Thiamine-responsive congenital lactic acidosis: clinical and biochemical studies

We studied six infants with thiamine-responsive congenital lactic acidosis and normal pyruvate dehydrogenase complex activity in vitro, through clinical and biochemical analysis. In addition to elevated lactate and pyruvate levels, the data revealed increased urinary excretion of alpha-ketoglutarate, alpha-ketoadipate, and branched chain ketoacids, indicating functional impairment of thiamine-requiring enzymes, such as pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, alpha-ketoadipate dehydrogenase, and branched chain amino acid dehydrogenase. The metabolism of thiamine has not been investigated in patients with thiamine-responsive congenital lactic acidosis. We evaluated two specific transport systems, THTR-1 (SLC19A2) and THTR-2 (SLC19A3), and a pyrophosphorylating enzyme of thiamine, thiamine pyrophosphokinase (hTPK 1), in addition to pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex activity; no abnormality was found. Although the clinical features of thiamine-responsive congenital lactic acidosis are heterogeneous and clinical responses to thiamine administration vary, we emphasize the importance of early diagnosis and initiation of thiamine therapy before the occurrence of permanent brain damage. Careful monitoring of lactate and pyruvate would be useful in determining thiamine dosage.     

Thiamine deficiency results in metabolic acidosis and energy failure in cerebellar granule cells: an in vitro model for the study of cell death mechanisms in Wernicke's encephalopathy

Thiamine deficiency (TD) in both humans and experimental animals results in severe compromise of mitochondrial function and leads to selective neuronal cell death in diencephalic and cerebellar structures. To examine further the influence of TD on neuronal survival in relation to metabolic changes, primary cultures of rat cerebellar granule cells were exposed to thiamine-deficient medium for up to 7 days in the absence or presence of the central thiamine antagonist pyrithiamine (Py). Exposure of cells for 7 days to thiamine-deficient medium alone resulted in no detectable cell death. On the other hand, 50 microM Py treatment led to reductions of thiamine phosphate esters, decreased activities of the thiamine-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase, a twofold increase in lactate release (P < 0.001), a lowering of pH, and significant (58%, P < 0.001) cell death. DNA fragmentation studies did not reveal evidence of apoptotic cell death. Addition of 50 microM alpha-tocopherol (vitamin E) or 100 microM of butylated hydroxyanisole (BHA) to Py-treated cells resulted in significant neuroprotection. On the other hand, addition of 10 microM MK-801, an NMDA receptor antagonist, was not neuroprotective. These results suggest that reactive oxygen species (ROS) play a major role in thiamine deficiency-induced neuronal cell death. Insofar as this experimental model recapitulates the metabolic and mitochondrial changes characteristic of thiamine deficiency in the intact animal, it might be useful in the elucidation of mechanisms involved in the neuronal cell death cascade resulting from thiamine deficiency.     

Pyruvate carboxylation in neurons.

Carboxylation of pyruvate in the brain was for many years thought to occur only in glia, an assumption that formed much of the basis for the concept of the glutamine cycle. It was shown recently, however, that carboxylation of pyruvate to malate occurs in neurons and that it supports formation of transmitter glutamate. The role of pyruvate carboxylation in neurons is to ensure tricarboxylic acid cycle activity by compensating for losses of alpha-ketoglutarate that occur through release of transmitter glutamate and GABA; these amino acids are alpha-ketoglutarate derivatives. Available data suggest that neuronal pyruvate carboxylation is quantitatively important. But because there is no net CO(2) fixation in the brain, pyruvate carboxylation must be balanced by decarboxylation of malate or oxaloacetate. Such decarboxylation occurs in both neurons and astrocytes. Several in vitro studies have shown a neuroprotective effect of pyruvate supplementation. Pyruvate carboxylation may be one mechanism through which such treatment is effective, because pyruvate carboxylation through malic enzyme is active during energy deficiency and leads to an increase in the level of dicarboxylates that can be metabolized through the tricarboxylic acid cycle for ATP production.     

Brain lactate synthesis in thiamine deficiency: a re-evaluation using 1H-13C nuclear magnetic resonance spectroscopy

Region-selective accumulation of brain lactate occurs in TD; however, the mechanisms responsible have not been elucidated fully. (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy were therefore used to investigate de novo lactate synthesis from [1-(13)C]glucose in vulnerable (medial thalamus) and nonvulnerable (frontal cortex) brain regions of rats made thiamine deficient by administration of the central thiamine antagonist pyrithiamine. De novo synthesis of lactate was increased in the medial thalamus to 148% and 226% of pair-fed control values at presymptomatic and symptomatic stages of thiamine deficiency, respectively, whereas no such changes were observed in the frontal cortex. Administration of a glucose load selectively worsened the changes in medial thalamus. Pyruvate recycling and peripherally derived lactate did not contribute significantly to the lactate increase within the thiamine-deficient brain. Increases in immunolabeling of the lactate dehydrogenase isoenzymes (LDH1 and LDH5) were observed in the medial thalamus of thiamine-deficient animals. Metabolic impairment due to thiamine deficiency thus results in increased glycolysis, increased LDH immunolabeling of neurons and astrocytes and increased de novo synthesis of lactate in brain regions vulnerable to thiamine deficiency. These results are consistent with the notion that focal lactate accumulation participates in the worsening of neurologic symptoms in thiamine-deficient patients.     

Biochemical and morphological studies of skeletal muscle in experimental chronic alcoholic myopathy

Experimental alcoholic myopathy was induced in rats by a combination of prolonged alcohol intake (mean 15.3 g ethanol/kg/day for up to 10 weeks) and a short fast. In view of literature evidence for impairment of both glycolytic and oxidative metabolism in alcoholic myopathy, we combined histological and histochemical observations with biochemical studies comprising assay of all glycolytic enzymes and measurement of respiration rates and cytochrome content in isolated intact mitochondria. The predominant histological finding was Type IIb fibre atrophy, while levels of the glycolytic enzymes aldolase, pyruvate kinase and lactate dehydrogenase were significantly depressed. Evidence of rhabdomyolysis was seen in a minority of animals. Mean mitochondrial respiratory rates were significantly lower with the Site I substrate glutamate in alcohol-treated animals. It is postulated that chronic alcoholic myopathy is associated with glycolytic deficiency and that acute rhabdomyolysis may arise from a superimposed mitochondrial failure, resulting in a severe energy crisis in muscle.     

Studies on the toxicity of 1-methyl-4-phenylpyridinium ion (MPP+) against mitochondria of mouse brain

Effects of 1-methyl-4-phenylpyridinium ion (MPP+) on cellular respiration were studied using mitochondria prepared from mouse brains. State 3 and state 4 respiration supported by glutamate plus malate or pyruvate plus malate were significantly inhibited by 0.05 mM MPP+. On the other hand, respirations supported by succinate or alpha-glycerophosphate were not inhibited at all. Activity of mitochondrial NADH-ubiquinone oxidoreductase was significantly inhibited by MPP+. This inhibition was markedly potentiated by preincubating mitochondria with MPP+ together with glutamate plus malate. The latter observation suggested accumulation of MPP+ within the mitochondria during preincubation. When mitochondria were pretreated with an uncoupling agent such as carbonylcyanide m-chlorophenylhydrazone (CCCP) or dinitrophenol, MPP+-induced inhibition of state 3 respiration or of activity of complex I could no longer be seen. A potassium ionophore, valinomycin, showed a similar effect. Adenosine triphosphate (ATP) synthesis was also inhibited by MPP+. Among the NAD+-linked dehydrogenases in the tricarboxylic acid cycle, alpha-ketoglutarate dehydrogenase complex was significantly inhibited by MPP+. This inhibition was reversible and competitive with NAD+. Energy crisis appears to be one of the most important mechanisms of neuronal degeneration in MPTP-induced parkinsonism. Biochemical mechanisms underlying MPP+-induced inhibition of mitochondrial respiration were discussed.     

Mitochondrial abnormalities in Alzheimer brain: mechanistic implications

Reductions in cerebral metabolism sufficient to impair cognition in normal individuals also occur in Alzheimer's disease (AD). The degree of clinical disability in AD correlates closely to the magnitude of the reduction in brain metabolism. Therefore, we tested whether impairments in tricarboxylic acid (TCA) cycle enzymes of mitochondria correlate with disability. Brains were from patients with autopsy-confirmed AD and clinical dementia ratings (CDRs) before death. Significant (p < 0.01) decreases occurred in the activities of the pyruvate dehydrogenase complex (-41%), isocitrate dehydrogenase (-27%), and the alpha-ketoglutarate dehydrogenase complex (-57%). Activities of succinate dehydrogenase (complex II) (+44%) and malate dehydrogenase (+54%) were increased (p < 0.01). Activities of the other four TCA cycle enzymes were unchanged. All of the changes in TCA cycle activities correlated with the clinical state (p < 0.01), suggesting a coordinated mitochondrial alteration. The highest correlation was with pyruvate dehydrogenase complex (r = 0.77, r2= 0.59). Measures to improve TCA cycle metabolism might benefit AD patients.     

Evaluation of brain mitochondrial glutamate and alpha-ketoglutarate transport under physiologic conditions

Some models of brain energy metabolism used to interpret in vivo (13)C nuclear magnetic resonance spectroscopic data assume that intramitochondrial alpha-ketoglutarate is in rapid isotopic equilibrium with total brain glutamate, most of which is cytosolic. If so, the kinetics of changes in (13)C-glutamate can be used to predict citric acid cycle flux. For this to be a valid assumption, the brain mitochondrial transporters of glutamate and alpha-ketoglutarate must operate under physiologic conditions at rates much faster than that of the citric acid cycle. To test the assumption, we incubated brain mitochondria under physiologic conditions, metabolizing both pyruvate and glutamate and measured rates of glutamate, aspartate, and alpha-ketoglutarate transport. Under the conditions employed (66% of maximal O(2) consumption), the rate of synthesis of intramitochondrial alpha-ketoglutarate was 142 nmol/min.mg and the combined initial rate of alpha-ketoglutarate plus glutamate efflux from the mitochondria was 95 nmol/min.mg. It thus seems that much of the alpha-ketoglutarate synthesized within the mitochondria proceeds around the citric acid cycle without equilibrating with cytosolic glutamate. Unless the two pools are in such rapid exchange that they maintain the same percent (13)C enrichment at all points, (13)C enrichment of glutamate alone cannot be used to determine tricarboxylic acid cycle flux. The alpha-ketoglutarate pool is far smaller than the glutamate pool and will therefore approach steady state faster than will glutamate at the metabolite transport rates measured.     

Chronic ketosis and cerebral metabolism

The effects of chronic ketosis on cerebral metabolism were determined in adult rats maintained on a high-fat diet for approximately three weeks and compared to a control group of animals. The fat-fed rats had statistically significantly lower blood glucose concentrations and higher blood beta-hydroxybutyrate and acetoacetate concentrations; higher brain concentrations of bound glucose, glucose 6-phosphate, pyruvate, lactate, beta-hydroxybutyrate, citrate, alpha-ketoglutarate, alanine, and adenosine triphosphate (ATP); lower brain concentrations of fructose 1,6-diphosphate, aspartate, adenosine diphosphate (ADP), creatine, cyclic nucleotides, succinyl coenzyme A (CoA), acid-insoluble CoA, and total CoA; and similar brain concentrations of glucose, malate, calculated oxaloacetate, glutamate, glutamine, adenosine monophosphate, phosphocreatine, reduced CoA, acetyl CoA, sodium, potassium, chloride, and water content. The metabolite data in the chronically ketotic rats demonstrate an increase in the cerebral energy reserve and energy charge. These data also suggest negative modification of the enzymes phosphofructokinase, pyruvic dehydrogenase, and alpha-ketoglutaric dehydrogenase; positive modification of glycogen synthase; and possible augmentation of the hexose transport system. There was no demonstrable difference in brain pH, water content, or electrolytes in the two groups of animals. We speculate that the increased brain ATP/ADP ratio is central to most, if not all, the observed metabolic perturbations and may account for the increased neuronal stability that accompanies chronic ketosis.     

Friedreich ataxia. II. Normal kinetics of lipoamide dehydrogenase.

Lipoamide dehydrogenase (LAD) kinetic values, Km and Vmax, were normal in 11 patients with Friedreich ataxia. Fibroblast activities of the pyruvate and alpha-ketoglutarate dehydrogenase complex, and LAD activities, were also normal. There was no reduction in oxidative decarboxylation of pyruvate, alpha-ketoglutarate, or several other substrates in intact fibroblasts. Methodologic differences may account for differences of opinion about putative abnormalities of the alpha-ketoacid dehydrogenase complexes.     

Alterations in uptake and metabolism of aspartate and glutamate in brain of thiamine deficient animals

The high affinity uptake systems of aspartate, glutamate, glycine and taurine were studied in synaptosomal preparations isolated from the cerebellum, medulla-pons and the telencephalon of rats made thiamine deficient (TD) by diet or pyrithiamine (PT). There was a significant enhancement in the uptake of asparate/glutamate (probably transported by the same carrier) by the synaptosomal preparations of the cerebellum only, in both groups of thiamine-deficient animals as compared to controls. This was due to an increase in the number of uptake sites and not to an alteration of the binding affinity. Aspartate levels decreased significantly in all three brain areas of PT-treated animals and this change was greatest in the medulla-pons and the cerebellum and least in the cortex. Glutamate and serine levels were significantly decreased only in the medulla-pons whilst the concentration of glutamine was significantly increased in the three brain regions studied. The changes in both uptake and levels of amino acids in TD rats were reversed by thiamine therapy. Though the uptake studies do not discriminate between an altered aspartergic or glutamergic system, the changes in the levels of aspartate in the cerebellum suggest that the aspartergic system is involved. Since earlier studies showed a selective impairment in the high affinity uptake of serotonin by cerebellar synaptosomes, thiamine deficiency could impair cerebellar function by inducing an imbalance in its neurotransmitter systems.     

Experimental thiamine deficiency. Neuropathic and mitochondrial changes induced in rat muscle.

Whether pure thiamine deficiency produces a neuropathy in Mammalia is still debated. Rats were pair-fed-synthetic diets with and without thiamine. When studied histochemically, soleus muscles from thiamine-deficient rats showed (1) small, angular fibers that had high NADH dehydrogenase activities; (2) a loss of 43% of type II (FOG) fibers; (3) decreased intensity of the reaction for betaOHB dehydrogenase; and (4) fibers with subsarcolemmal collections resembling "ragged-red" muscle. Electron microscopy revealed degeneration of some small myelin sheaths of distal and intramuscular nerves; atrophic, degenerating, hypoosmophilic muscle fibers in soleus and vastus medialis; and scattered muscle fibers with abnormal collections of deranged mitochondria accompanied by lipid droplets. These abnormalities, not found in control muscles, indicate that both motor neuropathy and mild mitochondrial changes, such as are seen in the "ragged-red" diseases, are induced by pure thiamine deficiency.     

Methylmalonate impairs mitochondrial respiration supported by NADH-linked substrates: involvement of mitochondrial glutamate metabolism

The neurodegeneration that occurs in methylmalonic acidemia is proposed to be associated with impairment of mitochondrial oxidative metabolism resulting from methylmalonate (MMA) accumulation. The present study evaluated the effects of MMA on oxygen consumption by isolated rat brain mitochondria in the presence of NADH-linked substrates (α-ketoglutarate, citrate, isocitrate, glutamate, malate, and pyruvate). Respiration supported either by glutamate or glutamate plus malate was significantly inhibited by MMA (1-10 mM), whereas no inhibition was observed when a cocktail of NADH-linked substrates was used. Measurements of glutamate transport revealed that the inhibitory effect of MMA on respiration maintained by this substrate is not due to inhibition of its mitochondrial uptake. In light of this result, the effect of MMA on the activity of relevant enzymes involved in mitochondrial glutamate metabolism was investigated. MMA had minor inhibitory effects on glutamate dehydrogenase and aspartate aminotransferase, whereas α-ketoglutarate dehydrogenase was significantly inhibited by this metabolite (K(i) = 3.65 mM). Moreover, measurements of α-ketoglutarate transport and mitochondrial MMA accumulation indicated that MMA/α-ketoglutarate exchange depletes mitochondria from this substrate, which may further contribute to the inhibition of glutamate-sustained respiration. To study the effect of chronic in vivo MMA treatment on mitochondrial function, young rats were intraperitoneally injected with MMA. No significant difference was observed in respiration between isolated brain mitochondria from control and MMA-treated rats, indicating that in vivo MMA treatment did not lead to permanent mitochondrial respiratory defects. Taken together, these findings indicate that the inhibitory effect of MMA on mitochondrial oxidative metabolism can be ascribed to concurrent inhibition of specific enzymes and lower availability of respiratory substrates.     

Role of thiamine in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia in elderly individuals and is associated with progressive neurodegeneration of the human neocortex. Thiamine levels and the activity of thiamine-dependent enzymes are reduced in the brains and peripheral tissues of patients with AD. Genetic studies have provided the opportunity to determine what proteins link thiamine to AD pathology (ie, transketolase, apolipoprotein E, α-1-antitrypsin, pyruvate dehydrogenase complex, p53, glycogen synthetase kinase-3β, c-Fos gene, the Sp1 promoter gene, and the poly(ADP-ribosyl) polymerase-1 gene). We reviewed the association between histopathogenesis and neurotransmitters to understand the relationship between thiamine and AD pathology. Oral thiamine trials have been shown to improve the cognitive function of patients with AD; however, absorption of thiamine is poor in elderly individuals. In the early stage of thiamine-deficient encephalopathy (Wernicke's encephalopathy), however, parental thiamine has been used successfully. Therefore, further studies are needed to determine the benefits of using parental thiamine as a treatment for AD.     

A biochemical and immunohistochemical study of central serotonin nerves in rats with chronic thiamine deficiency

An immunohistochemical and neurochemical investigation of central serotonin (5-HT) nerves was made in rats deprived of dietary thiamine at various stages of development. The classical symptoms of severe thiamine deficiency were produced in adult rats which had been maintained on a synthetic thiamine-free diet for 5-8 weeks and in young rats reared from birth to weaning by thiamine-deficient mothers. Offspring of rats which had been thiamine-deficient throughout pregnancy were also studied; there were no visible symptoms of thiamine deficiency in these rats after weaning. The number and distribution of 5-HT nerve cell bodies in the brainstem were compared in control and thiamine-deficient rats after visualizing the cells by immunofluorescence of endogenous 5-HT. 5-HT nerve terminals and axons were also compared in normal and deficient rats by immunofluorescence after loading with 5,7-dihydroxytryptamine. The immunohistochemical examination showed that central 5-HT nerves were not affected in any of the groups of thiamine-deficient rats studied. This was confirmed by measurements of tryptophan hydroxylase activity and 5-HT concentration in several brain regions. These results do not support earlier reports of a selective impairment of central 5-HT nerves in chronic diet-induced thiamine deficiency.     

Bioenergetic dysfunction in Huntington's disease human cybrids

In this work we studied the mitochondrial-associated metabolic pathways in Huntington's disease (HD) versus control (CTR) cybrids, a cell model in which the contribution of mitochondrial defects from patients is isolated. HD cybrids exhibited an interesting increase in ATP levels, when compared to CTR cybrids. Concomitantly, we observed increased glycolytic rate in HD cybrids, as revealed by increased lactate/pyruvate ratio, which was reverted after inhibition of glycolysis. A decrease in glucose-6-phosphate dehydrogenase activity in HD cybrids further indicated decreased rate of the pentose-phosphate pathway. ATP levels of HD cybrids were significantly decreased under glycolysis inhibition, which was accompanied by a decrease in phosphocreatine. Nevertheless, pyruvate supplementation could not recover HD cybrids' ATP or phosphocreatine levels, suggesting a dysfunction in mitochondrial use of that substrate. Oligomycin also caused a decrease in ATP levels, suggesting a partial support of ATP generation by the mitochondria. Nevertheless, mitochondrial NADH/NAD_t levels were decreased in HD cybrids, which was correlated with a decrease in pyruvate dehydrogenase activity and protein expression, suggesting decreased tricarboxylic acid cycle (TCA) input from glycolysis. Interestingly, the activity of alpha-ketoglutarate dehydrogenase, a critical enzyme complex that links the TCA to amino acid synthesis and degradation, was increased in HD cybrids. In accordance, mitochondrial levels of glutamate were increased and alanine was decreased, whereas aspartate and glutamine levels were unchanged in HD cybrids. Conversely, malate dehydrogenase activity from total cell extracts was unchanged in HD cybrids. Our results suggest that inherent dysfunction of mitochondria from HD patients affects cellular bioenergetics in an otherwise functional nuclear background.     

Lactate reverses insulin-induced hypoglycemic stupor in suckling-weanling mice: biochemical correlates in blood, liver, and brain

The recovery of weanling mice from insulin-induced hypoglycemic stupor-coma after injection of sodium -L(+)-lactate (18 mmol/kg) was as rapid (10 min) as in litter-mates treated with glucose (9 mmol/kg). Stimulated by this dramatic action, we studied the effects of lactate injection on brain carbohydrate and energy metabolism in normal and hypoglycemic mice; blood and liver tissue were also studied. Ten minutes after lactate injection in normal mice, plasma lactate levels increased by 15 mmol/L; plasma glucose levels were unchanged, but the beta-hydroxybutyrate concentration fell 59%. In the brains of these animals, glucose levels increased 2.3-fold, and there were significant increases in brain glycogen (10%), glucose-6-phosphate (27%), lactate (68%), pyruvate (37%), citrate (12%), and malate (19%); the increase in alpha-ketoglutarate (32%) was not significant. Lactate injection reduced the cerebral glucose-use rate 40%. These changes were not due to lactate-induced increases in blood [HCO-3] and pH (examined by injection of 15 mmol/kg sodium bicarbonate). Although lactate injection of hypoglycemic mice doubled levels of glucose in plasma and brain (not significant) and most of the cerebral glycolytic intermediates, values were far below normal (still in the range seen in hypoglycemic animals). By contrast, citrate and alpha-ketoglutarate levels returned to normal; the large increase in malate was not significant. Reduced glutamate levels increased to normal, and elevated aspartate levels fell below normal. Thus, recovery from hypoglycemic stupor does not necessarily depend on normal levels of plasma and/or brain glucose (or glycolytic intermediates).(ABSTRACT TRUNCATED AT 250 WORDS)     

Thiamine deficiency and brain atrophy in alcoholic patients]

CT brain scans of 65 alcohol-dependent inpatients were compared before and after 6 weeks of confirmed abstinence. Linear measurements revealed a significant reduction of the enlargement of the ventricular system in accordance with the re-expansion of the brain after alcohol abstinence (ANOVA, significant time effects). 22 patients showed a moderate or severe thiamine deficiency. CT findings on thiamine-deficient patients did not differ from those on patients without thiamine deficiency (ANOVA, no significant group effects). Correlations between thiamine deficiency and subcortical atrophy before treatment were not significant. The results are discussed in relation to the pathogenesis of reversible brain shrinkage in chronic alcoholics.     

Respiration and ROS production in brain and spinal cord mitochondria of transgenic rats with mutant G93a Cu/Zn-superoxide dismutase gene

Mitochondrial dysfunction is involved in the pathogenesis of motor neuron degeneration in the G93A mutant transgenic (tgmSOD1) animal model of ALS. However, it is unknown whether mitochondriopathy is a primary or secondary event. We isolated brain (BM) and spinal cord (SCM) mitochondria from 2 month old presymptomatic tgmSOD1 rats and studied respiration and generation of reactive oxygen species (ROS) using a new metabolic paradigm (Panov et al., Am. J. Physiol., Regul. Integr. Comp. Physiol., 2011). The yields of BM and SCM from tgmSOD1 rats were 27% and 58% lower than normal rats (WT). The rates of the State 3 and State 3U respiration of tgBM and tgSCM were normal with glutamate+pyruvate+malate as substrates but were inhibited with pyruvate+malate in tgBM and glutamate+malate in tgSCM. In tgSCM the State 4 respiration with all substrates was significantly (1.5-2 fold) increased as compared with WT-SCM. Western blot analysis showed that tgSCM had lower contents of complexes III (-60%) and IV (-35%), and the presence of mutated SOD1 protein in both tgBM and tgSCM. With glutamate+pyruvate+malate or succinate+glutamate+pyruvate+malate as substrates, tgBM and tgSCM generated 5-7 fold more ROS than normal mitochondria, and tgSCM generated two times more ROS than tgBM. We show that the major damaging ROS species in tgmSOD1 animals is H(2)O(2). It is known that mutated SOD1, damaged by H(2)O(2), associates with mitochondria, and we suggest that this further increases production of H(2)O(2). We also show that the total tissue calcium content remained normal in the brain but was diminished by 26% in the spinal cord of presymptomatic tgmSOD1 rats. CONCLUSION: In tgSCM abnormally high rates of ROS generation, associated with reverse electron transport, result in accelerated mitochondriopathy, and the Ca(2+)-dependent excitotoxic death of motor neurons. Thus mitochondrial dysfunction is a key early element in pathogenesis of motor neuron degeneration in tgmSOD1 rats.     

Glutamate and glutathione interplay in a motor neuronal model of amyotrophic lateral sclerosis reveals altered energy metabolism

Impairment of mitochondrial function might contribute to oxidative stress associated with neurodegeneration in amyotrophic lateral sclerosis (ALS). Glutamate levels in tissues of ALS patients are sometimes altered. In neurons, mitochondrial metabolism of exogenous glutamine is mainly responsible for the net synthesis of glutamate, which is a neurotransmitter, but it is also necessary for the synthesis of glutathione, the main endogenous antioxidant. We investigated glutathione synthesis and glutamine/glutamate metabolism in a motor neuronal model of familial ALS. In standard culture conditions (with glutamine) or restricting glutamine or cystine, the level of glutathione was always lower in the cell line expressing the mutant (G93A) human Cu, Zn superoxide dismutase (G93ASOD1) than in the line expressing wild-type SOD1. With glutamine the difference in glutathione was associated with a lower glutamate and impairment of the glutamine/glutamate metabolism as evidenced by lower glutaminase and cytosolic malate dehydrogenase activity. d-β-hydroxybutyrate, as an alternative to glutamine as energy substrate in addition to glucose, reversed the decreases of cytosolic malate dehydrogenase activity and glutamate and glutathione. However, in the G93ASOD1 cell line, in all culture conditions the expression of pyruvate dehydrogenase kinase l protein, which down-regulates pyruvate dehydrogenase activity, was induced, together with an increase in lactate release in the medium. These findings suggest that the glutathione decrease associated with mutant SOD1 expression is due to mitochondrial dysfunction caused by the reduction of the flow of glucose-derived pyruvate through the TCA cycle; it implies altered glutamate metabolism and depends on the different mitochondrial energy substrates.