Inactivation of beef brain alpha-ketoglutarate dehydrogenase complex by valproic acid and valproic acid metabolites. Possible mechanism of anticonvulsant and toxic actions.

The anticonvulsant valproic acid (VPA, 2-n-propylpentanoic acid) causes inhibition of the citric acid cycle and elevations of central nervous system (CNS) gamma-aminobutyric acid (GABA) levels, which correlates with anticonvulsant action. No unifying mechanism for these actions of VPA has won general acceptance. alpha-Ketoglutarate dehydrogenase complex (KDHC) is a critical control enzyme in the CNS. We hypothesized that VPA may be an inhibitor of this enzyme since decreased KDHC activity would reduce substrate flux through the citric acid cycle and may increase flux into GABA synthesis. To test this hypothesis, inhibition of purified beef brain KDHC by VPA and its metabolites 2-n-propylpent-2-enoic acid (delta 2,3 VPE) and their coenzyme A (CoA) derivatives were studied. Preincubation of the NADH-reduced enzyme with delta 2,3 VPE, VPA-CoA, and delta 2,3 VPE-CoA caused time-dependent inactivation, reversible by addition of CoA. Under steady-state conditions, delta 2,3 VPE and VPA-CoA were competitive inhibitors of KDHC and delta 2,3 VPE-CoA was a mixed inhibitor. These observations have implications for the molecular mechanisms of VPA action. VPA derivatives cause inactivation and inhibition of KDHC, which may explain the anticonvulsant and some toxic actions of VPA.

Myxothiazol resistance in human mitochondria.

We have investigated electron transfer activities of respiratory chain complexes in platelet mitochondria of a patient with intermittent ataxia and lactic acidosis who was previously reported to be deficient in the E1 (decarboxylase) component of the pyruvate dehydrogenase complex. Electron transfer from succinate to cytochrome c was normal, but the mitochondria exhibited moderately decreased (63% of control) quinol: cytochrome-c oxidoreductase activity, suggesting a defect in complex III. Consistent with some perturbation in complex III, electron flux through complex III was resistant to inhibition by myxothiazol compared to normal controls. In contrast, titration with antimycin revealed a less abnormal pattern of inhibition. The extreme specificity of myxothiazol binding at or near the quinol oxidase domain of mitochondrial cytochrome b, i.e., b-566, suggests a defect in this region of complex III which may perturb the kinetics or thermodynamics of quinol oxidation in the complex. These data suggest that the patient's illness results from a mutation in the quinol oxidase domain of mitochondrial cytochrome b (b-566).

The parkinsonian neurotoxin MPP+ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism.

The mitochondrial transition pore (MTP) is implicated as a mediator of cell injury and death in many situations. The MTP opens in response to stimuli including reactive oxygen species and inhibition of the electron transport chain. Sporadic Parkinson's disease (PD) is characterized by oxidative stress and specifically involves a defect in complex I of the electron transport chain. To explore the possible involvement of the MTP in PD models, we tested the effects of the complex I inhibitor and apoptosis-inducing toxin N-methyl-4-phenylpyridinium (MPP+) on cyclosporin A (CsA)-sensitive mitochondrial swelling and release of cytochrome c. In the presence of Ca2+ and Pi, MPP+ induced a permeability transition in both liver and brain mitochondria. MPP+ also caused release of cytochrome c from liver mitochondria. Rotenone, a classic non-competitive complex I inhibitor, completely inhibited MPP(+)-induced swelling and release of cytochrome c. The MPP(+)-induced permeability transition was synergistic with nitric oxide and the adenine nucleotide translocator inhibitor atractyloside, and additive with phenyl arsine oxide cross-linking of dithiol residues. MPP(+)-induced pore opening and cytochrome c release were blocked by CsA, the Ca2+ uniporter inhibitor ruthenium red, the hydrophobic disulfide reagent N-ethylmaleimide, butacaine, and the free radical scavenging enzymes catalase and superoxide dismutase. MPP+ neurotoxicity may derive from not only its inhibition of complex I and consequent ATP depletion, but also from its ability to open the MTP and to release mitochondrial factors including Ca2+ and cytochrome c known to be involved in apoptosis.

Elevated reactive oxygen species and antioxidant enzyme activities in animal and cellular models of Parkinson's disease.

The dopaminergic neurotoxin N-methyl,4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) causes a syndrome in primates and humans which mimics Parkinson's disease (PD) in clinical, pathological, and biochemical findings, including diminished activity of complex I in the mitochondrial electron transport chain. Reduced complex I activity is found in sporadic PD and can be transferred through mitochondrial DNA, suggesting a mitochondrial genetic etiology. We now show that MPTP treatment of mice and N-methylpyridinium (MPP+) exposure of human SH-SY5Y neuroblastoma cells increases oxygen free radical production and antioxidant enzyme activities. Cybrid cells created by transfer of PD mitochondria exhibit similar characteristics; however, PD cybrids' antioxidant enzyme activities are not further increased by MPP+ exposure, as are the activities in control cybrids. PD mitochondrial cybrids are subject to metabolic and oxidative stresses similar to MPTP parkinsonism and provide a model to determine mechanisms of oxidative damage and cell death in PD.

Preclinical detection of Parkinson's disease: biochemical approaches.

Dysfunction of NADH: ubiquinone oxidoreductase (complex I) of the mitochondrial electron transport chain has been linked to the pathogenesis of Parkinson's disease. While simple assays of complex I activity are unlikely to be useful in the preclinical detection of Parkinson's disease, other more sophisticated physical-chemical approaches including detection of free radical damage may have utility. Leber's hereditary optic neuropathy may provide a useful model system for development of such strategies.

Hypomelanosis of Ito: association with a chromosomal abnormality.

We report the clinical and neuroradiologic findings and the association of a chromosome abnormality, t(2,8), with a case of hypomelanosis of Ito (incontinentia pigmenti achromians). Chromosome anomalies have not been previously recognized in this genetically determined, neurocutaneous disorder.

Cytochrome c oxidase in Alzheimer's disease brain: purification and characterization.

Cytochrome c oxidase (COX) is deficient in both peripheral tissue and brain of Alzheimer's disease (AD) patients and may be of pathogenic significance in AD. We purified COX from AD brains (n = 3) and control brains (n = 3) and characterized the enzyme kinetically and spectrally. Purified AD brain COX displayed anomalous kinetic behavior compared with control brain COX in that the low Km binding site was kinetically unidentifiable. For purposes of comparison, we purified COX from a standard beef heart preparation and found normal kinetic behavior. AD brain COX may be structurally abnormal and may make an important contribution to the bioenergetic defect seen in AD.

Carnitine deficiency, organic acidemias, and Reye's syndrome.

Relative carnitine deficiency is important in the pathophysiology of several disorders, including Reye's syndrome and organic acidemias. In acute clinical crises, carnitine serves as a "buffer," trapping toxic acyl compounds. Mitochondrial failure develops in carnitine deficiency when there is insufficient tissue carnitine available to buffer toxic acyl-CoA metabolites. Toxic levels of acyl-CoA impair the citrate cycle, gluconeogenesis, the urea cycle, and fatty-acid oxidation. Carnitine replacement therapy is safe and induces excretion of toxic acyl groups in the urine.

Cytochrome oxidase deficiency in Alzheimer's disease.

We assayed cytochrome oxidase and other electron transport chain activities in platelet mitochondria isolated from patients with Alzheimer's disease (AD). Five of 6 patients had striking reductions of platelet cytochrome oxidase activity (patient mean, 83.72 +/- 82.99 nmol/min/mg; control mean, 167.14 +/- 36.21 nmol/min/mg; n = 8). Other electron transport chain catalytic activities were not significantly different than control values. AD may be a systemic illness, a primary defect in cytochrome oxidase may be pathogenically important in its production, and the mitochondrial genes encoding cytochrome oxidase subunits may be important in producing the defect.

Friedreich's disease: IV. Reduced mitochondrial malic enzyme activity in heterozygotes.

Friedreich's disease (FD) obligate heterozygotes have reduced mitochondrial malic enzyme (MEm) activity in cultured fibroblasts. This indicates that the MEm deficiency in homozygous affected patients is genetically determined. Heterozygote MEm activity was only 20% of the control mean activity, lower than the 50% expected in an autosomal-recessive disorder. This may result from negative interactions between mutant and normal subunits in the tetrameric enzyme. These data support the idea that MEm deficiency causes FD, but further studies are required to prove this hypothesis.

Effects of octanoate on rat brain and liver mitochondria.

Octanoate increased state 3 and state 4 respiration in rat liver mitochondria. The respiratory control rates decreased because state 4 was disproportionately affected. The ADP:O ratio was not affected. Octanoate produced a fall in the protonmotive force (delta p) of 30 mV during state 3 and state 4. The mitochondrial inner membrane proton conductance (Cm,H+) increased twofold during state 4. Similar effects were observed in polarographic assays of brain mitochondria, but measurements of delta p and Cm,H+ were not possible. Octanoate produces loose coupling in isolated mitochondria. This effect may play a role in the pathogenesis of Reye's syndrome.

Brain mitochondrial metabolism in experimental thiamine deficiency.

Thiamine deficiency causes Wernicke's encephalopathy, although the precise mechanism is unknown. We used a low-thiamine diet in conjunction with a thiamine analog, pyrithiamine, as a model of severe thiamine deficiency in rats. We investigated the function of intact, coupled mitochondria isolated from both brain and liver. State 4 respiration did not change in the thiamine-deficient animals. Brain state 3 rates fell in thiamine-deficient animals when pyruvate/malate, alpha-ketoglutarate, or glutamate were used as substrate. Liver state 3 rates were depressed only when pyruvate/malate was substrate. Activities of brain and liver pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were depressed in the thiamine-deficient group. We conclude that the mitochondrial abnormalities resulting from thiamine deficiency are secondary to depression of thiamine-mediated enzyme activity, rather than from a putative role of thiamine in chemiosmotic coupling, and that the resulting abnormalities in ATP synthesis and perhaps in glutamate catabolism result in the irreversible neurologic defect seen in this disease.

Evidence for a defect in NADH: ubiquinone oxidoreductase (complex I) in Huntington's disease.

We evaluated electron transport chain activity in platelet mitochondria taken from HD patients. All 5 patients studied had striking depressions of NADH:ubiquinone oxidoreductase activity (complex I) (5.36 +/- 2.91 nmol/min/mg; control mean, 19.12 +/- 5.64 nmol/min/mg). Other electron transport chain activities were not significantly different from control values. HD may be caused by a mutation in 1 of the nuclear coded subunits of NADH:ubiquinone oxidoreductase.

Electron transport chain defects in Alzheimer's disease brain.

Previous work suggested a deficiency in the terminal complex of the mitochondrial electron transport chain, cytochrome c oxidase (COX), in platelet mitochondria of Alzheimer's disease (AD) patients. The present study extends this observation to AD brain mitochondria through assay of electron transport chain activities in mitochondria isolated from autopsied brain samples from AD patients (n = 9) and from controls with and without known neurologic disease (n = 8). AD brain mitochondria demonstrated a generalized depression of activity of all electron transport chain complexes. This depression was most marked in COX activity (p < 0.001). Concentrations of cytochromes b, c1, and aa3 were similar in AD and controls. The electron transport chain is defective in AD brain, and the defect centers about COX.

Association of the mitochondrial 8344 MERRF mutation with maternally inherited spinocerebellar degeneration and Leigh disease.

We report previously undescribed or atypical clinical and biochemical manifestations of the mitochondrial DNA MERRF mutation at nucleotide 8344 in members of a multigenerational family with maternally inherited, highly variable neurodegenerative disorder. The more profound neurologic abnormalities include Leigh disease, spinocerebellar degeneration, and atypical Charcot-Marie-Tooth disease.

Cybrids in Alzheimer's disease: a cellular model of the disease?

The mitochondrial electron transport chain enzyme cytochrome c oxidase (COX) is defective in patients with sporadic Alzheimer's disease (AD). This defect arises from the mutation of mitochondrial DNA (mtDNA). To develop a tissue culture system that would express this genetically derived bioenergetic lesion and permit characterization of its functional consequences, we depleted Ntera2/D1 (NT2) teratocarcinoma cells of endogenous mtDNA and repopulated them with platelet mtDNA from AD patients. Cytochrome c oxidase activity was depressed in the resulting AD cytoplasmic hybrids (cybrids) compared with cybrids prepared with mtDNA from non-AD controls. Reactive oxygen species (ROS) production and free radical scavenging enzyme activities were significantly elevated in AD cybrids. A COX defect in NT2 AD cybrid lines indicates that AD patients possess mtDNA COX gene mutations that are sufficient for determining this biochemical lesion. Expression of unique functional characteristics (increased ROS production and free radical scavenging enzyme activities) relevant to neurodegeneration demonstrates the utility of these cells in defining AD pathophysiology at a cellular level. This in vitro tissue culture model of AD may prove useful in drug screening.

Reduced platelet cytochrome c oxidase activity in Alzheimer's disease.

We evaluated a simplified method for preparation and analysis of platelet cytochrome c oxidase activity in Alzheimer's disease (AD) and control patients. Mean cytochrome c oxidase activity in controls (n = 17) was 0.233 sec-1/mg whereas mean cytochrome c oxidase activity in Alzheimer patients (n = 19) was 0.193 sec-1/mg, p = 0.033. Complex III (ubiquinol:cytochrome c oxidoreductase), complex II (succinic dehydrogenase), and citrate synthase were all assayed as internal controls and were not significantly different in controls and Alzheimer patients. There is a relatively specific loss of platelet cytochrome c oxidase activity in Alzheimer disease patients.

Cytochrome oxidase deficiency in Alzheimer's disease.

Alzheimer's disease (AD) is a degenerative neurologic disorder that may be familial but is usually sporadic and not easily analyzable in terms of conventional Mendelian genetics. The mitochondrial electron transport chain contains 13 proteins that are encoded by mitochondrial genes rather than nuclear (chromosomal) genes. Disorders resulting from heteroplasmic mutations of mitochondrial genes may appear to be sporadic rather than familial. We evaluated electron transport chain activity in platelet mitochondria prepared from patients with AD and found a specific defect in cytochrome oxidase in five of six patients studied. The mitochondrial genome may play a role in the pathogenesis of AD.

Use of cytoplasmic hybrid cell lines for elucidating the role of mitochondrial dysfunction in Alzheimer's disease and Parkinson's disease.

There is substantial evidence of mitochondrial defects in neurodegenerative disorders such as Alzheimer's and Parkinson's diseases (AD and PD). We have probed the molecular implications of mitochondrial dysfunction in these diseases by transferring mitochondria from platelets obtained from disease and control donors into mitochondrial DNA-depleted recipient neuron-based cells (rho 0 cells). This process creates cytoplasmic hybrid (cybrid) cells where the mitochondrial DNA (mtDNA) from the donor is expressed in the nuclear and cellular background of the host rho 0 cell. Differences in phenotype between disease and control groups can thus be attributed to the exogenous mitochondria and mtDNA. Key methodological issues relating to this approach were addressed by demonstrating that recipient rho 0 cells have < 1 mtDNA copy/cell, and that exclusive repopulation with donor mtDNA occurs in cybrid cells. Further, we describe that sampling of heterogeneous cell populations is a valid approach for cybrid analysis. Our studies show that the focal respiratory chain defects reported in platelets of AD and PD cybrids can be recapitulated in AD and PD cybrids. In addition, both AD and PD cybrids display increased oxidative stress and perturbations in calcium homeostasis. These data suggest that the transfer of a mtDNA defect from disease donor platelets is the likely cause of the cybrid biochemical phenotype, and highlight the potential value of these cell lines as cellular disease models.

NMDA receptors increase OH radicals in vivo by using nitric oxide synthase and protein kinase C.

Prolonged activation of brain N-methyl-D-aspartic acid (NMDA) receptors increases intraneuronal (Ca2+) and nitric oxide (NO) synthesis, and may be responsible for neuronal death in acute brain insults and chronic neurodegenerative diseases. NO can be converted in vitro to toxic hydroxyl (OH) radical. Using microdialysis of striatum in awake animals, we found that local NMDA receptor activation increased outflow of OH radicals four-fold. NMDA-stimulated OH production was blocked by inhibitors of nitric oxide synthase (NOS) and protein kinase C (PKC). NMDA receptor-mediated neuronal death may derive from NOS- and PKC-dependent synthesis of OH radicals.