neurotoxin, MPP+, on mitochondrial function and superoxide anion generation, in vivo. Acetylsalicylic acid and acetaminophen prevented the MPP+-induced inhibition of the electron transport chain and complex I activity. In addition, acetylsalicylic acid and acetaminophen significantly attenuated the MPP+-induced superoxide anion generation. Furthermore the results provide novel data explaining the ability of these agents to prevent MPP+-induced mitochondrial dysfunction and subsequent reactive oxygen species generation. While these findings suggest the usefulness of non-narcotic analgesics in neuroprotective therapy in neurodegenerative diseases, acetylsalicylic acid appears to be a potential candidate in prophylactic as well as in adjuvant therapy in Parkinson's disease.     

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX₉C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX₉C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.     

Circumventing the Crabtree Effect: A method to induce lactate consumption and increase oxidative phosphorylation in cell culture

Most cells grown in glucose-containing medium generate almost all their ATP via glycolysis despite abundant oxygen supply and functional mitochondria, a phenomenon known as the Crabtree effect. By contrast, most cells within the body rely on mitochondrial oxidative phosphorylation (OXPHOS) to generate the bulk of their energy supply. Thus, when utilising the accessibility of cell culture to elucidate fundamental elements of mitochondria in health and disease, it is advantageous to adopt culture conditions under which the cells have greater reliance upon OXPHOS for the supply of their energy needs. Substituting galactose for glucose in the culture medium can provide these conditions, but additional benefit can be gained from alternate in vitro models. Herein we describe culture conditions in which complete autonomous depletion of medium glucose induces a lactate-consuming phase marked by increased MitoTracker Deep Red staining intensity, increased expression of Kreb’s cycle proteins, increased expression of electron transport chain subunits, and increased sensitivity to the OXPHOS inhibitor rotenone. We propose these culture conditions represent an alternate accessible model for the in vitro study of cellular processes and diseases involving the mitochondrion without limitations incurred via the Crabtree effect.     

In vivo labeling of mitochondrial complex I (NADH:ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone

Defects in mitochondrial energy metabolism have been implicated in several neurodegenerative disorders. Defective complex I (NADH:ubiquinone oxidoreductase) activity plays a key role in Leber's hereditary optic neuropathy and, possibly, Parkinson's disease, but there is no way to assess this enzyme in the living brain. We previously described an in vitro quantitative autoradiographic assay using [(3)H]dihydrorotenone ([(3)H]DHR) binding to complex I. We have now developed an in vivo autoradiographic assay for complex I using [(3)H]DHR binding after intravenous administration. In vivo [(3)H]DHR binding was regionally heterogeneous, and brain uptake was rapid. Binding was enriched in neurons compared with glia, and white matter had the lowest levels of binding. In vivo [(3)H]DHR binding was markedly reduced by local and systemic infusion of rotenone and was enhanced by local NADH administration. There was an excellent correlation between regional levels of in vivo [(3)H]DHR binding and the in vitro activities of complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase), suggesting that the stoichiometry of these components of the electron transport chain is relatively constant across brain regions. The ability to assay complex I in vivo should provide a valuable tool to investigate the status of this mitochondrial enzyme in the living brain and suggests potential imaging techniques for complex I in humans.     

Mitochondrial respiratory chain disorders in childhood: Insights into diagnosis and management in the new era of genomic medicine

Background

Mitochondrial respiratory chain disorders (MRCDs) are some of the most common metabolic disorders presenting in childhood, however because of it clinical heterogeneity, diagnosis is often challenging. Being a multisystemic disorder with variable and non-specific presentations, definitive diagnosis requires a combination of investigative approaches, and is often a laborious process.

Scope of review

In this review we provide a broad overview of the clinical presentations of MRCDs in childhood, evaluating the different diagnostic approaches and treatment options, and highlighting the recent research advances in this area.

Major conclusions

Extensive research over the years has significantly increased the frequency with which accurate diagnosis is being made, including the identification of new biomarkers and next generation sequencing (NGS) technologies. NGS has provided a breakthrough in unravelling the genetic basis of MRCDs, especially considering the complexity of mitochondrial genetics with its dual genetic contributions.

General significance

With an increased understanding of the pathophysiology of this group of disorders, clinical trials are now being established using a number of different therapeutic approaches, with the hope of changing the focus of treatment from being largely supportive to potentially having a positive effect on the natural history of the disorder.This article is part of a Special Issue entitled: Special Issue: Frontiers of Mitochondria IG000218.     

The mitochondria-targeted antioxidant MitoQ extends lifespan and improves healthspan of a transgenic Caenorhabditis elegans model of Alzheimer disease

β-Amyloid (Aβ)-induced toxicity and oxidative stress have been postulated to play critical roles in the pathogenic mechanism of Alzheimer disease (AD). We investigated the in vivo ability of a mitochondria-targeted antioxidant, MitoQ, to protect against Aβ-induced toxicity and oxidative stress in a Caenorhabditis elegans model overexpressing human Aβ. Impairment of electron transport chain (ETC) enzymatic activity and mitochondrial dysfunction are early features of AD. We show that MitoQ extends lifespan, delays Aβ-induced paralysis, ameliorates depletion of the mitochondrial lipid cardiolipin, and protects complexes IV and I of the ETC. Despite its protective effects on lifespan, healthspan, and ETC function, we find that MitoQ does not reduce DCFDA fluorescence, protein carbonyl levels or modulate steadystate ATP levels or oxygen consumption rate. Moreover, MitoQ does not attenuate mitochondrial DNA (mtDNA) oxidative damage. In agreement with its design, the protective effects of MitoQ appear to be targeted specifically to the mitochondrial membrane and our findings suggest that MitoQ may have therapeutic potential for Aβ- and oxidative stress-associated neurodegenerative disorders, particularly AD.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: I. EQUILIBRIUM AND MODELING STUDIES*

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c₁ in detergent-solubilized bc₁ complex from Rhodobacter sphaeroides. Imidazole binding to cyt c₁ substantially lowers the midpoint potential of the heme and fully inhibits bc₁ complex activity. Temperature dependences showed that binding of Im (K_d ≈ 330 μm, 25 °C, pH 8) is enthalpically driven (ΔH⁰ = −56 kJ/mol, ΔS⁰ = −121 J/mol/K), whereas binding of MeIm is 30 times weaker (K_d ≈ 9.3 mm) and is entropically driven (ΔH⁰ = 47 kJ/mol, ΔS⁰° = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c₁ triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c₂ suggested that Im binding to cyt c₁ is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c₂, and c₁, which showed hydrogen bonds formed between the N_δH of Im and the cyt c₁ protein, or with a water molecule sequestered with the ligand in the heme cleft.     

Kinetic and Structural Characterization of the Interaction between the FMN Binding Domain of Cytochrome P450 Reductase and Cytochrome c

Cytochrome P450 reductase (CPR) is a diflavin enzyme that transfers electrons to many protein partners. Electron transfer from CPR to cyt c has been extensively used as a model reaction to assess the redox activity of CPR. CPR is composed of multiple domains, among which the FMN binding domain (FBD) is the direct electron donor to cyt c. Here, electron transfer and complex formation between FBD and cyt c are investigated. Electron transfer from FBD to cyt c occurs at distinct rates that are dependent on the redox states of FBD. When compared with full-length CPR, FBD reduces cyt c at a higher rate in both the semiquinone and hydroquinone states. The NMR titration experiments reveal the formation of dynamic complexes between FBD and cyt c on a fast exchange time scale. Chemical shift mapping identified residues of FBD involved in the binding interface with cyt c, most of which are located in proximity to the solvent-exposed edge of the FMN cofactor along with other residues distributed around the surface of FBD. The structural model of the FBD-cyt c complex indicates two possible orientations of complex formation. The major complex structure shows a salt bridge formation between Glu-213/Glu-214 of FBD and Lys-87 of cyt c, which may be essential for the formation of the complex, and a predicted electron transfer pathway mediated by Lys-13 of cyt c. The findings provide insights into the function of CPR and CPR-cyt c interaction on a structural basis.     

Reactivity of Nitric Oxide with Cytochrome c Oxidase: Interactions with the Binuclear Centre and Mechanism of Inhibition

Nitric oxide (NO) has recently been recognized as an important biological mediator that inhibits respiration at cytochrome c oxidase (CcO). This inhibition is reversible and shows competition with oxygen, the K _i being lower at low oxygen concentrations. Although the species that binds NO in turnover has been suggested to contain a partially reduced binuclear center, the exact mechanism of the inhibition is not clear. Recently, rapid (ms) redox reactions of NO with the binuclear center have been reported, e.g., the ejection of an electron to cytochrome a and the depletion of the intermediates P and F. These observations have been rationalized within a scheme in which NO reacts with oxidized Cu_B leading to the reduction of this metal center and formation of nitrite in a very fast reaction. Electron migration from Cu_B to other redox sites within the enzyme is proposed to explain the optical transitions observed. The relevance of these reactions to the inhibition of CcO and metabolism of NO are discussed.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: II. KINETICS AND MECHANISM OF BINDING*

The kinetics of imidazole (Im) and N-methylimidazole (MeIm) binding to oxidized cytochrome (cyt) c₁ of detergent-solubilized bc₁ complex from Rhodobacter sphaeroides are described. The rate of formation of the cyt c₁-Im complex exhibited three separated regions of dependence on the concentration of imidazole: (i) below 8 mm Im, the rate increased with concentration in a parabolic manner; (ii) above 20 mm, the rate leveled off, indicating a rate-limiting conformational step with lifetime ∼1 s; and (iii) at Im concentrations above 100 mm, the rate substantially increased again, also parabolically. In contrast, binding of MeIm followed a simple hyperbolic concentration dependence. The temperature dependences of the binding and release kinetics of Im and MeIm were also measured and revealed very large activation parameters for all reactions. The complex concentration dependence of the Im binding rate is not consistent with the popular model for soluble c-type cytochromes in which exogenous ligand binding is preceded by spontaneous opening of the heme cleft, which becomes rate-limiting at high ligand concentrations. Instead, binding of ligand to the heme is explained by a model in which an initial and superficial binding facilitates access to the heme by disruption of hydrogen-bonded structures in the heme domain. For imidazole, two separate pathways of heme access are indicated by the distinct kinetics at low and high concentration. The structural basis for ligand entry to the heme cleft is discussed.     

Regulation of Cytochrome c Oxidase Subunit mRNA and Enzyme Activity in Rat Brain Reward Regions During Withdrawal from Chronic Cocaine

Abstract: A subtractive hybridization and differential screening procedure was used to detect up-regulation of cytochrome c oxidase (CO) subunits I, III, and IV mRNA in the nucleus accumbens (NAc) of rats chronically treated with cocaine. Northern blot analyses of mRNA isolated from individual rats confirmed that CO subunit I was up-regulated by chronic, but not acute, cocaine in two brain regions, the NAc (33%) and caudate-putamen (CP)(35%). CO activity, used as a measure of metabolic activity, was increased by 88% in the NAc, and decreased by 20% in the medial prefrontal cortex (mPFC), the day after chronic treatment was terminated. CO enzyme activity was not regulated in the CP, or in other brain regions not involved in drug reward. CO activity in both the NAc and mPFC showed unique time-dependent patterns of regulation during the week after chronic cocaine treatment.     

Effects of Selective Monoamine Oxidase Inhibitors on the In Vivo Release and Metabolism of Dopamine in the Rat Striatum

Brain microdialysis was used to examine the in vivo efflux and metabolism of dopamine (DA) in the rat striatum following monoamine oxidase (MAO) inhibition. Relevant catecholamines and indoleamines were quantified by HPLC coupled with a electrochemical detection system. The MAO-B inhibitor selegiline only affected DA deamination at a dose shown to inhibit partially type A MAO. Alterations in DA and metabolite efflux were not observed when using the MAO-B-selective dose of 1 mg/kg of selegiline. At 10 mg/kg, selegiline reduced the efflux of DA metabolites to approximately 70% of basal values without affecting DA efflux. K(+)- and veratrine-stimulated DA efflux was not affected by selegiline. Experiments using amphetamine and the DA uptake inhibitor nomifensine demonstrated that the effect of selegiline on DA metabolism was unlikely to be mediated either by inhibition of DA uptake or by an indirect effect of its metabolite amphetamine. The possibility that the effect of selegiline is mediated via a nonspecific inhibition of MAO is discussed. In contrast, the MAO-A inhibitor clorgyline inhibited basal DA metabolism and increased basal and depolarisation-induced DA efflux. A 1 mg/kg dose of clorgyline reduced basal DA metabolite efflux (40-60% of control values) without affecting DA efflux. At 10 mg/kg of clorgyline, DA efflux increased to 253 +/- 19% of basal values, whereas efflux of DA metabolites was reduced to between 15 and 26% of control values. The release of DA induced by K+ and veratrine was not affected by 1 mg/kg of clorgyline but was increased by approximately 200% following pretreatment with 10 mg/kg of clorgyline. The nonselective MAO inhibitor pargyline caused similar but more pronounced alterations in these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.     

The effects of mitochondrial inhibitors on Ca2+ signalling and electrical conductances required for pacemaking in interstitial cells of Cajal in the mouse small intestine

Interstitial cells of Cajal (ICC-MY) are pacemakers that generate and propagate electrical slow waves in gastrointestinal (GI) muscles. Slow waves appear to be generated by the release of Ca²⁺ from intracellular stores and activation of Ca²⁺-activated Cl⁻ channels (Ano1). Conduction of slow waves to smooth muscle cells coordinates rhythmic contractions. Mitochondrial Ca²⁺ handling is currently thought to be critical for ICC pacemaking. Protonophores, inhibitors of the electron transport chain (FCCP, CCCP or antimycin) or mitochondrial Na⁺/Ca²⁺ exchange blockers inhibited slow waves in several GI muscles. Here we utilized Ca²⁺ imaging of ICC in small intestinal muscles in situ to determine the effects of mitochondrial drugs on Ca²⁺ transients in ICC. Muscles were obtained from mice expressing a genetically encoded Ca²⁺ indicator (GCaMP3) in ICC. FCCP, CCCP, antimycin, a uniporter blocker, Ru360, and a mitochondrial Na⁺/Ca²⁺ exchange inhibitor, CGP-37157 inhibited Ca²⁺ transients in ICC-MY. Effects were not due to depletion of ATP, as oligomycin did not affect Ca²⁺ transients. Patch-clamp experiments were performed to test the effects of the mitochondrial drugs on key pacemaker conductances, Ano1 and T-type Ca²⁺ (Ca_V3.2), in HEK293 cells. Antimycin blocked Ano1 and reduced Ca_V3.2 currents. CCCP blocked Ca_V3.2 current but did not affect Ano1 current. Ano1 and Cav3.2 currents were inhibited by CGP-37157. Inhibitory effects of mitochondrial drugs on slow waves and Ca²⁺ signalling in ICC can be explained by direct antagonism of key pacemaker conductances in ICC that generate and propagate slow waves. A direct obligatory role for mitochondria in pacemaker activity is therefore questionable.