Role of mitochondrial dysfunction and oxidative stress in the pathogenesis of selective neuronal loss in Wernicke’s encephalopathy

Thiamine deficiency results in Wernicke’s encephalopathy and is commonly encountered in chronic alcoholism, gastrointestinal diseases, and HIV AIDS. The earliest metabolic consequence of thiamine deficiency is a selective loss in activity of the thiamine diphosphate-dependent enzyme α-ketoglutarate dehydrogenase (α-KGDH), a rate-limiting tricarboxylic acid cycle enzyme. Thiamine deficiency is characterized neuropathologically by selective neuronal cell death in the thalamus, pons, and cerebellum. The cause of this region-selective neuronal loss is unknown, but mechanisms involving cellular energy failure, focal lactic acidosis, and NMDA receptor-mediated excitotoxicity have classically been implicated. More recently, evidence supports a role for oxidative stress. Evidence includes increased endothelial nitric oxide synthase, nitrotyrosine deposition, microglial activation, and lipid peroxidation. Reactive oxygen species production results in decreased expression of astrocytic glutamate transporters and decreased activities of α-KGDH, resulting in an amplification of cell death mechanisms in thiamine deficiency.     

Region-selective alterations of glucose oxidation and amino acid synthesis in the thiamine-deficient rat brain: a re-evaluation using 1H/13C nuclear magnetic resonance spectroscopy

Thiamine deficiency provides an effective model of selective neuronal cell death. ¹H and ¹³C-NMR was used to investigate the effects of thiamine deficiency on the synthesis of amino acids derived from [1-¹³C]glucose in vulnerable (medial thalamus; MT) compared to non-vulnerable (frontal cortex; FC) brain regions. Following 11 days of thiamine deficiency, a time-point associated with the absence of significant neuronal cell death, regional concentrations of glutamate, glutamine and GABA remained unaffected in FC and MT; however, decreased levels of aspartate in MT at this time-point were a predictor of regional vulnerability. De novo synthesis of glutamate and GABA were unaffected at 11 days of thiamine deficiency, while synthesis of [2-¹³C]aspartate was significantly impaired. Glucose loading, which has been shown to exacerbate symptoms in patients with thiamine deficiency, resulted in further decreases of TCA cycle flux and reduced de novo synthesis of glutamate, aspartate and GABA in thiamine-deficient (TD) rats. Isotopomer analysis revealed that impaired TCA cycle flux and decreased aspartate synthesis due to thiamine deficiency occurred principally in neurons. Glucose loading deteriorated TD-related decreases in TCA cycle flux, and concomitantly reduced synthesis of aspartate and glutamate in MT.     

Alpha-Phenyl-N-Tert-Butylnitrone Attenuates Excitotoxicity in Rat Striatum by Preventing Hydroxyl Radical Accumulation

Various in vitro experiments have indicated that oxygen-derived free radicals may contribute to excitotoxic neuronal death. In the present study we induced excitotoxicity in rat striatum by perfusing glutamate at a high concentration through a microdialysis probe. We observed an increased formation of hydroxyl radicals (^˙OH) during the perfusion of the excitotoxin and an extensive striatal lesion 24 h after the insult. The spin trap, -phenyl-N-tert-butylnitrone (PBN), attenuated both hydroxyl radical levels and the volume of the lesion. This result suggests that the neuroprotection may be due to a free radical scavenging mechanism. It also implies that PBN may be used in pathological situations involving excitotoxicity such as stroke, brain trauma, and chronic neurologic diseases.     

Chronic elevation of extracellular glutamate due to transport blockade is innocuous for spinal motoneurons in vivo

Glutamate-mediated excitotoxicity has been considered to play an important role in the mechanism of spinal motoneuron death in amyotrophic lateral sclerosis (ALS), and some reports suggest that this excitotoxicity may be due to a decreased glutamate transport and the consequent elevation of its extracellular level. We have previously shown that short lasting increments in extracellular glutamate due to administration of the non-selective glutamate transport blocker l-2,4-trans-pyrrolidine-dicarboxylate (PDC) by microdialysis in the rat spinal cord do not induce motoneuron damage. In the present work we examined the potential involvement of chronic glutamate transport blockade as a causative factor of spinal motoneuron death and paralysis in vivo. Using osmotic minipumps, we infused directly in the spinal cord for up to 10 days PDC and another glutamate transport blocker, dl-threo-β-benzyloxyaspartate (TBOA), and we measured by means of microdialysis and HPLC the extracellular concentration of glutamate and other amino acids. We found that after the infusion of both PDC and TBOA the concentration of endogenous extracellular glutamate was 3–4-fold higher than that of the controls. Nevertheless, in spite of this elevation no motoneuron degeneration or gliosis were observed, assessed by histological examination and choline acetyltransferase and glial fibrillary acidic protein immunocytochemistry. In accord with this lack of toxic effect, no motor deficits, assessed by three motor activity tests, were observed.Because we had previously shown that under identical experimental conditions the infusion of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) induced progressive motoneuron death and paralysis, we conclude that prolonged elevation of extracellular glutamate due to its transport blockade in vivo is innocuous for spinal motoneurons and therefore that these results do not support the hypothesis that glutamate transport deficiency plays a crucial role as a causal factor of spinal motoneuron degeneration in ALS.     

Loss of [3H]kainate and of NMDA-displaceable [3H]glutamate binding sites in brain in thiamine deficiency: Results of a quantitative autoradiographic study

Previous studies suggest that alterations of brain glutamate synthesis and release occur in experimental thiamine deficiency. In order to assess the integrity of post-synaptic glutamatergic receptors in thiamine deficiency, binding sites for [³H]glutamate (displaced by NMDA), [³H]-kainate, and [³H]quisqualate (AMPA sites) were evaluated using Quantitative Receptor Autoradiography in rat brain following 14 days of treatment with the central thiamine antagonist pyrithiamine. Compared to pair-fed controls, brains of symptomatic thiamine-deficient animals contained significantly fewer NMDA-displaceable binding sites in cerebral cortex, medial septum and hippocampus. It has been suggested that NMDA-receptor mediated glutamate excitotoxicity plays a role in the pathogenesis of neuronal loss in thiamine deficiency. If such is the case, the selective loss of NMDA binding sites in cerebral cortex and hippocampus offers a possible explanation for the relative nonvulnerability of these brain regions to pyrithiamine-induced thiamine deficiency. [³H]quisqualate (AMPA) binding sites were unchanged in all brain regions of pyrithiamine-treated rats whereas [³H]kainate sites were significantly reduced in density in medial and lateral thalamus. The decline in these binding sites may be due to neuronal loss in pyrithiamine-induced thiamine deficiency. Alterations of glutamatergic synaptic function involving both NMDA and kainate receptor subclasses could contribute to the pathogenesis of neurological dysfunction in Wernicke's Encephalopathy in humans.     

Inhibition of Drp1 provides neuroprotection in vitro and in vivo

Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.     

Genetic evidence for adenylyl cyclase 1 as a target for preventing neuronal excitotoxicity mediated by N-methyl-D-aspartate receptors

The excessive activation of N-methyl-D-aspartate (NMDA) receptors by glutamate results in neuronal excitotoxicity. cAMP is a key second messenger and contributes to NMDA receptor-dependent synaptic plasticity. Adenylyl cyclases 1 (AC1) and 8 (AC8) are the two major calcium-stimulated ACs in the central nervous system. Previous studies demonstrate AC1 and AC8 play important roles in synaptic plasticity, memory, and persistent pain. However, little is known about the possible roles of these two ACs in glutamate-induced neuronal excitotoxicity. Here, we report that genetic deletion of AC1 significantly attenuated neuronal death induced by glutamate in primary cultures of cortical neurons, whereas AC8 deletion did not produce a significant effect. AC1, but not AC8, contributes to intracellular cAMP production following NMDA receptor activation by glutamate in cultured cortical neurons. AC1 is involved in the dynamic modulation of cAMP-response element-binding protein activity in neuronal excitotoxicity. To explore the possible roles of AC1 in cell death in vivo, we studied neuronal excitotoxicity induced by an intracortical injection of NMDA. Cortical lesions induced by NMDA were significantly reduced in AC1 but not in AC8 knock-out mice. Our findings provide direct evidence that AC1 plays an important role in neuronal excitotoxicity and may serve as a therapeutic target for preventing excitotoxicity in stroke and neurodegenerative diseases.     

MFN2 Couples Glutamate Excitotoxicity and Mitochondrial Dysfunction in Motor Neurons

Mitochondrial dysfunction plays a central role in glutamate-evoked neuronal excitotoxicity, and mitochondrial fission/fusion dynamics are essential for mitochondrial morphology and function. Here, we establish a novel mechanistic linker among glutamate excitotoxicity, mitochondrial dynamics, and mitochondrial dysfunction in spinal cord motor neurons. Ca²⁺-dependent activation of the cysteine protease calpain in response to glutamate results in the degradation of a key mitochondrial outer membrane fusion regulator, mitofusin 2 (MFN2), and leads to MFN2-mediated mitochondrial fragmentation preceding glutamate-induced neuronal death. MFN2 deficiency impairs mitochondrial function, induces motor neuronal death, and renders motor neurons vulnerable to glutamate excitotoxicity. Conversely, MFN2 overexpression blocks glutamate-induced mitochondrial fragmentation, mitochondrial dysfunction, and/or neuronal death in spinal cord motor neurons both in vitro and in mice. The inhibition of calpain activation also alleviates glutamate-induced excitotoxicity of mitochondria and neurons. Overall, these results suggest that glutamate excitotoxicity causes mitochondrial dysfunction by impairing mitochondrial dynamics via calpain-mediated MFN2 degradation in motor neurons and thus present a molecular mechanism coupling glutamate excitotoxicity and mitochondrial dysfunction.     

d-β-Hydroxybutyrate Prevents Glutamate-Mediated Lipoperoxidation and Neuronal Damage Elicited during Glycolysis Inhibition In Vivo

Excitotoxic neuronal death mediated by over-activation of glutamate receptors has been implicated in ischemia, hypoglycemia and some neurodegenerative diseases. It involves oxidative stress and is highly facilitated during impairment of energy metabolism. We have shown previously that in vivo systemic glycolysis inhibition with iodoacetate (IOA), exacerbates glutamate excitotoxicity. We have now investigated whether this effect involves oxidative damage to membrane lipids, as evaluated by the presence of thiobarbituric acid-reactive substances. We have also tested whether the ketone body, d-β-hydroxybutyrate (d-BHB), prevents lipoperoxidation and tissue damage. Results show that glutamate intrastriatal injection in control rats transiently enhances lipoperoxidation, while in IOA-treated animals increased lipoperoxidation is sustained. Treatment with d-BHB significantly reduces striatal lesions and lipoperoxidation. Vitamin E also reduced neuronal damage and lipoperoxidation. Results suggest that glycolysis impairment favors a pro-oxidant condition and situates oxidative damage as an important mediator of in vivo induced excitotoxicity. Results provide evidence for the neuroprotective effect of d-BHB against glutamate toxicity.     

The role of Bax in glutamate-induced nerve cell death

The role of the Bax gene product was examined in three forms of cortical nerve cell death in primary cultures. These include spontaneous cell death, oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake resulting in toxic oxidative stress, and ionotropic glutamate receptor-mediated excitotoxicity following a brief exposure to 10 microM glutamate. Primary cortical and hippocampal neuron cultures were established from embryos of Bax -/+ x Bax -/+ matings and the embryos genotyped and assayed for cell death in the three experimental paradigms. Cell death induced by oxidative glutamate toxicity and glutamate-mediated excitotoxicity was not altered in the Bax -/- homozygous knockout animals. In contrast, there was an approximately 50% inhibition of spontaneous cell death. These results suggest that a classical Bax-dependent apoptotic pathway contributes to the spontaneous cell death that takes place when nerve cells are initially exposed to cell culture conditions. A Bax-dependent programmed cell death pathway is not, however, utilized in oxidative glutamate toxicity and NMDA receptor-mediated excitotoxicity following a brief exposure to low concentrations of glutamate.     

Increased brain endothelial nitric oxide synthase expression in thiamine deficiency: relationship to selective vulnerability

Thiamine deficiency results in selective neuronal cell death in thalamic structures. Previous studies provide evidence for a role implicating nitric oxide (NO) in the pathogenesis of cell death due to thiamine deficiency. In order to ascertain the origin of increased NO in the thiamine deficient brain, expression of endothelial nitric oxide synthase isoform (eNOS), was measured in the medial thalamus and in the inferior colliculus and compared to the frontal cortex (a spared region) of rats in which thiamine deficiency was induced through a feeding protocol of thiamine-deficient diet concomitant with daily administration of pyrithiamine, a central thiamine antagonist. eNOS mRNA and protein expression were significantly increased as a function of the severity of neurological impairment and the degree of neuronal cell loss in the medial thalamus and in the inferior colliculus. These findings suggest that the vascular endothelium is a major site of NO production in the brain in thiamine deficiency and that eNOS-derived NO could account for the selective damage to the thalamic structures that are observed in this particular disorder.     

Cerebral ischemic preconditioning reduces glutamate excitotoxicity by up-regulating the uptake activity of GLT-1 in rats

Our previous study has shown that cerebral ischemic preconditioning (CIP) can up-regulate the expression of glial glutamate transporter-1 (GLT-1) during the induction of brain ischemic tolerance in rats. The present study was undertaken to further explore the uptake activity of GLT-1 in the process by observing the changes in the concentration of extracellular glutamate with cerebral microdialysis and high-performance liquid chromatography. The results showed that a significant pulse of glutamate concentration reached the peak value of sevenfold of the basal level after lethal ischemic insult, which was associated with delayed neuronal death in the CA1 hippocampus. When the rats were pretreated 2 days before the lethal ischemic insult with CIP which protected the pyramidal neurons against delayed neuronal death, the peak value of glutamate concentration decreased to 3.9 fold of the basal level. Furthermore, pre-administration of dihydrokainate, an inhibitor of GLT-1, prevented the protective effect of CIP on ischemia-induced CA1 cell death. At the same time, compared with the CIP + Ischemia group, the peak value of glutamate concentration significantly increased and reached sixfold of the basal level. These results indicate that CIP induced brain ischemic tolerance via up-regulating GLT-1 uptake activity for glutamate and then decreasing the excitotoxicity of glutamate.     

Long-lasting changes in regional brain amino acids and monoamines in recovered pyrithiamine treated rats

Rats were subjected to a severe bout of thiamine deficiency induced by daily pyrithiamine +a thiamine deficient diet, reversed by thiamine administration and allowed to recover. Pyrithiamine treated animals demonstrated impaired retention of a 24 h recall of passive avoidance. Regional brain concentration of norepinephrine, dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid, GABA, glutamate, aspartate, glutamine, and glycine were determined after 2 and 9 weeks of nutritional recovery. A significant increase in NE content of cerebellum from the pyrithiamine treated animals was observed at both 2 and 9 week recovery periods. The concentrations of serotonin and its metabolite were signifciantly elevated in midbrain-thalamus and striatum. Significant reductions of GABA and glutamate were also observed in midbrain-thalamus. Amino acid levels in all other brain areas were unchanged from pair-fed controls. These results suggest regionally specific, chronic alterations in GABA, glutamate, serotonin, and norepinephrine activity following recovery from an acute bout of pyrithiamine-induced thiamine deficiency. The absence of a permanent reduction of cortical norepinephrine similar to that observed in an earlier study is discussed.     

NMDA and non-NMDA receptors stimulation causes differential oxidative stress in rat cortical slices

Glutamate receptor activated neuronal cell death is attributed to a massive influx of Ca(2+) and subsequent formation of reactive oxygen species (ROS) but the relative contribution of NMDA and non-NMDA sub-types of glutamate receptors in excitotoxicity is not known. In the present study, we have examined the role of NMDA and non-NMDA receptors in glutamate-induced neuronal injury in cortical slices from young (20+/-2 day) and adult (80+/-5 day) rats. Treatment of slices with glutamate receptor agonists NMDA, AMPA and KA elicited the formation of reactive oxygen species (ROS) and neuronal cell death. In young slices, NMDA receptor stimulation caused a higher ROS formation and neurotoxicity, but KA was more effective in producing ROS and cell death in adult slices. AMPA exhibited an intermediate effect on ROS formation and toxicity in both the age groups. A significant protection in glutamate mediated ROS formation and neurotoxicity was observed in presence of NMDA or/and non-NMDA receptors antagonists APV and NBQX, respectively. This further confirms the involvement of both NMDA and non-NMDA receptors in glutamate mediated neurotoxicity. In adult slices, we did not find positive correlation between ligand induced neurotoxicity and mitochondrial depolarization. Though, NMDA and KA stimulation produced differential effect on ROS formation and neurotoxicity in young and adult slices, the mitochondrial depolarization was higher and comparable on NMDA stimulation in both the age groups as compared to KA, suggesting that the mitochondrial depolarization may not be a good indicator for neurotoxicity. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors are involved in glutamate mediated neurotoxicity but their relative contribution is highly dependent on the age of the animal.     

p53-dependent neuronal cell death in a DJ-1-deficient zebrafish model of Parkinson's disease

Mutations in DJ-1 lead to early onset Parkinson's disease (PD). The aim of this study was to elucidate further the underlying mechanisms leading to neuronal cell death in DJ-1 deficiency in vivo and determine whether the observed cell loss could be prevented pharmacologically. Inactivation of DJ-1 in zebrafish, Danio rerio, resulted in loss of dopaminergic neurons after exposure to hydrogen peroxide and the proteasome inhibitor MG132. DJ-1 knockdown by itself already resulted in increased p53 and Bax expression levels prior to toxin exposure without marked neuronal cell death, suggesting subthreshold activation of cell death pathways in DJ-1 deficiency. Proteasome inhibition led to a further increase of p53 and Bax expression with widespread neuronal cell death. Pharmacological p53 inhibition either before or during MG132 exposure in vivo prevented dopaminergic neuronal cell death in both cases. Simultaneous knockdown of DJ-1 and the negative p53 regulator mdm2 led to dopaminergic neuronal cell death even without toxin exposure, further implicating involvement of p53 in DJ-1 deficiency-mediated neuronal cell loss. Our study demonstrates the utility of zebrafish as a new animal model to study PD gene defects and suggests that modulation of downstream mechanisms, such as p53 inhibition, may be of therapeutic benefit.     

Relationships among seizures, extracellular amino acid changes, and neurodegeneration induced by 4-aminopyridine in rat hippocampus: a microdialysis and electroencephalographic study

4-Aminopyridine is a powerful convulsant that induces the release of neurotransmitters, including glutamate. We report the effect of intrahippocampal administration of 4-aminopyridine at six different concentrations through microdialysis probes on EEG activity and on concentrations of extracellular amino acids and correlate this effect with histological changes in the hippocampus. 4-Aminopyridine induced in a concentration-dependent manner intense and frequent epileptic discharges in both the hippocampus and the cerebral cortex. The three highest concentrations used induced also a dose-dependent enhancement of extracellular glutamate, aspartate, and GABA levels and profound hippocampal damage. Neurodegenerative changes occurred in CA1, CA3, and CA4 subfields, whereas CA2 was spared. In contrast, microdialysis administration of a depolarizing K+ concentration and of tetraethylammonium resulted in increased amino acid levels but no epileptic activity and no or moderate neuronal damage. These results suggest that seizure activity induced by 4-aminopyridine is due to a combined action of excitatory amino acid release and direct stimulation of neuronal firing, whereas neuronal death is related to the increased glutamate release but is independent of seizure activity. In addition, it is concluded that the glutamate release-inducing effect of 4-aminopyridine results in excitotoxicity because it occurs at the level of nerve endings, thus permitting the interaction of glutamate with its postsynaptic receptors, which is probably not the case after K+ depolarization.     

A highly sulfated chondroitin sulfate preparation, CS-E, prevents excitatory amino acid-induced neuronal cell death

Chondroitin sulfate (CS) is a major microenvironmental molecule in the CNS, and there have been few reports about its neuroprotective activity. As neuronal cell death by excitotoxicity is a crucial phase in many neuronal diseases, we examined the effect of various CS preparations on neuronal cell death induced by the excitotoxicity of glutamate analogs. CS preparations were added to cultured neurons before and after the administration of glutamate analogs. Then, the extents of both neuronal cell death and survival were estimated. Pre-administration of a highly sulfated CS preparation, CS-E, significantly reduced neuronal cell death induced by not only NMDA but also ( S )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate. Neither CS preparations other than CS-E nor other highly sulfated polysaccharides such as heparin and dextran sulfate exerted any neuroprotective effects. NMDA-induced current in neurons was not changed by pre-administration of CS-E, but the pattern of protein-tyrosine phosphorylation was changed. In addition, the elevation of caspase 3 activity was significantly suppressed in CS-E-treated neurons. These results indicate that CS-E prevents neuronal cell death mediated by various glutamate receptors, and suggest that phosphorylation-related intracellular signals and the suppression of caspase 3 activation are implicated in neuroprotection by CS-E.     

Ascorbate and Glutamate Release in the Rat Hippocampus After Perforant Path Stimulation: A "Dialysis Electrode" Study

Abstract: Excitatory amino acids have been proposed to play a critical role in the development and maintenance of epileptic seizures and in the development of neuronal damage. Previous animal studies of glutamate during seizures, however, have often failed to measure any rise in glutamate. We have overcome many of the problems of these studies by using an animal model in which epileptic afterdischarges are induced by stimulation of the perforant path, and glutamate and ascorbate are measured using a newly developed microdialysis electrode that combines the advantages of microdialysis and in vivo electrochemistry. We have successfully shown (1) a rise in glutamate after an epileptic afterdischarge, (2) a concomitant initial fall and then a later rise in ascorbate, and (3) progressive dwindling of this effect when afterdischarges are repeated within minutes, despite similar electroencephalographic responses. The possible mechanisms of these effects are discussed and include ascorbate/glutamate heteroexchange, reversal of the glutamate uptake mechanism, and augmentation of glutamate uptake after a seizure.