Alan S. Hazell

Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury

Glutamate excitotoxicity plays an important role in the development of secondary injuries that occur following traumatic brain injury (TBI), and contributes significantly to expansion of the total volume of injury. Acute increases in extracellular glutamate levels have been detected in both experimental brain trauma models and in human patients, and can lead to over-stimulation of glutamate receptors, resulting in a cascade of excitotoxic-related mechanisms culminating in neuronal damage. These elevated levels of glutamate can be effectively controlled by the astrocytic glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2). However, evidence indicate these transporters and splice variant are downregulated shortly following the insult, which then precipitates glutamate-mediated excitotoxic conditions. Lack of success with glutamate receptor antagonists as a potential source of clinical intervention treatment following TBI has resulted in the necessity for a better understanding of the mechanisms that underlie the process of excitotoxicity, including the function and regulation of glutamate transporters. Such new insight should improve the likelihood of development of novel avenues for therapeutic intervention following TBI.

Excitotoxic mechanisms in stroke: An update of concepts and treatment strategies

Cerebral damage as a consequence of glutamate-mediated excitotoxicity represents a major consequence of stroke. However, the development of effective clinical treatments for this potentially devastating condition has been largely unsuccessful to date, despite promising basic research. This review will focus on the latest advances in our understanding of the excitotoxic process including the release of glutamate as a neurotransmitter and the potential contribution of complexins, the important role of astrocytes, including its involvement in glutamate uptake, alterations in glutamate transporter levels, reversed glutamate uptake, and the vesicular release of glutamate. Recent progress in our understanding of the involvement of excitotoxicity in white matter injury following ischemic insults is also discussed, as is oxidative stress and ischemic tolerance, along with an update on the use of treatment strategies with potential therapeutic benefit including stimulation of neurogenesis. Such key issues are at the heart of future interventions directed at limiting the extent of the excitotoxic process, and remain a viable consideration for effective stroke management.

Manganese Neurotoxicity: An Update of Pathophysiologic Mechanisms

The central nervous system, and the basal ganglia in particular, is an important target in manganese neurotoxicity, a disorder producing neurological symptoms similar to that of Parkinsons disease. Increasing evidence suggests that astrocytes are a site of early dysfunction and damage; chronic exposure to manganese leads to selective dopaminergic dysfunction, neuronal loss, and gliosis in basal ganglia structures together with characteristic astrocytic changes known as Alzheimer type II astrocytosis. Astrocytes possess a high affinity, high capacity, specific transport system for manganese facilitating its uptake, and sequestration in mitochondria, leading to a disruption of oxidative phosphorylation. In addition, manganese causes a number of other functional changes in astrocytes including an impairment of glutamate transport, alterations of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, production of nitric oxide, and increased densities of binding sites for the “peripheral-type” benzodiazepine receptor (a class of receptor predominantly localized to mitochondria of astrocytes and involved in oxidative metabolism, mitochondrial proliferation, and neurosteroid synthesis). Such effects can lead to compromised energy metabolism, resulting in altered cellular morphology, production of reactive oxygen species, and increased extracellular glutamate concentration. These consequences may result in impaired astrocytic–neuronal interactions and play a major role in the pathophysiology of manganese neurotoxicity.

Effects of ammonia on glutamate transporter (GLAST) protein and mRNA in cultured rat cortical astrocytes

Ammonia is a neurotoxic substance which accumulates in brain in liver failure and it has been suggested that ammonia plays a key role in contributing to the astrocytic dysfunction characteristic of hepatic encephalopathy. In particular, the effects of ammonia may be responsible for the reduced astrocytic uptake of neuronally-released glutamate and high extracellular glutamate levels consistently seen in experimental models of hepatic encephalopathy. To further address this issue, [(3)H]-D-aspartate uptake was examined in primary rat cortical astrocyte cultures exposed to 5 mM ammonium chloride for a period of 7 days. In addition, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot studies were performed to examine the mRNA and protein expression respectively of the glutamate transporter GLAST in ammonia-treated cells. Studies revealed a 57% (p<0.05) decrease in [(3)H]-D-aspartate uptake and a concomitant significant decrease in GLAST transporter protein (43%, p<0.05) and mRNA (32%, p<0.05) expression. The reduced capacity of astrocytes to reuptake glutamate following ammonia exposure may result in compromised neuron-astrocyte trafficking of glutamate and could thus contribute to the pathogenesis of the cerebral dysfunction characteristic of hyperammonemic syndromes such as hepatic encephalopathy.

Mechanisms of Neuronal Cell Death in Wernicke's Encephalopathy

Wernickes Encephalopathy (WE) is a serious neurological disorder resulting from thiamine deficiency, encountered in chronic alcoholics and in patients with grossly impaired nutritional status. Neuropathologic studies as well as Magnetic Resonance Imaging reveal selective diencephalic and brainstem lesions in patients with WE. The last decade has witnessed major advances in the understanding of pathophysiologic mechanisms linking thiamine deficiency to the selective brain lesions characteristic of WE. Activities of the thiamine-dependent enzyme α-ketoglutarate dehydrogenase, a rate-limiting tricarboxylic acid cycle enzyme are significantly reduced in autopsied brain tissue from patients with WE and from rats treated with the central thiamine antagonist, pyrithiamine. In the animal studies, evidence suggests that such enzyme deficits result in focal lactic acidosis, cerebral energy impairment and depolarization resulting from increased release of glutamate in vulnerable brain structures. It has been proposed that this depolarization may result in N-Methyl-D-Aspartate receptor-mediated excitotoxicity as well as increased expression of immediate early genes such as c-fos and c-jun resulting in apoptotic cell death. Other mechanisms involved in thiamine deficiency-induced cell loss may involve free radicals and alterations of the blood-brain barrier. Additional studies are still required to identify the site of the initial cellular insult and to explain the predilection of diencephalic and brainstem structures due to thiamine deficiency.

Energy metabolism in astrocytes and neurons treated with manganese: relation among cell-specific energy failure, glucose metabolism, and intercellular trafficking using multinuclear NMR-spectroscopic analysis.

A central question in manganese neurotoxicity concerns mitochondrial dysfunction leading to cerebral energy failure. To obtain insight into the underlying mechanism(s), the authors investigated cell-specific pathways of [1–13C]glucose metabolism by high-resolution multinuclear NMR-spectroscopy. Five-day treatment of neurons with 100-μmol/L MnCl2 led to 50% and 70% decreases of ATP/ADP and phosphocreatine–creatine ratios, respectively. An impaired flux of [1-13C]glucose through pyruvate dehydrogenase, which was associated with Krebs cycle inhibition and hence depletion of [4–13C]glutamate, [2–13C]GABA, and [13C]glutathione, hindered the ability of neurons to compensate for mitochondrial dysfunction by oxidative glucose metabolism and further aggravated neuronal energy failure. Stimulated glycolysis and oxidative glucose metabolism protected astrocytes against energy failure and oxidative stress, leading to twofold increased de novo synthesis of [3–13C]lactate and fourfold elevated [4–13C]glutamate and [13C]glutathione levels. Manganese, however, inhibited the synthesis and release of glutamine. Comparative NMR data obtained from cocultures showed disturbed astrocytic function and a failure of astrocytes to provide neurons with substrates for energy and neurotransmitter metabolism, leading to deterioration of neuronal antioxidant capacity (decreased glutathione levels) and energy metabolism. The results suggest that, concomitant to impaired neuronal glucose oxidation, changes in astrocytic metabolism may cause a loss of intercellular homeostatic equilibrium, contributing to neuronal dysfunction in manganese neurotoxicity.

Update of Cell Damage Mechanisms in Thiamine Deficiency: Focus on Oxidative Stress, Excitotoxicity and Inflammation

Thiamine deficiency (TD) is a well-established model of Wernickes encephalopathy. Although the neurologic dysfunction and brain damage resulting from the biochemical consequences of TD is well characterized, the mechanism(s) that lead to the selective histological lesions characteristic of this disorder remain a mystery. Over the course of many years, various structural and functional changes have been identified that could lead to cell death in this disorder. However, despite a concerted effort to explain the consequences of TD in terms of these changes, our understanding of the pathophysiology of this disorder remains unclear. This review will focus on three of these processes, i.e. oxidative stress, glutamate-mediated excitotoxicity and inflammation and their role in selective vulnerability in TD. Since TD inhibits oxidative metabolism, a feature of many neurodegenerative disease states, it represents a model system with which to explore pathological mechanisms inherent in such maladies, with the potential to yield new insights into their possible treatment and prevention.

Selective down‐regulation of the astrocyte glutamate transporters GLT‐1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT‐1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT‐1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non‐vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT‐3 were unaffected in the medial thalamus. In addition, astrocyte‐specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT‐1 or GLAST protein was not observed on day 12 of treatment, indicating that down‐regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT‐1 content was positively correlated with levels of the neurofilament protein α‐internexin, suggesting that early neuronal drop‐out may contribute to the down‐regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT‐1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency‐induced cell death.

Astrocytes are a major target in thiamine deficiency and Wernicke's encephalopathy

Thiamine deficiency (TD) is the underlying cause, and an established model, of Wernickes encephalopathy (WE). Although the neurologic dysfunction and brain damage that results from TD has been well-described, the precise mechanisms that lead to the selective histological lesions characteristic of this disorder remain a mystery. Over the course of many years, various processes have been proposed that could lead to focal neuronal cell death in this disorder. But despite a concerted effort to relate these processes to a clear sequelae of events culminating in development of the focal neuropathology, little success has resulted. In recent years, however, a role for astrocytes in the pathophysiology of TD has been emerging. Here, alterations in glutamate uptake, and levels of the astrocytic glutamate transporters EAAT1 and EAAT2 in TD and WE, are discussed in terms of an excitotoxic event, along with the GABA transporter subtype GAT-3, and changes in other astrocytic proteins including GFAP and glutamine synthetase. Lactic acidosis, changes in the water channel protein AQP-4 and brain edema are also a focus of attention in relation to astrocyte dysfunction, while involvement of oxidative stress and inflammatory processes, along with white matter injury in terms of excitotoxicity are other key issues considered. In summary, a new appraisal of the extent of involvement of astrocytes in TD and WE is presented, with the evidence suggesting these cells represent a major target for damage during the disease process.

Alzheimer type II astrocytic changes following sub-acute exposure to manganese and its prevention by antioxidant treatment

Exposure to manganese in an industrial or clinical setting can lead to manganism, a neurological disorder with similarities to Parkinsons disease. Although the pathogenetic basis of this disorder is unclear, studies indicate this metal is highly accumulated in astrocytes, suggesting an involvement of these glial cells. To investigate this issue, we have used a recently characterized, sub-acute model of manganese neurotoxicity. Treatment of rats with manganese (II) chloride (50 mg/kg body weight, i.p.) once daily for 1 or 4 days led to increases in manganese levels of up to 232, 523, and 427% in the cerebral cortex, globus pallidus, and cerebellum, respectively, by instrumental neutron activation analysis. These changes were accompanied by development of pathological changes in glial morphology identified as Alzheimer type II astrocytosis in both cortical and sub-cortical structures. Co-treatment with either the antioxidant N-acetylcysteine or the manganese chelator 1,2-cyclohexylenedinitrilotetraacetic acid completely blocked this pathology, indicating the cellular transformation may be mediated by oxidative stress associated with the presence of this metal. These findings represent, to our knowledge, the first report of early induction of this pathological hallmark of manganese neurotoxicity, an event previously considered a consequence of chronic exposure to manganese in primates and in human cases of manganism. Our results also indicate that use of this rodent model may provide a novel opportunity to examine the nature and role of the Alzheimer type II astrocyte in the pathophysiology of this disorder as well as in other disease processes in which cerebral accumulation of manganese occurs.