The α-Ketoglutarate Dehydrogenase Complex as a Hub of Plasticity in Neurodegeneration and Regeneration.

Abnormal glucose metabolism is central to neurodegeneration, and considerable evidence suggests that abnormalities in key enzymes of the tricarboxylic acid (TCA) cycle underlie the metabolic deficits. Significant recent advances in the role of metabolism in cancer provide new insight that facilitates our understanding of the role of metabolism in neurodegeneration. Research indicates that the rate-limiting step of the TCA cycle, the α-ketoglutarate dehydrogenase complex (KGDHC) and its substrate alpha ketoglutarate (KG), serve as a signaling hub that regulates multiple cellular processes: (1) is the rate-limiting step of the TCA cycle, (2) is sensitive to reactive oxygen species (ROS) and produces ROS, (3) determines whether KG is used for energy or synthesis of compounds to support growth, (4) regulates the cellular responses to hypoxia, (5) controls the post-translational modification of hundreds of cell proteins in the mitochondria, cytosol, and nucleus through succinylation, (6) controls critical aspects of transcription, (7) modulates protein signaling within cells, and (8) modulates cellular calcium. The primary focus of this review is to understand how reductions in KGDHC are translated to pathologically important changes that underlie both neurodegeneration and cancer. An understanding of each role is necessary to develop new therapeutic strategies to treat neurodegenerative disease.

Mild metabolic perturbations alter succinylation of mitochondrial proteins.

Succinylation of proteins is widespread, modifies both the charge and size of the molecules, and can alter their function. For example, liver mitochondrial proteins have 1,190 unique succinylation sites representing multiple metabolic pathways. Succinylation is sensitive to both increases and decreases of the NAD+ -dependent desuccinylase, SIRT5. Although the succinyl group for succinylation is derived from metabolism, the effects of systematic variation of metabolism on mitochondrial succinylation are not known. Changes in succinylation of mitochondrial proteins following variations in metabolism were compared against the mitochondrial redox state as estimated by the mitochondrial NAD+ /NADH ratio using fluorescent probes. The ratio was decreased by reduced glycolysis and/or glutathione depletion (iodoacetic acid; 2-deoxyglucose), depressed tricarboxylic acid cycle activity (carboxyethyl ester of succinyl phosphonate), and impairment of electron transport (antimycin) or ATP synthase (oligomycin), while uncouplers of oxidative phosphorylation (carbonyl cyanide m-chlorophenyl hydrazine or tyrphostin) increased the NAD+ /NADH ratio. All of the conditions decreased succinylation. In contrast, reducing the oxygen from 20% to 2.4% increased succinylation. The results demonstrate that succinylation varies with metabolic states, is not correlated to the mitochondrial NAD+ /NADH ratio, and may help coordinate the response to metabolic challenge.

Reductions in the mitochondrial enzyme α-ketoglutarate dehydrogenase complex in neurodegenerative disease - beneficial or detrimental?

Reductions in metabolism and excess oxidative stress are prevalent in multiple neurodegenerative diseases. The activity of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) appears central to these abnormalities. KGDHC is diminished in multiple neurodegenerative diseases. KGDHC can not only be rate limiting for NADH production and for substrate level phosphorylation, but is also a source of reactive oxygen species (ROS). The goal of these studies was to determine how changes in KGDHC modify baseline ROS, the ability to buffer ROS, baseline glutathionylation, calcium modulation and cell death in response to external oxidants. In vivo, reducing KGDHC with adeno virus diminished neurogenesis and increased oxidative stress. In vitro, treatments of short duration increased ROS and glutathionylation and enhanced the ability of the cells to diminish the ROS from added oxidants. However, long-term reductions lessened the ability to diminish ROS, diminished glutathionylation and exaggerated oxidant-induced changes in calcium and cell death. Increasing KGDHC enhanced the ability of the cells to diminish externally added ROS and protected against oxidant-induced changes in calcium and cell death. The results suggest that brief periods of diminished KGDHC are protective, while prolonged reductions are harmful. Furthermore, elevated KGDHC activities are protective. Thus, mitogenic therapies that increase KGDHC may be beneficial in neurodegenerative diseases. Read the Editorial Highlight for this article on Page 689.

Mild mitochondrial metabolic deficits by α-ketoglutarate dehydrogenase inhibition cause prominent changes in intracellular autophagic signaling: Potential role in the pathobiology of Alzheimer's disease.

Brain activities of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) are reduced in Alzheimer's disease and other age-related neurodegenerative disorders. The goal of the present study was to test the consequences of mild impairment of KGDHC on the structure, protein signaling and dynamics (mitophagy, fusion, fission, biogenesis) of the mitochondria. Inhibition of KGDHC reduced its in situ activity by 23-53% in human neuroblastoma SH-SY5Y cells, but neither altered the mitochondrial membrane potential nor the ATP levels at any tested time-points. The attenuated KGDHC activity increased translocation of dynamin-related protein-1 (Drp1) and microtubule-associated protein 1A/1B-light chain 3 (LC3) from the cytosol to the mitochondria, and promoted mitochondrial cytochrome c release. Inhibition of KGDHC also increased the negative surface charges (anionic phospholipids as assessed by Annexin V binding) on the mitochondria. Morphological assessments of the mitochondria revealed increased fission and mitophagy. Taken together, our results suggest the existence of the regulation of the mitochondrial dynamism including fission and fusion by the mitochondrial KGDHC activity via the involvement of the cytosolic and mitochondrial protein signaling molecules. A better understanding of the link among mild impairment of metabolism, induction of mitophagy/autophagy and altered protein signaling will help to identify new mechanisms of neurodegeneration and reveal potential new therapeutic approaches.

The RNA-binding protein HuR is essential for the B cell antibody response.

Post-transcriptional regulation of mRNA by the RNA-binding protein HuR (encoded by Elavl1) is required in B cells for the germinal center reaction and for the production of class-switched antibodies in response to thymus-independent antigens. Transcriptome-wide examination of RNA isoforms and their abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interactions, revealed that HuR-dependent splicing of mRNA affected hundreds of transcripts, including that encoding dihydrolipoamide S-succinyltransferase (Dlst), a subunit of the 2-oxoglutarate dehydrogenase (α-KGDH) complex. In the absence of HuR, defective mitochondrial metabolism resulted in large amounts of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for the proliferation and differentiation of B cells.

Deficits in the mitochondrial enzyme α-ketoglutarate dehydrogenase lead to Alzheimer's disease-like calcium dysregulation.

Understanding the molecular sequence of events that culminate in multiple abnormalities in brains from patients that died with Alzheimer's disease (AD) will help to reveal the mechanisms of the disease and identify upstream events as therapeutic targets. The activity of the mitochondrial α-ketoglutarate dehydrogenase complex (KGDHC) in homogenates from autopsy brain declines with AD. Experimental reductions in KGDHC in mouse models of AD promote plaque and tangle formation, the hallmark pathologies of AD. We hypothesize that deficits in KGDHC also lead to the abnormalities in endoplasmic reticulum (ER) calcium stores and cytosolic calcium following K(+) depolarization that occurs in cells from AD patients and transgenic models of AD. The activity of the mitochondrial enzyme KGDHC was diminished acutely (minutes), long-term (days), or chronically (weeks). Acute inhibition of KGDHC produced effects on calcium opposite to those in AD, while the chronic or long-term inhibition of KGDHC mimicked the AD-related changes in calcium. Divergent changes in proteins released from the mitochondria that affect endoplasmic reticulum calcium channels may underlie the selective cellular consequences of acute versus longer term inhibition of KGDHC. The results suggest that the mitochondrial abnormalities in AD can be upstream of those in calcium.

Mild reduction in the activity of the alpha-ketoglutarate dehydrogenase complex elevates GABA shunt and glycolysis.

Diminished energy metabolism and reduced activity of brain alpha-ketoglutarate dehydrogenase complex (KGDHC) occur in a number of neurodegenerative diseases. The relation between diminished KGDHC activity and altered energy metabolism is unknown. The present study tested whether a reduction in the KGDHC activity would alter cellular metabolism by comparing metabolism of [U-13C]glucose in a human embryonic kidney cell line (E2k100) to one in which the KGDHC activity was about 70% of control (E2k67). After a 2 h incubation of the cells with [U-13C]glucose, the E2k67 cells showed a greater increase in 13C labeling of alanine compared with the E2k100 cells. This suggested an increase in glycolysis. Furthermore, an increase in labeled lactate after 12 h incubation supported the suggestion of an increased glycolysis in the E2k67 cells. Increased GABA shunt in the E2k67 cells was indicated by increased 13C labeling of GABA at both 2 and 12 h compared with the control cells. GABA concentration as determined by HPLC was also increased in the E2k67 cells compared with the control cells. However, the GABA shunt was not sufficient to normalize metabolism in the E2k67 cells compared with control at 2 or 12 h. However, by 24 h metabolism had normalized (i.e. labeling was similar in E2k67 and E2k100). Thus, the data are consistent with an enhanced glycolysis and GABA shunt in response to a mild reduction in KGDHC. Our findings indicate that a mild change in KGDHC activity can lead to large changes in metabolism. The changes may maintain normal energy metabolism but make the cells more vulnerable to perturbations such as occur with oxidants.

Dietary supplementation with resveratrol reduces plaque pathology in a transgenic model of Alzheimer's disease.

Resveratrol, a polyphenol found in red wine, peanuts, soy beans, and pomegranates, possesses a wide range of biological effects. Since resveratrol's properties seem ideal for treating neurodegenerative diseases, its ability to diminish amyloid plaques was tested. Mice were fed clinically feasible dosages of resveratrol for forty-five days. Neither resveratrol nor its conjugated metabolites were detectable in brain. Nevertheless, resveratrol diminished plaque formation in a region specific manner. The largest reductions in the percent area occupied by plaques were observed in medial cortex (-48%), striatum (-89%) and hypothalamus (-90%). The changes occurred without detectable activation of SIRT-1 or alterations in APP processing. However, brain glutathione declined 21% and brain cysteine increased 54%. The increased cysteine and decreased glutathione may be linked to the diminished plaque formation. This study supports the concept that onset of neurodegenerative disease may be delayed or mitigated with use of dietary chemo-preventive agents that protect against beta-amyloid plaque formation and oxidative stress.

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.

Cisplatin-induced toxicity is associated with platinum deposition in mouse kidney mitochondria in vivo and with selective inactivation of the alpha-ketoglutarate dehydrogenase complex in LLC-PK1 cells.

The anticancer drug cisplatin is nephrotoxic and neurotoxic. Previous data support the hypothesis that cisplatin is bioactivated to a nephrotoxicant. The final step in the proposed bioactivation is the formation of a platinum-cysteine S-conjugate followed by a pyridoxal 5'-phosphate (PLP)-dependent cysteine S-conjugate beta-lyase reaction. This reaction would generate pyruvate, ammonium, and a highly reactive platinum (Pt)-thiol compound in vivo that would bind to proteins. In this work, the cellular location and identity of the PLP-dependent cysteine S-conjugate beta-lyase were investigated. Pt was shown to bind to proteins in kidneys of cisplatin-treated mice. The concentration of Pt-bound proteins was higher in the mitochondrial fraction than in the cytosolic fraction. Treatment of the mice with aminooxyacetic acid (AOAA, a PLP enzyme inhibitor), which had previously been shown to block the nephrotoxicity of cisplatin, decreased the binding of Pt to mitochondrial proteins but had no effect on the amount of Pt bound to proteins in the cytosolic fraction. These data indicate that a mitochondrial enzyme catalyzes the PLP-dependent cysteine S-conjugate beta-lyase reaction. PLP-dependent mitochondrial aspartate aminotransferase (mitAspAT) is a mitochondrial enzyme that catalyzes beta-elimination reactions with cysteine S-conjugates of halogenated alkenes. We reasoned that the enzyme might also catalyze a beta-lyase reaction with the cisplatin-cysteine S-conjugate. In this study, mitAspAT was stably overexpressed in LLC-PK(1) cells. Cisplatin was significantly more toxic in confluent monolayers of LLC-PK(1) cells that overexpressed mitAspAT than in control cells containing vector alone. AOAA completely blocked the cisplatin toxicity in confluent mitAspAT-transfected cells. The Pt-thiol compound could rapidly bind proteins and inactivate enzymes in close proximity of the PLP-dependent cysteine S-conjugate beta-lyase. Treatment with 50 or 100 microM cisplatin for 3 h, followed by removal of cisplatin from the medium for 24 h, resulted in a pronounced loss of alpha-ketoglutarate dehydrogenase complex (KGDHC) activity in both mitAspAT-transfected cells and control cells. Exposure to 100 microM cisplatin resulted in a significantly greater loss of KGDHC activity in the cells overexpressing mitAspAT than in control cells. Aconitase activity was diminished in both cell types, but only at the higher level of exposure to cisplatin. AspAT activity was also significantly decreased by cisplatin treatment. By contrast, several other enzymes (both cytosolic and mitochondrial) involved in energy/amino acid metabolism were not significantly affected by cisplatin treatment in the LLC-PK(1) cells, whether or not mitAspAT was overexpressed. The susceptibility of KGDHC and aconitase to inactivation in kidney cells exposed to cisplatin metabolites may be due to the proximity of mitAspAT to KGDHC and aconitase in mitochondria. These findings support the hypothesis that a mitochondrial cysteine S-conjugate beta-lyase converts the cisplatin-cysteine S-conjugate to a toxicant, and the data are consistent with the hypothesis that mitAspAT plays a role in the bioactivation of cisplatin.

Mitochondrial aconitase is a transglutaminase 2 substrate: transglutamination is a probable mechanism contributing to high-molecular-weight aggregates of aconitase and loss of aconitase activity in Huntington disease brain.

Transglutaminase activity was found to be present in highly purified non-synaptosomal rat brain mitochondria. A 78-kDa protein in these organelles was shown to be a transglutaminase 2 substrate, and incubation of a non-synaptosomal mitochondrial lysate with transglutaminase 2 yielded high-Mr proteins. The 78-kDa protein was identified as mitochondrial aconitase by MALDI-TOF analysis. Aconitase activity was decreased in a dose-dependent manner when non-synaptosomal rat brain mitochondria were incubated with transglutaminase 2. Transglutaminase activity is increased about 2-fold in the mitochondrial fraction of HD caudate. Moreover, Western blotting of the mitochondrial fraction revealed that most of the mitochondrial aconitase in HD caudate is present as high-Mr aggregates. Aconitase activity was previously shown to be decreased in Huntington disease (HD) caudate (a region severely damaged by the disease). The present findings suggest that an increase of transglutaminase activity in HD caudate may contribute to mitochondrial dysfunction by incorporating aconitase into inactive polymers.

CD40-CD40L interactions promote neuronal death in a model of neurodegeneration due to mild impairment of oxidative metabolism.

Abnormalities in oxidative processes, region-selective neuron loss, inflammation and diminished activity of thiamine-dependent enzymes characterize age-related neurodegenerative diseases. Thiamine deficiency (TD) models the selective neurodegeneration that accompanies mild impairment of oxidative metabolism. As in human neurodegenerative diseases, alterations in multiple cell types accompany the TD-induced neurodegeneration. The current studies demonstrate that CD40 and CD40 ligand (CD40L), two co-stimulatory immune molecules, are involved in TD-induced selective neuronal death. TD induced CD40 immunoreactivity in microglia and CD40L immunoreactivity in astrocytes. Both CD40-positive microglia and CD40L-positive astrocytes increased during the progressive TD-induced neuronal death. In early stages of TD, targeted deletion of CD40 diminished the number of CD40L-positive astrocytes and reduced neuronal death by 35%. The number of CD40L-positive astrocytes increased whenever the number of NeuN-positive neurons decreased. In early stages of TD, deletion of CD40L diminished CD40-positive microglia and reduced the neuronal death by 64%. In advanced phases of TD, neither CD40 nor CD40L deletion protected against neuronal death. The data show for the first time that TD induces expression of CD40 by the microglia and CD40L by astrocytes. The results indicate that CD40-CD40L interactions promote neuronal death in early stages of TD, but that at later phases the protective effects of the diminished CD40 or CD40L are over-ridden by other mechanisms.

CD40L deletion delays neuronal death in a model of neurodegeneration due to mild impairment of oxidative metabolism.

Inflammatory/immune processes are important in the pathogenesis of neurodegenerative diseases. Thiamine deficiency (TD) models the region selective neuronal loss in brain that accompanies mild impairment of oxidative metabolism. TD induces well-defined alterations in neurons, microglia, astrocytes, and endothelial cells. To test the role of inflammatory/immune mechanisms in TD-induced neurodegeneration, the temporal profile of neurodegeneration was compared to the activation of CD68-positive microglia and ICAM-1-positive endothelial cells during TD in wild type mice and in CD40L-/- mice. CD40L-/- delayed the onset of TD-induced neuronal death as well as the activation of microglia and endothelial cells. The current results suggest that CD40L-mediated immune and inflammatory responses have a role in TD-induced neuronal death.

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.

Mitochondrial heterogeneity within and between different cell types.

Mitochondrial membrane potentials (MMP) reflect the functional status of mitochondria within cells. Our recently published method provides a semiquantitative estimate of the MMP of populations of mitochondrial-like particles within living cells at 37 degrees C using a combination of conventional fluorescence microscopy and three-dimensional deconvolution by exhaustive photon reassignment. The current studies demonstrate variations in the mean MMP among six different cell types (i.e., human skin fibroblasts, naive and differentiated PC12 cells, SH-SY5Y cells, dopaminergic cells, and primary cultured neurons) and MMP in different parts of the same cells (i.e., growth cones vs. cell bodies). The largest MMP was in nontransformed fibroblasts (mean MMP was -112 +/- 2 mV), while the lowest was in transformed neuroblastoma SH-SY5Y cells (-87 +/- 2 mV). This method revealed large variations in mean MMP among cells of the same type within a single culture dish. The percent area of the cell occupied by mitochondrial-like particles differed among different cell types, and ranged from 4% in SH-SY5Y to 24% in differentiated PC12 cells. The data can also be analyzed by calculating the sum potential of all of the pixels in a cell. The sum MMP per cell revealed a large range between cell types from -2238 +/- 355 mV/microm2 in SH-Y5Y to -15445 +/- 1039 mV/microm2 in PC12 cells. Although biological implications of heterogeneity of MMP are not clear, this approach provides a tool to address this question.

Inhibition of alpha-ketoglutarate dehydrogenase complex promotes cytochrome c release from mitochondria, caspase-3 activation, and necrotic cell death.

Mitochondrial dysfunction has been implicated in cell death in many neurodegenerative diseases. Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a key and arguably rate-limiting enzyme of the Krebs cycle, occurs in these disorders and may underlie decreased brain metabolism. The present studies used alpha-keto-beta-methyl-n-valeric acid (KMV), a structural analogue of alpha-ketoglutarate, to inhibit KGDHC activity to test effects of reduced KGDHC on mitochondrial function and cell death cascades in PC12 cells. KMV decreased in situ KGDHC activity by 52 +/- 7% (1 hr) or 65 +/- 4% (2 hr). Under the same conditions, KMV did not alter the mitochondrial membrane potential (MMP), as assessed with a method that detects changes as small as 5%. KMV also did not alter production of reactive oxygen species (ROS). However, KMV increased lactate dehydrogenase (LDH) release from cells by 100 +/- 4.7%, promoted translocation of mitochondrial cytochrome c to the cytosol, and activated caspase-3. Inhibition of the mitochondrial permeability transition pore (MPTP) by cyclosporin A (CsA) partially blocked this KMV-induced change in cytochrome c (-40%) and LDH (-15%) release, and prevented necrotic cell death. Thus, impairment of this key mitochondrial enzyme in PC12 cells may lead to cytochrome c release and caspase-3 activation by partial opening of the MPTP before the loss of mitochondrial membrane potentials.

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.

The role of the metabolic lesion in Alzheimer's disease.

This paper discusses the hypothesis that the cerebrometabolic deficiency in Alzheimer's disease(AD) is the proximate cause of the clinical disability. Several sets of observations support this hypothesis. (1) Impaired brain metabolism essentially always occurs in clinically significant AD, and the degree of clinical disability is proportional to the degree of metabolic impairment. The earliest, mildest changes in brain metabolism occur even before the onset of measurable cognitive impairment or atrophy. This observation disproves the now outdated assumption that the decreased metabolism is simply a consequence of decreased mental function or of atrophy. One of the important mechanisms reducing brain metabolism in AD appears to be damage to key mitochondrial components. Another appears to relate to inappropriate responses to insulin, i.e. to diabetes of the brain. (2) Inducing impairments of brain metabolism causes changes in mentation that mimic the clinical disabilities in AD, in both humans and experimental animals. (3) Preliminary results from several units suggest that treatment directed at the impairment of brain metabolism can improve neuropsychological functions in AD patients. The hypothesis presented here in no way negates the importance of other mechanisms in AD, such as amyloid accumulation, vascular compromise, and free radical action. However, those other abnormalities including amyloidosis can occur in people whose mentation is still clinically unimpaired. In contrast, once significant decrease in the rate of brain metabolism occurs, mentation becomes defective.

Oxidative stress increases internal calcium stores and reduces a key mitochondrial enzyme.

Fibroblasts from patients with genetic and non-genetic forms of Alzheimer's disease (AD) show many abnormalities including increased bombesin-releasable calcium stores (BRCS), diminished activities of the mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC), and an altered ability to handle oxidative stress. The link between genetic mutations (and the unknown primary event in non-genetic forms) and these other cellular abnormalities is unknown. To determine whether oxidative stress could be a convergence point that produces the other AD-related changes, these experiments tested in fibroblasts the effects of H(2)O(2), in the presence or absence of select antioxidants, on BRCS and KGDHC. H(2)O(2) concentrations that elevated carboxy-dichlorofluorescein (c-H(2)DCF)-detectable ROS increased BRCS and decreased KGDHC activity. These changes are in the same direction as those in fibroblasts from AD patients. Acute treatments with the antioxidants Trolox, or DMSO decreased c-H(2)DCF-detectable ROS by about 90%, but exaggerated the H(2)O(2)-induced increases in BRCS by about 4-fold and did not alter the reduction in KGDHC. Chronic pretreatments with Trolox more than doubled the BRCS, tripled KGDHC activities, and reduced the effects of H(2)O(2). Pretreatment with DMSO or N-acetyl cysteine diminished the BRCS and either had no effect, or exaggerated the H(2)O(2)-induced changes in these variables. The results demonstrate that BRCS and KGDHC are more sensitive to H(2)O(2) derived species than c-H(2)DCF, and that oxidized derivatives of the antioxidants exaggerate the actions of H(2)O(2). The findings support the hypothesis that select abnormalities in oxidative processes are a critical part of a cascade that leads to the cellular abnormalities in cells from AD patients.