Immunochemical Characterization of the Deficiency of the α-Ketoglutarate Dehydrogenase Complex in Thiamine-Deficient Rat Brain

Abstract: Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. The metabolic encephalopathy caused by thiamine deficiency (TD) is a classic example in which an impairment of cerebral oxidative metabolism leads to selective cell death. In experimental TD in rodents, a reduction in the activity of the thiamine diphosphate-dependent, mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) occurs before the onset of pathologic lesions and is among the earliest biochemical deficits found. To understand the molecular basis and the significance of the deficiency of KGDHC in TD-induced brain damage, the enzyme activity and protein levels of KGDHC were analyzed. The effect of TD on the subregional/cellular distribution of KGDHC and the anatomic relation of KGDHC with selective cell death were also tested by immunocytochemistry. Consistent with several previous studies, TD dramatically reduced KGDHC activity in both anatomically damaged (thalamus and inferior colliculus) and spared (cerebral cortex) regions. Immunocytochemistry revealed no apparent correlation of regional KGDHC immunoreactivity or its response to TD with affected regions in TD. The basis of the enzymatic and immunocytochemical behavior of KGDHC was further assessed by quantitative immunoblots, using antibodies specific for each of the three KGDHC components. Despite the marked decrease of KGDHC activity in TD, no reduction of any of the three KGDHC protein levels was found. Thus, TD impairs the efficacy of the KGDHC catalytic machinery, whereas the concentration of protein molecules persists. The generalized decline of KGDHC activity with no apparent anatomic selectivity is consistent with the notion that the compromised mitochondrial oxidation sensitizes the brain cells to various other insults that precipitate the cell death. The current TD model provides a relevant experimental system to understand the molecular basis of many neurodegenerative conditions in which mitochondrial dysfunction and KGDHC deficiency are prominent features.     

Quantitative alpha-ketoglutarate dehydrogenase activity staining in brain sections and in cultured cells

The activity of a key mitochondrial enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in the brains of patients with neurodegenerative diseases such as Alzheimer's disease, as well as in thiamine-deficient (TD) animals. The decreased activity often occurs without a reduction in enzyme protein, which negates the use of immunocytochemistry to study cellular or regional changes in enzyme activity within the brain. To overcome this limitation, an activity staining method using nitroblue tetrazolium was developed. The histochemical activity staining was standardized in cultured cells. The assay was linear with time and was highly specific for KGDHC. The dark-blue reaction product (formazan) formed a pattern that was consistent with mitochondrial localization. Treatment of the cultured cells with both reversible and irreversible inhibitors decreased formazan production, whereas conventional enzyme assays on cell lysates only revealed loss of KGDHC activity with irreversible inhibitors. The activity staining was also linear with time and highly specific for KGDHC activity in mouse brain sections. Staining occurred throughout the brain, and discrete neuronal populations exhibited particularly intense staining. The pattern of staining differed markedly from the distribution of KGDHC protein by immunocytochemistry. Generalized decreases in the intensity of activity staining that occurred in the TD brains compared to controls were comparable with the loss of KGDHC activity by conventional enzyme assay. Thus, the present study introduces a new histochemical method to measure KGDHC activity at the cellular and regional level, which will be useful to determine changes of in situ enzyme activity.     

The alpha-ketoglutarate dehydrogenase complex in neurodegeneration

Altered energy metabolism is characteristic of many neurodegenerative disorders. Reductions in the key mitochondrial enzyme complex, the alpha-ketoglutarate dehydrogenase complex (KGDHC), occur in a number of neurodegenerative disorders including Alzheimer's Disease (AD). The reductions in KGDHC activity may be responsible for the decreases in brain metabolism, which occur in these disorders. KGDHC can be inactivated by several mechanisms, including the actions of free radicals (Reactive Oxygen Species, ROS). Other studies have associated specific forms of one of the genes encoding KGDHC (namely the DLST gene) with AD, Parkinson's disease, as well as other neurodegenerative diseases. Reductions in KGDHC activity can be plausibly linked to several aspects of brain dysfunction and neuropathology in a number of neurodegenerative diseases. Further studies are needed to assess mechanisms underlying the sensitivity of KGDHC to oxidative stress and the relation of KGDHC deficiency to selective vulnerability in neurodegenerative diseases.     

Brain α-Ketoglutarate Dehydrogenase Complex: Kinetic Properties, Regional Distribution, and Effects of Inhibitors

The substrate and cofactor requirements and some kinetic properties of the alpha-ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for alpha-ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg2+ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex greater than cerebellum greater than or equal to midbrain greater than striatum = hippocampus greater than hypothalamus greater than pons and medulla greater than olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2-2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both beta-methylene-D,L-aspartate and D,L-vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 microM, calcium slightly stimulated (by 10-15%) the brain mitochondrial KGDHC. At concentrations above 100 microM, calcium (IC50 = 1 mM) inhibited both brain mitochondrial and purified heart KGDHC. The present results suggest that some of the kinetic properties of the rat brain mitochondrial KGDHC differ from those of the purified bovine heart and rat heart mitochondrial enzyme complexes. They also suggest that the inhibition of KGDHC by ammonia and the consequent effect on the citric acid cycle fluxes may be of pathophysiological and/or pathogenetic importance in hyperammonemia and in diseases (e.g., hepatic encephalopathy, inborn errors of urea metabolism, Reye's syndrome) where hyperammonemia is a consistent feature. Brain accumulation of calcium occurs in a number of pathological conditions. Therefore, it is possible that such a calcium accumulation may have a deleterious effect on KGDHC activity.     

Regionally selective alterations in enzymatic activities and metabolic fluxes during thiamin deficiency

To further elucidate the molecular basis of the selective damage to various brain regions by thiamin deficiency, changes in enzymatic activities were compared to carbohydrate flux through various pathways from vulnerable (mammillary bodies and inferior colliculi) and nonvulnerable (cochlear nuclei) regions after 11 or 14 days of pyrithiamin-induced thiamin deficiency. After 11 days,large decreases (–43 to –59%) in transketolase (TK) occurred in all 3 regions; 2-ketoglutarate dehydrogenase (KGDHC) declined (–45%), but only in mammillary bodies; pyruvate dehydrogenase (PDHC) was unaffected. By day 14, TK remained reduced by 58%–66%; KGDHC was now reduced in all regions (–48 to –55%); PDHC was also reduced (–32%), but only in the mammillary bodies. Thus, the enzyme changes did not parallel the pathological vulnerability of these regions to thiamin deficiency.¹⁴CO₂ production from¹⁴C-glucose labeled in various positions was utilized to assess metabolic flux. After 14 days, CO₂ production in the vulnerable regions declined severely (–46 to 70%) and approximately twice as much as those in the cochlear nucleus. Also by day 14, the ratio of enzymatic activity to metabolic flux increased as much as 56% in the vulnerable regions, but decreased 18 to 30% in the cochlear nuclei. These differences reflect a greater decrease in flux than enzyme activities in the two vulnerable regions. Thus, selective cellular responses to thiamin deficiency can be demonstrated ex vivo, and these changes can be directly related to alterations in metabolic flux. Since they cannot be related to enzymatic alterations in the three regions, factors other than decreases in the activity of these TPP-dependent enzymes must underlie selective vulnerability in this model of thiamin deficiency.Abbreviations KGDHC 2-ketoglutarate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.61, EC 1.6.4.3. - PDHC pyruvate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.12, EC 1.6.4.3 - TK transketolase (EC 2.2.1.1) - TPP thiamin pyrophosphate     

Metabolic impairment elicits brain cell type-selective changes in oxidative stress and cell death in culture

Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases. Thiamine deficiency (TD) is an animal model in which chronic oxidative stress and inflammation lead to selective neuronal death, whereas other cell types show an inflammatory response. Therefore, the current studies determined the response of different brain cell types to TD and/or inflammation in vitro and tested whether their responses reflect inherent properties of the cells. The cells that have been implicated in TD-induced neurotoxicity, including neurons, microglia, astrocytes, and brain endothelial cells, as well as neuroblastoma and BV-2 microglial cell lines, were cultured in either thiamine-depleted media or in normal culture media with amprolium, a thiamine transport inhibitor. The activity levels of a key mitochondrial enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), were uniquely distributed among different cell types: The highest activity was in the endothelial cells, and the lowest was in primary microglia and neurons. The unique distribution of the activity did not account for the selective response to TD. TD slightly inhibited general cellular dehydrogenases in all cell types, whereas it significantly reduced the activity of KGDHC exclusively in primary neurons and neuroblastoma cells. Among the cell types tested, only in neurons did TD induce apoptosis and cause the accumulation of 4-hydroxy-2-nonenal, a lipid peroxidation product. On the other hand, chronic lipopolysaccharide-induced inflammation significantly inhibited cellular dehydrogenase and KGDHC activities in microglia and astrocytes but not in neurons or endothelial cells. The results demonstrate that the selective cell changes during TD in vivo reflect inherent properties of the different brain cell types.     

Inhibition of the alpha-ketoglutarate dehydrogenase complex by the myeloperoxidase products, hypochlorous acid and mono-N-chloramine

Abstract alpha-Ketoglutarate dehydrogenase (KGDHC) complex activity is diminished in a number of neurodegenerative disorders and its diminution in Alzheimer Disease (AD) is thought to contribute to the major loss of cerebral energy metabolism that accompanies this disease. The loss of KGDHC activity appears to be predominantly due to post-translation modifications. Thiamine deficiency also results in decreased KGDHC activity and a selective neuronal loss. Recently, myeloperoxidase has been identified in the activated microglia of brains from AD patients and thiamine-deficient animals. Myeloperoxidase produces a powerful oxidant, hypochlorous acid that reacts with amines to form chloramines. The aim of this study was to investigate the ability of hypochlorous acid and chloramines to inhibit the activity of KGDHC activity as a first step towards investigating the role of myeloperoxidase in AD. Hypochlorous acid and mono-N-chloramine both inhibited purified and cellular KGDHC and the order of inhibition of the purified complex was hypochlorous acid (1x) > mono-N-chloramine (approximately 50x) > hydrogen peroxide (approximately 1,500). The inhibition of cellular KGDHC occurred with no significant loss of cellular viability at all exposure times that were examined. Thus, hypochlorous acid and chloramines have the potential to inactivate a major target in neurodegeneration.     

Inactivation and reactivation of the mitochondrial α-ketoglutarate dehydrogenase complex

Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.     

Mice deficient in dihydrolipoamide dehydrogenase show increased vulnerability to MPTP, malonate and 3-nitropropionic acid neurotoxicity

Altered energy metabolism, including reductions in activities of the key mitochondrial enzymes alpha-ketoglutarate dehydrogenase complex (KGDHC) and pyruvate dehydrogenase complex (PDHC), are characteristic of many neurodegenerative disorders including Alzheimer's Disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Dihydrolipoamide dehydrogenase is a critical subunit of KGDHC and PDHC. We tested whether mice that are deficient in dihydrolipoamide dehydrogenase (Dld+/-) show increased vulnerability to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), malonate and 3-nitropropionic acid (3-NP), which have been proposed for use in models of PD and HD. Administration of MPTP resulted in significantly greater depletion of tyrosine hydroxylase-positive neurons in the substantia nigra of Dld+/- mice than that seen in wild-type littermate controls. Striatal lesion volumes produced by malonate and 3-NP were significantly increased in Dld+/- mice. Studies of isolated brain mitochondria treated with 3-NP showed that both succinate-supported respiration and membrane potential were suppressed to a greater extent in Dld+/- mice. KGDHC activity was also found to be reduced in putamen from patients with HD. These findings provide further evidence that mitochondrial defects may contribute to the pathogenesis of neurodegenerative diseases.     

Metabolic impairment induces oxidative stress, compromises inflammatory responses, and inactivates a key mitochondrial enzyme in microglia

Microglial activation, oxidative stress, and dysfunctions in mitochondria, including the reduction of cytochrome oxidase activity, have been implicated in neurodegeneration. The current experiments tested the effects of reducing cytochrome oxidase activity on the ability of microglia to respond to inflammatory insults. Inhibition of cytochrome oxidase by azide reduced oxygen consumption and increased reactive oxygen species (ROS) production but did not affect cell viability. Azide also attenuated microglial activation, as measured by nitric oxide (NO.) production in response to lipopolysaccharide (LPS). It is surprising that the inhibition of cytochrome oxidase also diminished the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a Krebs cycle enzyme. This reduction was exaggerated when the azide-treated microglia were also treated with LPS. The combination of the azide-stimulated ROS and LPS-induced NO. would likely cause peroxynitrite formation in microglia. Thus, the possibility that KGDHC was inactivated by peroxynitrite was tested. Peroxynitrite inhibited the activity of isolated KGDHC, nitrated tyrosine residues of all three KGDHC subunits, and reduced immunoreactivity to antibodies against two KGDHC components. Thus, our data suggest that inhibition of the mitochondrial respiratory chain diminishes aerobic energy metabolism, interferes with microglial inflammatory responses, and compromises mitochondrial function, including KGDHC activity, which is vulnerable to NO. and peroxynitrite that result from microglial activation. Thus, activation of metabolically compromised microglia can further diminish their oxidative capacity, creating a deleterious spiral that may contribute to neurodegeneration.     

The α-ketoglutarate-dehydrogenase complex

Damage from oxidative stress and mitochondrial dysfunction occur together in many common neurodegenerative diseases. The enzymes that form the mitochondrial alpha-ketoglutarate- dehydrogenase complex (KGDHC), a key and arguably rate-limiting enzyme system of the tricarboxylic acid cycle, might mediate the interaction of these processes. KGDHC activity is reduced in numerous age-related neurodegenerative diseases and is diminished by oxidative stress. In Alzheimer's disease (AD), the reduction correlates highly to diminished mental performance. Thus, research has focused on the mechanisms by which select oxidants reduce KGDHC and the consequences of such a reduction. Diminished KGDHC in cells is associated with apoptosis without changes in the mitochondrial membrane potential. Studies of isolated mitochondria and of animal models suggest that a reduction in KGDHC can predispose to damage by other toxins that promote neurodegeneration. Diminished oxidative metabolism can be plausibly linked to pathological features of neurodegenerative diseases (e.g., reduced mental function, the plaques and tangles in AD). Thus, reductions in KGDHC might be central to the pathophysiology of these diseases. Studies of proteins, cells, animal models, and humans suggest that treatments to diminish, or bypass, the reduction in KGDHC might be beneficial in age-related neurodegenerative disorders.     

Inhibition of the alpha-ketoglutarate dehydrogenase complex alters mitochondrial function and cellular calcium regulation

Mitochondrial dysfunction occurs in many neurodegenerative diseases. The alpha-ketoglutarate dehydrogenase complex (KGDHC) catalyzes a key and arguably rate-limiting step of the tricarboxylic acid cycle (TCA). A reduction in the activity of the KGDHC occurs in brains and cells of patients with many of these disorders and may underlie the abnormal mitochondrial function. Abnormalities in calcium homeostasis also occur in fibroblasts from Alzheimer's disease (AD) patients and in cells bearing mutations that lead to AD. Thus, the present studies test whether the reduction of KGDHC activity can lead to the alterations in mitochondrial function and calcium homeostasis. alpha-Keto-beta-methyl-n-valeric acid (KMV) inhibits KGDHC activity in living N2a cells in a dose- and time-dependent manner. Surprisingly, concentration of KMV that inhibit in situ KGDHC by 80% does not alter the mitochondrial membrane potential (MMP). However, similar concentrations of KMV induce the release of cytochrome c from mitochondria into the cytosol, reduce basal [Ca(2+)](i) by 23% (P<0.005), and diminish the bradykinin (BK)-induced calcium release from the endoplasmic reticulum (ER) by 46% (P<0.005). This result suggests that diminished KGDHC activities do not lead to the Ca(2+) abnormalities in fibroblasts from AD patients or cells bearing PS-1 mutations. The increased release of cytochrome c with diminished KGDHC activities will be expected to activate other pathways including cell death cascades. Reductions in this key mitochondrial enzyme will likely make the cells more vulnerable to metabolic insults that promote cell death.     

Novel functions of the alpha-ketoglutarate dehydrogenase complex may mediate diverse oxidant-induced changes in mitochondrial enzymes associated with Alzheimer's disease

Measures in autopsied brains from Alzheimer's Disease (AD) patients reveal a decrease in the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity. The present experiments tested whether both changes could be caused by the common oxidant H(2)O(2) and to probe the mechanism underlying these changes. Since the response to H(2)O(2) is modified by the level of the E2k subunit of KGDHC, the interaction of MDH and KGDHC was studied in cells with varying levels of E2k. In cells with only 23% of normal E2k protein levels, one-hour treatment with H(2)O(2) decreased KGDHC and increased MDH activity as well as the mRNA level for both cytosolic and mitochondrial MDH. The increase in MDH did not occur in cells with 100% or 46% of normal E2k. Longer treatments with H(2)O(2) inhibited the activity of both enzymes. Glutathione is a major regulator of cellular redox state and can modify enzyme activities. H(2)O(2) converts reduced glutathione (GSH) to oxidized glutathione (GSSG), which reacts with protein thiols. Treatment of purified KGDHC with GSSG leads to glutathionylation of all three KGDHC subunits. Thus, cellular glutathione level was manipulated by two means to determine the effect on KGDHC and MDH activities. Both buthionine sulfoximine (BSO), which inhibits glutathione synthesis without altering redox state, and H(2)O(2) diminished glutathione to a similar level after 24 h. However, H(2)O(2), but not BSO, reduced KGDHC and MDH activities, and the reduction was greater in the E2k-23 line. These findings suggest that the E2k may mediate diverse responses of KGDHC and MDH to oxidants. In addition, the differential response of activities to BSO and H(2)O(2) together with the in vitro interaction of KGDHC with GSSG suggests that glutathionylation is one possible mechanism underlying oxidative stress-induced inhibition of the TCA cycle enzymes.     

PINK1 kinase dysfunction triggers neurodegeneration in the primate brain without impacting mitochondrial homeostasis

In vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson's disease(PD).However,difficulty in detecting endogenous PINK1 protein in rodent brains and cell lines has prevented the rigorous investigation of the in vivo role of PINK1.Here we report that PINK1 kinase form is selectively expressed in the human and monkey brains.CRISPR/Cas9-mediated deficiency of PINK1 causes similar neurodegeneration in the brains of fetal and adult monkeys as well as cultured monkey neurons without affecting mitochondrial protein expression and morphology.Importantly,PINK1 mutations in the primate brain and human cells reduce protein phosphorylation that is important for neuronal function and survival.Our findings suggest that PINK1 kinase activity rather than its mitochondrial function is essential for the neuronal survival in the primate brains and that its kinase dysfunction could be involved in the pathogenesis of PD.     

Mitochondrial involvement in Parkinson's disease, Huntington's disease, hereditary spastic paraplegia and Friedreich's ataxia

Respiratory chain dysfunction has been identified in several neurodegenerative disorders. In Friedreich's ataxia (FA) and Huntington's disease (HD), where the respective mutations are in nuclear genes encoding non-respiratory chain mitochondrial proteins, the defects in oxidative phosphorylation are clearly secondary. In Parkinson's disease (PD) the situation is less clear, with some evidence for a primary role of mitochondrial DNA in at least a proportion of patients. The pattern of the respiratory chain defect may provide some clue to its cause; in PD there appears to be a selective complex I deficiency; in HD and FA the deficiencies are most severe in complex II/III with a less severe defect in complex IV. Aconitase activity in HD and FA is severely decreased in brain and muscle, respectively, but appears to be normal in PD brain. Free radical generation is thought to be of importance in both HD and FA, via excitotoxicity in HD and abnormal iron handling in FA. The oxidative damage observed in PD may be secondary to the mitochondrial defect. Whatever the cause(s) and sequence of events, respiratory chain deficiencies appear to play an important role in the pathogenesis of neurodegeneration. The mitochondrial abnormalities induced may converge on the function of the mitochondrion in apoptosis. This mode of cell death is thought to play an important role in neurodegenerative diseases and it is tempting to speculate that the observed mitochondrial defects in PD, HD and FA result directly in apoptotic cell death, or in the lowering of a cell's threshold to undergo apoptosis. Clarifying the role of mitochondria in pathogenesis may provide opportunities for the development of treatments designed to reverse or prevent neurodegeneration.     

Tricarboxylic acid cycle enzymes following thiamine deficiency

Thiamine (Vitamin B1) deficiency (TD) leads to memory deficits and neurological disease in animals and humans. The thiamine-dependent enzymes of the tricarboxylic acid (TCA) cycle are reduced following TD and in the brains of patients that died from multiple neurodegenerative diseases. Whether reductions in thiamine or thiamine-dependent enzymes leads to changes in all TCA cycle enzymes has never been tested. In the current studies, the pyruvate dehydrogenase complex (PDHC) and all of enzymes of the TCA cycle were measured in the brains of TD mice. Non-thiamine-dependent enzymes such as succinate dehydrogenase (SDH), succinate thiokinase (STH) and malate dehydrogenase (MDH) were altered as much or more than thiamine-dependent enzymes such as the alpha-ketoglutarate dehydrogenase complex (KGDHC) (-21.5%) and PDHC (-10.5%). Succinate dehydrogenase (SDH) activity decreased by 27% and succinate thiokinase (STH) decreased by 24%. The reductions in these other enzymes may result from oxidative stress because of TD or because these other enzymes of the TCA cycle are part of a metabolon that respond as a group of enzymes. The results suggest that other TCA cycle enzymes should be measured in brains from patients that died from neurological disease in which thiamine-dependent enzymes are known to be reduced. The diminished activities of multiple TCA cycle enzymes may be important in our understanding of how metabolic lesions alter brain function in neurodegenerative disorders.     

Aberrant BCAA and glutamate metabolism linked to regional neurodegeneration in a mouse model of Leigh syndrome

The dysfunction of respiratory chain complex I (CI) is the most common form of mitochondrial disease that most often presents as Leigh syndrome (LS) in children — a severe neurometabolic disorder defined by progressive focal lesions in specific brain regions. The mechanisms underlying this region-specific vulnerability to CI deficiency, however, remain elusive. Here, we examined brain regional respiratory chain enzyme activities and metabolic profiles in a mouse model of LS with global CI deficiency to gain insight into regional vulnerability to neurodegeneration. One lesion-resistant and three lesion-prone brain regions were investigated in Ndufs4 knockout (KO) mice at the late stage of LS. Enzyme assays confirmed significantly decreased (60–80%) CI activity in all investigated KO brain regions, with the lesion-resistant region displaying the highest residual CI activity (38% of wild type). A higher residual CI activity, and a less perturbed NADH/NAD⁺ ratio, correlate with less severe metabolic perturbations in KO brain regions. Moreover, less perturbed BCAA oxidation and increased glutamate oxidation seem to distinguish lesion-resistant from -prone KO brain regions, thereby identifying key areas of metabolism to target in future therapeutic intervention studies.     

Mitochondrial Enzymes and Endoplasmic Reticulum Calcium Stores as Targets of Oxidative Stress in Neurodegenerative Diseases

Considerable evidence indicates that oxidative stress accompanies age-related neurodegenerative diseases. Specific mechanisms by which oxidative stress leads to neurodegeneration are unknown. Two targets of oxidative stress that are known to change in neurodegenerative diseases are the mitochondrial enzyme alpha-ketoglutarate dehydrogenase complex (KGDHC) and endoplasmic reticulum calcium stores. KGDHC activities are diminished in all common neurodegenerative diseases and the changes are particularly well documented in Alzheimer's disease (AD). A second change that occurs in cells from AD patients is an exaggerated endoplasmic reticulum calcium store [i.e., bombesin-releasable calcium stores (BRCS)]. H(2)O(2), a general oxidant, changes both variables in the same direction as occurs in disease. Other oxidants selectively alter these variables. Various antioxidants were used to help define the critical oxidant species that modifies these responses. All of the antioxidants diminish the oxidant-induced carboxy-dichlorofluorescein (cDCF) detectable reactive oxygen species (ROS), but have diverse actions on these cellular processes. For example, alpha-keto-beta-methyl-n-valeric acid (KMV) diminishes the H(2)O(2) effects on BRCS, while trolox and DMSO exaggerate the response. Acute trolox treatment does not alter H(2)O(2)-induced changes in KGDHC, whereas chronic treatment with trolox increases KGDHC almost threefold. The results suggest that KGDHC and BRCS provide targets by which oxidative stress may induce neurodegeneration and a useful tool for selecting antioxidants for reversing age-related neurodegeneration.     

Reduction in the E2k subunit of the alpha-ketoglutarate dehydrogenase complex has effects independent of complex activity

The activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC) declines in brains of patients with several neurodegenerative diseases. KGDHC consists of multiple copies of E1k, E2k, and E3. E1k and E2k are unique to KGDHC and may have functions independent of the complex. The present study tested the consequences of different levels of diminished E2k mRNA on protein levels of the subunits, KGDHC activity, and physiological responses. Human embryonic kidney cells were stably transfected with an E2k sense or antisense expression vector. Sense control (E2k-mRNA-100) was compared with two clones in which the mRNA was reduced to 67% of control (E2k-mRNA-67) or to 30% of control (E2k-mRNA-30). The levels of the E2k protein in clones paralleled the reduction in mRNA, and E3 proteins were unaltered. Unexpectedly, the clone with the greatest reduction in E2k protein (E2k-mRNA-30) had a 40% increase in E1k protein. The activity of the complex was only 52% of normal in E2k-mRNA-67 clone, but was near normal (90%) in E2k-mRNA-30 clone. Subsequent experiments tested whether the physiological consequences of a reduction in E2k mRNA correlated more closely to E2k protein or to KGDHC activity. Growth rate, increased DCF-detectable reactive oxygen species, and cell death in response to added oxidant were proportional to E2k proteins, but not complex activity. These results were not predicted because subunits unique to KGDHC have never been manipulated in mammalian cells. These results suggest that in addition to its essential role in metabolism, the E2k component of KGDHC may have other novel roles.     

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.