Systems biology identifies preserved integrity but impaired metabolism of mitochondria due to a glycolytic defect in Alzheimer's disease neurons

Mitochondrial dysfunction is implicated in most neurodegenerative diseases, including Alzheimer's disease (AD). We here combined experimental and computational approaches to investigate mitochondrial health and bioenergetic function in neurons from a double transgenic animal model of AD (PS2APP/B6.152H). Experiments in primary cortical neurons demonstrated that AD neurons had reduced mitochondrial respiratory capacity. Interestingly, the computational model predicted that this mitochondrial bioenergetic phenotype could not be explained by any defect in the mitochondrial respiratory chain (RC), but could be closely resembled by a simulated impairment in the mitochondrial NADH flux. Further computational analysis predicted that such an impairment would reduce levels of mitochondrial NADH, both in the resting state and following pharmacological manipulation of the RC. To validate these predictions, we utilized fluorescence lifetime imaging microscopy (FLIM) and autofluorescence imaging and confirmed that transgenic AD neurons had reduced mitochondrial NAD(P)H levels at rest, and impaired power of mitochondrial NAD(P)H production. Of note, FLIM measurements also highlighted reduced cytosolic NAD(P)H in these cells, and extracellular acidification experiments showed an impaired glycolytic flux. The impaired glycolytic flux was identified to be responsible for the observed mitochondrial hypometabolism, since bypassing glycolysis with pyruvate restored mitochondrial health. This study highlights the benefits of a systems biology approach when investigating complex, nonintuitive molecular processes such as mitochondrial bioenergetics, and indicates that primary cortical neurons from a transgenic AD model have reduced glycolytic flux, leading to reduced cytosolic and mitochondrial NAD(P)H and reduced mitochondrial respiratory capacity.     

Reversible and irreversible oxidant injury to PC12 cells by hydrogen peroxide.

A simple and reproducible model to identify biochemical changes associated with the transition from reversible to irreversible oxidant injury and cell death was established using rat pheochromocytoma PC12 cells. Cells were subjected to a transient oxidative stress induced by exposure to hydrogen peroxide (H2O2). Reversible loss of high-energy phosphates, induced by exposing cells to 0.2 mM H2O2, was preceded by transient increases in cytosolic calcium with no loss of plasma membrane integrity, as indexed by release of cytosolic enzymes. In contrast, permanent loss of high-energy phosphates, induced by treating cells with 0.5 mH H2O2, was associated with sustained rises in cytosolic-free calcium and increased oxidation of pyruvate and palmitate, two mitochondrial substrates. Initial production of pyruvate and lactate was inhibited by exposure to 0.5 mM H2O2 but returned to values comparable to control values at one hour after treatment with H2O2. Compromise of the plasma membrane was a late event, occurring between 1 and 2 hours after exposure to 0.5 mM H2O2. Collectively, these data indicate that irreversible loss of high-energy phosphates and cell death caused by oxidative stress is more closely associated with altered mitochondrial function than with impaired glycolysis.     

Mitochondrial activation and the pyruvate paradox in a human cell line

Pyruvate promotes hyperpolarization of the inner mitochondrial membrane. However, in isolated mitochondria, pyruvate could participate in a futile cycle leading to mitochondrial depolarization. Here, we investigated this paradox in intact human cells by measuring parameters reflecting mitochondrial activation in response to 1 mM pyruvate and 5 mM glucose. NAD(P)H levels were elevated similarly by both substrates. Conversely, pyruvate induced a first transient phase of mitochondrial depolarization before the establishment of the expected sustained hyperpolarization. This correlated with kinetics of cytosolic ATP levels exhibiting a first phase decrease followed by an increase. Therefore, pyruvate transiently depolarizes mitochondria and reduces ATP in intact cells.     

The role of an astrocytic NADPH oxidase in the neurotoxicity of amyloid beta peptides

Amyloid beta peptide (Abeta) accumulates in the CNS in Alzheimer's disease. Both the full peptide (1-42) or the 25-35 fragment are toxic to neurons in culture. We have used fluorescence imaging technology to explore the mechanism of neurotoxicity in mixed asytrocyte/neuronal cultures prepared from rat or mouse cortex or hippocampus, and have found that Abeta acts preferentially on astrocytes but causes neuronal death. Abeta causes sporadic transient increases in [Ca2+]c in astrocytes, associated with a calcium dependent increased generation of reactive oxygen species (ROS) and glutathione depletion. This caused a slow dissipation of mitochondrial potential on which abrupt calcium dependent transient depolarizations were superimposed. The mitochondrial depolarization was reversed by mitochondrial substrates glutamate, pyruvate or methyl succinate, and by NADPH oxidase (NOX) inhibitors, suggesting that it reflects oxidative damage to metabolic pathways upstream of mitochondrial complex I. The Abeta induced increase in ROS and the mitochondrial depolarization were absent in cells cultured from transgenic mice lacking the NOX component, gp91phox. Neuronal death after 24 h of Abeta exposure was dramatically reduced both by NOX inhibitors and in gp91phox knockout mice. Thus, by raising [Ca2+]c in astrocytes, Abeta activates NOX, generating oxidative stress that is transmitted to neurons, causing neuronal death.     

Interaction of oxamate with the gluconeogenic pathway in rat liver

Oxamate, a structural analog of pyruvate, known as a potent inhibitor of lactic dehydrogenase, lactic dehydrogenase, produces an inhibition of gluconeogenic flux in isolated perfused rat liver or hepatocyte suspensions from low concentrations of pyruvate (less than 0.5 mM) or substrates yielding pyruvate. The following observations indicate that oxamate inhibits flux through pyruvate carboxylase: accumulation of substrates and decreased concentration of all metabolic intermediates beyond pyruvate; decreased levels of aspartate, glutamate, and alanine; and enhanced ketone body production, which is a sensitive indicator of decreased mitochondrial free oxaloacetate levels. The decreased pyruvate carboxylase flux does not seem to be the result of a direct inhibitory action of oxamate on this enzyme but is secondary to a decreased rate of pyruvate entry into the mitochondria. This assumption is based on the following observations: Above 0.4 mM pyruvate, no significant inhibitory effect of oxamate on gluconeogenesis was observed. The competitive nature of oxamate inhibition is in conflict with its effect on isolated pyruvate carboxylase which is noncompetitive for pyruvate. Fatty acid oxidation was effective in stimulating gluconeogenesis in the presence of oxamate only at concentrations of pyruvate above 0.4 mM. Since only at low pyruvate concentrations its entry into the mitochondria occurs via the monocarboxylate translocator, from these observations it follows that pyruvate transport across the mitochondrial membrane, and not its carboxylation, is the first nonequilibrium step in the gluconeogenic pathway. In the presence of oxamate, fatty acid oxidation inhibited gluconeogenesis from lactate, alanine, and low pyruvate concentrations (less than 0.5 mM), and the rate of transfer of reducing equivalents to the cytosol was significantly decreased. Whether fatty acids stimulate or inhibit gluconeogenesis appears to correlate with the rate of flux through pyruvate carboxylase which ultimately seems to rely on pyruvate availability. Unless adequate rates of oxaloacetate formation are maintained, the shift of the mitochondrial NAD couple to a more reduced state during fatty acid oxidation seems to decrease mitochondrial oxaloacetate resulting in a decreased rate of transfer of carbon and reducing power to the cytosol.     

Increased expression of SERCA in the hearts of transgenic mice results in increased oxidation of glucose

While several transgenic mouse models exhibit improved contractile characteristics in the heart, less is known about how these changes influence energy metabolism, specifically the balance between carbohydrate and fatty acid oxidation. In the present study we examine glucose and fatty acid oxidation in transgenic mice, generated to overexpress sarco(endo)plasmic reticulum calcium-ATPase (SERCA), which have an enhanced contractile phenotype. Energy substrate metabolism was measured in isolated working hearts using radiolabeled glucose and palmitate. We also examined oxygen consumption to see whether SERCA overexpression is associated with increased oxygen utilization. Since SERCA is important in calcium handling within the cardiac myocyte, we examined cytosolic calcium transients in isolated myocytes using indo-1, and mitochondrial calcium levels using pericam, an adenovirally expressed, mitochondrially targeted ratiometric calcium indicator. Oxygen consumption did not differ between wild-type and SERCA groups; however, we were able to show an increased utilization of glucose for oxidative metabolism and a corresponding decreased utilization of fatty acids in the SERCA group. Cytosolic calcium transients were increased in myocytes isolated from SERCA mice, and they show a faster rate of decay of the calcium transient. With these observations we noted increased levels of mitochondrial calcium in the SERCA group, which was associated with an increase in the active form of the pyruvate dehydrogenase complex. Since an increase in mitochondrial calcium levels leads to activation of the pyruvate dehydrogenase complex (the rate-limiting step for carbohydrate oxidation), the increased glucose utilization observed in isolated perfused hearts in the SERCA group may reflect a higher level of mitochondrial calcium.     

Cytosolic and mitochondrial malic enzyme isoforms differentially control insulin secretion

In islet beta-cells and INS-1 cells both the high activity of malic enzyme and the correlation of insulin secretion rates with pyruvate carboxylase (PC) flux suggest that a pyruvate-malate cycle is functionally relevant to insulin secretion. Expression of the malic enzyme isoforms in INS-1 cells and rat islets was measured, and small interfering RNA was used to selectively reduce isoform mRNA expression in INS-1 cells to evaluate its impact on insulin secretion. The cytosolic NADP(+)-specific isoform (ME1) was the most abundant, with the mitochondrial isoforms NAD(+)-preferred (ME2) expressed at approximately 50%, and the NADP(+)-specific (ME3) at approximately 10% compared with ME1. Selective reduction (89 +/- 2%) of cytosolic ME1 mRNA expression and enzyme activity significantly reduced glucose (15 mM:41 +/- 6%, p < 0.01) and amino acid (4 mM glutamine +/- 10 mM leucine: 39 +/- 6%, p < 0.01)-stimulated insulin secretion. Selective small interfering RNA reduction (51 +/- 6%) of mitochondrial ME2 mRNA expression did not impact glucose-induced insulin secretion, but decreased amino acid-stimulated insulin secretion by 25 +/- 4% (p < 0.01). Modeling of the metabolism of [U-(13)C]glucose by its isotopic distribution in glutamate indicates a second pool of pyruvate distinct from glycolytically derived pyruvate in INS-1 cells. ME1 knockdown decreased flux of both pools of pyruvate through PC. In contrast, ME2 knockdown affected only PC flux of the pyruvate derived from glutamate metabolism. These results suggest a physiological basis for two metabolically and functionally distinct pyruvate cycles. The cycling of pyruvate by ME1 generates cytosolic NADPH, whereas mitochondrial ME2 responds to elevated amino acids and serves to supply sufficient pyruvate for increased Krebs cycle flux when glucose is limiting.     

Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca2+

In cardiac muscle, mitochondrial ATP synthesis is driven by demand for ATP through feedback from the products of ATP hydrolysis. However, in skeletal muscle at higher workloads there is an apparent contribution of open-loop stimulation of ATP synthesis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the pathway. The role of calcium, which is known to stimulate the activity of mitochondrial dehydrogenases, as an open-loop controller, was investigated in isolated cardiac and skeletal muscle mitochondria. The kinetics of NADH synthesis and respiration, feedback from ATP hydrolysis products, and stimulation by calcium were characterized in isolated mitochondria to test the hypothesis that calcium has a stimulatory role in skeletal muscle mitochondria not apparent in cardiac mitochondria. A range of respiratory states were obtained in cardiac and skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate and malate, and flux of respiration, NAD(P)H fluorescence, and rhodamine 123 fluorescence were measured over a range of extra mitochondrial calcium concentrations. We found that under these conditions calcium stimulates NADH synthesis in skeletal muscle mitochondria but not in cardiac mitochondria.     

Effects of homocysteine on metabolic pathways in cultured astrocytes

Homocysteine is an amino acid that is an important risk factor for several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Increased homocysteine levels induce neuronal cell death in a variety of neuronal types. However, very few studies have probed the effects of homocysteine in astrocytes. The present study investigated the effects of homocysteine on primary cultures of astrocytes by exposing astrocytes to 400 microM homocysteine for 20 h. Metabolic extracts of cells were prepared following a 4-h incubation in minimum medium with 5.5 mM [U-(13)C]glucose in the presence or absence of homocysteine and analysed using (13)C NMR. The expression level of pyruvate dehydrogenase kinase isoform 2 (PDK-2), NAD(P)H levels and mitochondrial membrane potential responses were investigated following culture with homocysteine. Metabolomic analysis was performed using (1)H NMR spectroscopy and pattern recognition analysis. Following incubation with homocysteine there was a significant decrease (48%) in the ratio of flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) which was due to an increased flux through PDH. In addition, homocysteine culture resulted in a significant reduction in PDK-2 protein expression. Following stimulation with glucose there was a significant increase in NAD(P)H levels and an impaired hyperpolarisation of the mitochondrial membrane in homocysteine-treated cells. Metabolomic analysis showed that the most discriminating metabolites following homocysteine treatment were choline and hypotaurine. In summary, the results demonstrated that sub-lethal concentrations of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes.     

Mitochondrial free radical production induced by glucose deprivation in cerebellar granule neurons

Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 microM) and NBQX (10 microM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase--oligomycin, an uncoupler of oxidative phosphorylation--CCCP, a chelator of intracellular Ca2+--BAPTA, an inhibitor of electrogenic mitochondrial Ca2+ transport--ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca2+](i). Pyruvate, BAPTA, and ruthenium red lowered the GD-induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca2+ overload. GD-induced mitochondrial Ca2+ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.     

Pyruvate Induces Transient Tumor Hypoxia by Enhancing Mitochondrial Oxygen Consumption and Potentiates the Anti-Tumor Effect of a Hypoxia-Activated Prodrug TH-302

Background

TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.

Methodology/Results

The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O₂), with minimal effect under aerobic conditions (21% O₂). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm³. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm³), significantly delayed tumor growth.

Conclusions/Significance

Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.     

The effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver

The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of 14CO2 from infused [1-14C] pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Also, phenylephrine caused no significant effect on tissue NADH/NAD+ and acetyl-CoA/CoASH ratios or on the kinetics of pyruvate decarboxylation in 14CO2 washout experiments. Phenylephrine inhibition of [1-14C]pyruvate decarboxylation was, however, closely associated with a decrease in the specific radioactivity of perfusate lactate, suggesting that the pyruvate decarboxylation response simply reflected dilution of the labeled pyruvate pool due to phenylephrine-stimulated glycogenolysis. This suggestion was confirmed in additional experiments which showed that the alpha-adrenergic-mediated inhibitory effect on pyruvate decarboxylation was reduced in livers perfused with a high concentration of pyruvate (1 mM) and was absent in livers from starved rats. Thus, alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.     

Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.     

Effect of histone H1 on the cytosolic calcium levels in human breast cancer MCF 7 cells

In human breast cancer MCF 7 cells, the effect of exogenous histone H1 on intracellular calcium ([Ca2+]i) levels was measured using Fura 2AM. The dose and time dependent assessment revealed significant cell killing effect of histone H1 on MCF 7 cells. Histone H1 induced a sustained concentration dependent increase in [Ca2+]i levels in the presence of calcium in the medium, but the increase was reduced in the absence of extra cellular calcium. The effect of histone H1 on intracellular calcium flux measured using 45Ca radiolabel revealed significant inhibition of calcium uptake in endoplasmic reticulum, whereas the rate of uptake was unaltered in the mitochondria. The activities of phospholipase A2 showed a significant transient increase at 1 minute which by the end of 5 minutes decreased, whereas the activities of phospholipase C which showed a transient increase at the end of 1 minute, was maintained at basal levels in histone H1 treated cells compared to control cells. These findings suggest that histone H1 increases [Ca2+]i in MCF 7 cells by stimulating both extra cellular calcium influx and intracellular calcium release at higher concentrations exhibiting cytotoxic effect.     

Role of mitochondrial DNA in calcium signaling abnormality in bipolar disorder

Altered intracellular calcium levels are a consistent finding in studies of bipolar disorder, and recent studies point to the role of mitochondrial dysfunction, leading to the possibility that mitochondrial calcium dysregulation is involved in the pathophysiology of the disease. Although the mitochondrion is a key organelle for calcium accumulation, initial calcium signaling studies in bipolar disorder did not focus on the role of mitochondria. Later, neuroimaging and molecular genetic studies suggested the possibility that altered mitochondrial calcium regulation due to mitochondrial DNA (mtDNA) polymorphisms/mutations might be involved in the pathophysiology of bipolar disorder. Recent studies show that certain mtDNA polymorphisms alter mitochondrial calcium levels. Mutant mtDNA polymerase (Polg) transgenic mice carrying mtDNA mutations in forebrain cells show an increased calcium uptake rate in isolated mitochondria. This was found to be mediated by downregulation of cyclophilin D, a component of the mitochondrial permeability transition pore. In addition, agonist-stimulated calcium response is attenuated in hippocampal neurons of these transgenic mice. The finding that mtDNA polymorphisms and mutations affect mitochondrial calcium regulation supports the idea that mitochondrial calcium dysregulation may be involved in the pathophysiology of bipolar disorder. In this review, the history and recent findings of studies elucidating the role of mitochondrial calcium signaling in bipolar disorder are summarized.     

The effect of pyruvate transport inhibitors on the regulation of pyruvate dehydrogenase in the perfused rat heart.

The effect of the mitochondrial pyruvate transport inhibitors, α-cyanocinnamate and α-cyano-4-hydroxycinnamate, on the regulation of the pyruvate dehydrogenase multienzyme complex was investigated in the isolated perfused rat heart. Metabolic flux through pyruvate dehydrogenase was monitored by measuring ¹⁴CO₂ production from [1-¹⁴C]pyruvate infused into the heart. A stepwise increase in the concentration of the inhibitor in the influent perfusate effected a stepwise reduction of the flux through the enzyme complex at all pyruvate concentrations tested. However, the magnitude of the α-cyanocinnamate-insensitive flux through pyruvate dehydrogenase increased markedly as the infused pyruvate concentration was elevated. The inhibition of pyruvate decarboxylation in the heart was nearly completely reversed following cessation of the inhibitor infusion. α-Cyanocinnamate was nearly 10 times more potent than α-cyano-4-hydroxycinnamate as an inhibitor of the flux through pyruvate dehydrogenase. Maximally inhibiting levels of α-cyano-4-hydroxycinnamate caused an increase in the ratio of the active form of pyruvate dehydrogenase to the total extractable enzyme complex from a value of 0.5 at 1 mm infused pyruvate (in the absence of the inhibitor) to a value of near unity. This result indicated that the intramitochondrial pyruvate concentration was severely depleted by the infusion of the inhibitor and that the enzyme complex was interconverted to its active form under these conditions. Removal of the inhibitor from the perfusion medium again lowered the ratio of the active/total pyruvate dehydrogenase to near its original level of 0.5 and restored the original flux through the enzyme complex indicating that mitochondrial pyruvate transport has been restored. The results of this study indicate that α-cyanocinnamate and its derivatives are effective inhibitors of pyruvate transport in the perfused heart and that carrier-mediated pyruvate transport can be an important parameter in the regulation of the activation state and the metabolic flux through the pyruvate dehydrogenase multienzyme complex in the heart.     

Mitochondrial permeability transition pore induces mitochondria injury in Huntington disease

Background

Mitochondrial impairment has been implicated in the pathogenesis of Huntington’s disease (HD). However, how mutant huntingtin impairs mitochondrial function and thus contributes to HD has not been fully elucidated. In this study, we used striatal cells expressing wild type (STHdh^Q7/Q7) or mutant (STHdh^Q111/Q111) huntingtin protein, and cortical neurons expressing the exon 1 of the huntingtin protein with physiological or pathological polyglutamine domains, to examine the interrelationship among specific mitochondrial functions.

Results

Depolarization induced by KCl resulted in similar changes in calcium levels without compromising mitochondrial function, both in wild type and mutant cells. However, treatment of mutant cells with thapsigargin (a SERCA antagonist that raises cytosolic calcium levels), resulted in a pronounced decrease in mitochondrial calcium uptake, increased production of reactive oxygen species (ROS), mitochondrial depolarization and fragmentation, and cell viability loss. The mitochondrial dysfunction in mutant cells was also observed in cortical neurons expressing exon 1 of the huntingtin protein with 104 Gln residues (Q104-GFP) when they were exposed to calcium stress. In addition, calcium overload induced opening of the mitochondrial permeability transition pore (mPTP) in mutant striatal cells. The mitochondrial impairment observed in mutant cells and cortical neurons expressing Q104-GFP was prevented by pre-treatment with cyclosporine A (CsA) but not by FK506 (an inhibitor of calcineurin), indicating a potential role for mPTP opening in the mitochondrial dysfunction induced by calcium stress in mutant huntingtin cells.

Conclusions

Expression of mutant huntingtin alters mitochondrial and cell viability through mPTP opening in striatal cells and cortical neurons.

     

Reduced calcium-dependent mitochondrial damage underlies the reduced vulnerability of excitotoxicity-tolerant hippocampal neurons

In central neurons, over-stimulation of NMDA receptors leads to excessive mitochondrial calcium accumulation and damage, which is a critical step in excitotoxic death. This raises the possibility that low susceptibility to calcium overload-induced mitochondrial damage might characterize excitotoxicity-resistant neurons. In this study, we have exploited two complementary models of preconditioning-induced excitotoxicity resistance to demonstrate reduced calcium-dependent mitochondrial damage in NMDA-tolerant hippocampal neurons. We have further identified adaptations in mitochondrial calcium handling that account for enhanced mitochondrial integrity. In both models, enhanced tolerance was associated with improved preservation of mitochondrial membrane potential and structure. In the first model, which exhibited modest neuroprotection, mitochondria-dependent calcium deregulation was delayed, even though cytosolic and mitochondrial calcium loads were quantitatively unchanged, indicating that enhanced mitochondrial calcium capacity accounts for reduced injury. In contrast, the second model, which exhibited strong neuroprotection, displayed further delayed calcium deregulation and reduced mitochondrial damage because downregulation of NMDA receptor surface expression depressed calcium loading. Reducing calcium entry also modified the chemical composition of the calcium-buffering precipitates that form in calcium-loaded mitochondria. It thus appears that reduced mitochondrial calcium loading is a major factor underlying the robust neuroprotection seen in highly tolerant cells.     

Early Deficits in Glycolysis Are Specific to Striatal Neurons from a Rat Model of Huntington Disease

In Huntington disease (HD), there is increasing evidence for a link between mutant huntingtin expression, mitochondrial dysfunction, energetic deficits and neurodegeneration but the precise nature, causes and order of these events remain to be determined. In this work, our objective was to evaluate mitochondrial respiratory function in intact, non-permeabilized, neurons derived from a transgenic rat model for HD compared to their wild type littermates by measuring oxygen consumption rates and extracellular acidification rates. Although HD striatal neurons had similar respiratory capacity as those from their wild-type littermates when they were incubated in rich medium containing a supra-physiological glucose concentration (25 mM), pyruvate and amino acids, respiratory defects emerged when cells were incubated in media containing only a physiological cerebral level of glucose (2.5 mM). According to the concept that glucose is not the sole substrate used by the brain for neuronal energy production, we provide evidence that primary neurons can use lactate as well as pyruvate to fuel the mitochondrial respiratory chain. In contrast to glucose, we found no major deficits in HD striatal neurons’ capacity to use pyruvate as a respiratory substrate compared to wild type littermates. Additionally, we used extracellular acidification rates to confirm a reduction in anaerobic glycolysis in the same cells. Interestingly, the metabolic disturbances observed in striatal neurons were not seen in primary cortical neurons, a brain region affected in later stages of HD. In conclusion, our results argue for a dysfunction in glycolysis, which might precede any defects in the respiratory chain itself, and these are early events in the onset of disease.     

Control of gluconeogenesis in rat liver cells. I. Kinetics of the individual enzymes and the effect of glucagon

Control of gluconeogenesis from lactate was studied by titrating rat liver cells with lactate and pyruvate in a ratio of 10:1 in a perifusion system. At different steady states of glucose formation, the concentration of key gluconeogenic intermediates was measured and plotted against gluconeogenic flux (J glucose). Complete saturation was observed only in the plot relating J glucose to the extracellular pyruvate concentration. Measurement of pyruvate distribution in the cell showed that the mitochondrial pyruvate translocator operates close to equilibrium at high lactate and pyruvate concentrations. It can therefore be concluded that pyruvate carboxylase limits maximal gluconeogenic flux. Addition of glucagon did not cause a shift in the plots relating J glucose to glucose 6-phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, and phosphoenolpyruvate. It can thus be concluded that glucagon does not affect the kinetic parameters of the enzymes involved in the conversion of phosphoenolpyruvate to glucose. Addition of glucagon led to a shift in the curves relating J glucose to the concentration of cytosolic oxalacetate and extracellular pyruvate. The shift in the curve relating J glucose to oxalacetate is due to glucagon-induced inhibition of pyruvate kinase. The stimulation of gluconeogenesis by glucagon can be accounted for almost completely by inhibition of pyruvate kinase. There was almost no stimulation by glucagon of pyruvate carboxylation. In the absence of glucagon, control on gluconeogenesis from lactate is distributed among different steps including pyruvate carboxylase and pyruvate kinase. Assuming that in the presence of glucagon all pyruvate kinase flux is inhibited, the control of gluconeogenesis in the presence of the hormone is confined exclusively to pyruvate carboxylase.