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.     

Estrogen Induction of Cytochrome c Oxidase Subunit III in Rat Hippocampus

Differential screening of a cDNA library prepared from mRNA of the hippocampus of estrogen-stimulated ovariectomized female rats led to the identification of a single estrogen-induced clone. Analysis of the sequence identified this cDNA as the gene coding for subunit III of the enzyme cytochrome c oxidase. Cytochrome c oxidase subunit III mRNA levels significantly increased as early as 3 h following the administration of a single dose of hormone. This effect was visible in the hippocampus and in the hypothalamus, but not in the other brain areas examined. Because subunit III of the cytochrome c oxidase is of mitochondrial origin, the mechanism involved in the estrogenic effect is still unknown. The observation that the activity of cytochrome c oxidase can also be induced by estrogens in the hippocampus indicates that this induction may be secondary to the increased expression of the other subunits of cytochrome c oxidase or to the general increase of neuronal activity.     

Oligomerization of Heme o Synthase in Cytochrome Oxidase Biogenesis Is Mediated by Cytochrome Oxidase Assembly Factor Coa2

The synthesis of the heme a cofactor used in cytochrome c oxidase (CcO) is dependent on the sequential action of heme o synthase (Cox10) and heme a synthase (Cox15). The active state of Cox10 appears to be a homo-oligomeric complex, and formation of this complex is dependent on the newly synthesized CcO subunit Cox1 and the presence of an early Cox1 assembly intermediate. Cox10 multimerization is triggered by progression of Cox1 from the early assembly intermediate to downstream intermediates. The CcO assembly factor Coa2 appears important in coupling the presence of newly synthesized Cox1 to Cox10 oligomerization. Cells lacking Coa2 are impaired in Cox10 complex formation as well as the formation of a high mass Cox15 complex. Increasing Cox1 synthesis in coa2Δ cells restores respiratory function if Cox10 protein levels are elevated. The C-terminal segment of Cox1 is important in triggering Cox10 oligomerization. Expression of the C-terminal 54 residues of Cox1 appended to a heterologous matrix protein leads to efficient Cox10 complex formation in coa2Δ cells, but it fails to induce Cox15 complex formation. The state of Cox10 was evaluated in mutants, which predispose human patients to CcO deficiency and the neurological disorder Leigh syndrome. The presence of the D336V mutation in the yeast Cox10 backbone results in a catalytically inactive enzyme that is fully competent to oligomerize. Thus, Cox10 oligomerization and catalytic activation are separate processes and can be uncoupled.     

Diabetic Modulation of the Temperature Kinetics Properties of Cytochrome Oxidase Activity in Rat Brain Mitochondria

The effects of alloxan-diabetes and subsequent treatment with insulin on temperature kinetics properties of cytochrome oxidase activity from rat brain mitochondria were examined. The enzyme activity decreased only at the late stage of diabetes which was not normalized by insulin treatment; however at early stage of diabetes hyper-stimulation occurred. In the control animals the Arrhenius plot was chair shaped with three energies of (E₁, E₂ and E₃) and two phase transition temperatures (T_t1 and T_t2). At early diabetic stage the Arrhenius plot became biphasic and E₁ and E₂ decreased_; insulin treatment reversed chair-shaped pattern with increase in E₂. These changes correlated with transient changes in the phospholipids profiles especially decreased acidic phospholipids. The temperature kinetics parameters were minimally affected at the late stage of diabetes or by insulin treatment. Thus at the late stage the brain tissue seems to have readjusted to its insulin homeostasis.     

Pathways for Electron Tunneling in Cytochrome c Oxidase

Warburg showed in 1929 that the photochemical action spectrum for CO dissociation from cytochrome c oxidase is that of a heme protein. Keilin had shown that cytochrome a does not react with oxygen, so he did not accept Warburg's view until 1939, when he discovered cytochrome a ₃. The dinuclear cytochrome a ₃-Cu_B unit was found by EPR in 1967, whereas the dinuclear nature of the Cu_A site was not universally accepted until oxidase crystal structures were published in 1995. There are negative redox interactions between cytochrome a and the other redox sites in the oxidase, so that the reduction potential of a particular site depends on the redox states of the other sites. Calculated electron-tunneling pathways for internal electron transfer in the oxidase indicate that the coupling-limited rates are 9×10⁵ (Cu _A a) and 7×10⁶ s^–1 (a a ₃); these calculations are in reasonable agreement with experimental rates, after corrections are made for driving force and reorganization energy. The best Cu_A-a pathway starts from the ligand His204 and not from the bridging sulfur of Cys196, and an efficient a-a ₃ path involves the heme ligands His378 and His376 as well as the intervening Phe377 residue. All direct paths from Cu_A to a ₃ are poor, indicating that direct Cu_A a ₃ electron transfer is much slower than the Cu_A a reaction. The pathways model suggests a means for gating the electron flow in redox-linked proton pumps.     

Phosphatidylinositol Kinase Is Reduced in Alzheimer''s Disease

Phosphatidylinositol (PI) kinase and PI phosphate (PIP) kinase activities were measured in postmortem samples of brain tissue from patients with Alzheimer's disease and nondemented control subjects. A membrane-free cytosolic fraction from four neocortical locations, with exogenous inositol lipids as the substrate, was used. Tissue from patients with Alzheimer's disease was characterized by reduced PIP formation; the reduction was 50% in prefrontal cortex, temporal cortex, and parietal cortex and 40% in precentral gyrus. In contrast, no alterations were found in PI bisphosphate formation in these four neocortical locations. The specific changes in PI kinase but not PIP kinase activity suggest that the findings may have functional relevance to the involvement of brain membrane processes in Alzheimer's disease.     

Nitric Oxide Causes Glutamate Release from Brain Synaptosomes

Abstract: We determined the ability of pathological levels of nitric oxide (NO) to cause glutamate release from isolated rat brain nerve terminals using a fluorometric assay. It was found that NO (0.7 and 2 µ M ) produced (4 and 10 nmol/mg of synaptosomal protein) Ca²⁺-independent glutamate release from synaptosomes (after 1 min of exposure). Spermine/NO complex (spermine NONOate; a slow NO donor) and potassium cyanide (an inhibitor of cytochrome oxidase) also caused Ca²⁺-independent glutamate release. Preincubation of synaptosomes with 5 µ M 1 H -[1,2,4]oxadiazole[4,3- a ]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) had no effect on NO-induced Ca²⁺-independent glutamate release. Ca²⁺-independent glutamate release produced by NO was greater in a low-oxygen medium. NO, spermine NONOate, and potassium cyanide inhibited synaptosomal respiration with a similar order of potency with respect to their ability to cause glutamate release. Because NO has been shown previously to inhibit reversibly cytochrome oxidase in competition with oxygen, our findings in this study suggest that NO (and cyanide) causes glutamate release following inhibition of mitochondrial respiration at the level of cytochrome oxidase. Thus, elevated NO production leading to mitochondrial dysfunction, glutamate release, and excitotoxicity may contribute to neuronal death in neurological diseases.     

Anoxia-Induced Changes in Pyridine Nucleotide Redox State in Cortical Neurons and Astrocytes

NAD(P)H autofluorescence was used to verify establishment of metabolic anoxia using primary cultures of cortical neurons and astrocytes. Cells on cover slips were placed in a chamber and O₂ was displaced by continuous infusion of argon. Perfusion with medium at PO₂ < 0.4 mm Hg caused an increase in NAD(P)H fluorescence, albeit to levels lower than that obtained with cyanide. Addition of the nitric oxide-generating agent DETA-NO to the hypoxic medium further increased fluorescence to the level with cyanide. Fluorescence under anoxia remained high in the presence of glucose, but declined in neurons and not in astrocytes when glucose was substituted with 2-deoxyglucose. Reoxygenation of neurons resulted in a decline in fluorescence and a loss in fluorescent gradient between fully reduced and fully oxidized (plus respiratory uncoupler). We conclude that (1) DETA-NO is useful for generating metabolic anoxia in the presence of argon (2) Exogenous glucose is necessary to maintain NAD(P)H in a reduced state during metabolic anoxia in neurons but not astrocytes (3) Neurons undergo a partially irreversible decline in NAD(P)H fluorescence during metabolic anoxia and reoxygenation that could contribute to prolonged metabolic failure. Special issue dedicated to John P. Blass.     

Brain Cytochrome Oxidase in Alzheimer''s Disease

A recent demonstration of markedly reduced (-50%) activity of cytochrome oxidase (CO; complex 4), the terminal enzyme of the mitochondrial enzyme transport chain, in platelets of patients with Alzheimer's disease (AD) suggested the possibility of a systemic and etiologically fundamental CO defect in AD. To determine whether a CO deficiency occurs in AD brain, we measured the activity of CO in homogenates of autopsied brain regions of 19 patients with AD and 30 controls matched with respect to age, postmortem time, sex, and, as indices of agonal status, brain pH and lactic acid concentration. Mean CO activity in AD brain was reduced in frontal (-26%: p less than 0.01), temporal (-17%; p less than 0.05), and parietal (-16%; not significant, p = 0.055) cortices. In occipital cortex and putamen, mean CO levels were normal, whereas in hippocampus, CO activity, on average, was nonsignificantly elevated (20%). The reduction of CO activity, which is tightly coupled to neuronal metabolic activity, could be explained by hypofunction of neurons, neuronal or mitochondrial loss, or possibly by a more primary, but region-specific, defect in the enzyme itself. The absence of a CO activity reduction in all of the examined brain areas does not support the notion of a generalized brain CO abnormality. Although the functional significance of a 16-26% cerebral cortical CO deficit in human brain is not known, a deficiency of this key energy-metabolizing enzyme could reduce energy stores and thereby contribute to the brain dysfunction and neurodegenerative processes in AD.     

Entorhinal Cortex Lesions Transiently Alter Glucocorticoid but Not Mineralocorticoid Receptor Gene Expression in the Rat Hippocampus

Abstract: The mechanisms of delayed onset and cell death in Huntington's disease (HD) are unknown. One possibility is that a genetic defect in energy metabolism may result in slow excitotoxic neuronal death. Therefore, we examined the effects of age on striatal lesions produced by local administration of the mitochondrial toxin 3-nitropropionic acid in rats. In vivo chemical shift magnetic resonance imaging showed marked increases in striatal lactate concentrations that significantly correlated with increasing age. Histologic and neurochemical studies showed a striking age dependence of the lesions, with 4- and 12-month-old animals being much more susceptible than 1-month-old animals. Continuous systemic administration of low doses of 3-nitropropionic acid for 1 month resulted in striatal lesions showing growth-related changes in dendrites of striatal spiny neurons using the Golgi technique. These results show that a known mitochondrial toxin can produce selective axon-sparing striatal lesions showing both the age dependence and striatal spiny neuron dendritic changes that characterize HD.     

心磷脂对细胞色素C氧化酶质子泵功能的影响

 用胆酸盐透析法将猪心线粒体细胞色素C氧化酶重组在含心磷脂和二肉豆寇磷脂酰胆碱的脂质体上,以还原态细胞色素C作为酶反应底物,记录脂酶体囊泡外介质液pH的变化,pH下降幅度可以反映细胞色素C氧化酶质子泵的功能。 心磷脂含量不同的细胞色素C氧化酶脂酶体质子泵功能不同。心磷脂含量在10％—40％(w/w)范围内,随心磷脂含量增高,该酶质子泵功能增强;当心磷艏含量超过50％时,该酶质子泵功能却随心磷脂含量的增加表现出下降的趋势。阿霉素可以与心磷脂紧密结合,抑制细胞色素C氧化酶的质子泵功能。然而,少量阿霉素却能增强含70％心磷脂的脂酶体的质子泵功能。     

Galanin-Like Immunoreactivity Is Increased in the Postmortem Cerebral Cortex from Patients with Alzheimer's Disease

Abstract: Galanin is a peptide that is associated with cholinergic neurons of the basal forebrain and, thus, of interest for the neuropathology of Alzheimer's disease. In the present study, human galanin-like immunoreactivity was measured in postmortem human cerebral cortical tissues by using a homologous radioimmunoassay. In an initial study, six cerebral cortical regions were evaluated from nine elderly controls, 13 neuropathologically verified Alzheimer's disease patients, and 19 elderly schizophrenics. A significant 65% increase in galanin was found in frontal cortex Brodmann area 8 of Alzheimer's disease patients compared with controls. In contrast, cerebral cortical tissues from elderly schizophrenics were not different from those from elderly controls in any region. In a second study, 10 cerebral cortical regions were evaluated from 50 neuropathologically verified Alzheimer's disease patients and nine elderly controls. Concentrations of galanin were increased significantly 26–61% in six of 10 cerebral cortical regions examined (Brodmann areas F8, F44, T20, T21, T36, and P22). Purification of brain extracts by size-exclusion Sephadex G-50 chromatography revealed that human galanin-like immunoreactivity eluted in two peaks of different molecular weights. These studies reveal increased concentrations of galanin in the cerebral cortex of Alzheimer's disease, similar to previous findings in basal forebrain tissue. Because galanin inhibits cholinergic neurotransmission, these findings may have important implications in the understanding of Alzheimer's disease neuropathology and associated cognitive deficits.     

Angiotensin Converting Enzyme in Alzheimer''s Disease: Increased Activity in Caudate Nucleus and Cortical Areas

The activity of the dipeptidyl carboxypeptidase, angiotensin converting enzyme, was assayed in several brain regions of patients dying with Alzheimer's disease and compared to that of appropriately age-matched controls. Enzyme activity was found to be elevated by 44% and 41% in the medial hippocampus and parahippocampal gyrus, respectively, and by 27% and 29% in the frontal cortex (area 10 of Brodman) and caudate nucleus, respectively, in Alzheimer's disease patients. Converting enzyme activity did not differ from controls in the nucleus accumbens, substantia nigra, temporal cortex, anterior or posterior hippocampus, amydgala, and septal nuclei.     

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.     

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.     

Striatal GABAergic Neuronal Activity Is Not Reduced in Parkinson''s Disease

The content of gamma-aminobutyric acid (GABA) and the activities of glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) were measured in whole putamen obtained at autopsy from 13 patients dying with idiopathic Parkinson's disease and 13 appropriate control subjects. Mean GABA content was significantly elevated (by 28%) in the putamen of the Parkinson's disease patients. TH activity was markedly reduced, while there was no significant reduction of GAD activity in the putamen of these patients. GABA content was also measured in both sides of the striatum in rats which had received unilateral injections of 6-hydroxydopamine (6-OHDA) in the vicinity of the axons of the nigrostriatal projection. Mean GABA content was found significantly elevated (by 33%) in the ipsilateral striatum. Loss of dopaminergic nigrostriatal neurons, in both human Parkinson's disease and in the rat 6-OHDA model, is accompanied by increased striatal GABA content. The assumption that GABAergic neurotransmission is reduced in the striatum in Parkinson's disease may not be correct.     

Cortical Substance P-Like Immunoreactivity in Cases of Alzheimer's Disease and Senile Dementia of the Alzheimer Type

Abstract: The concentration of substance P-like immunoreactive material (SPLI) and somatostatin-like immunoreactive material (SLI) and the activity of acetyl-CoA: choline- O -acetyltransferase (ChAT; EC 2.3.1.6.) were measured in eight brain regions of 13 normal patients and 12 patients with Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT). SPLI was significantly lower in five of eight regions in the patients with AD/SDAT. Younger patients with AD/SDAT had significantly lower SLI in the parietal cortex than older patients. ChAT activity and SPLI in the parietal cortex of the presenile patients with ADISDAT were not significantly different from values found in older patients.