Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: A perspective on the contributions of apoptosis and necrosis

In the human brain and spinal cord, neurons degenerate after acute insults (e.g., stroke, cardiac arrest, trauma) and during progressive, adult-onset diseases [e.g., amyotrophic lateral sclerosis, Alzheimer's disease]. Glutamate receptor-mediated excitotoxicity has been implicated in all of these neurological conditions. Nevertheless, effective approaches to prevent or limit neuronal damage in these disorders remain elusive, primarily because of an incomplete understanding of the mechanisms of neuronal death in in vivo settings. Therefore, animal models of neurodegeneration are crucial for improving our understanding of the mechanisms of neuronal death. In this review, we evaluate experimental data on the general characteristics of cell death and, in particular, neuronal death in the central nervous system (CNS) following injury. We focus on the ongoing controversy of the contributions of apoptosis and necrosis in neurodegeneration and summarize new data from this laboratory on the classification of neuronal death using a variety of animal models of neurodegeneration in the immature or adult brain following excitotoxic injury, global cerebral ischemia, and axotomy/target deprivation. In these different models of brain injury, we determined whether the process of neuronal death has uniformly similar morphological characteristics or whether the features of neurodegeneration induced by different insults are distinct. We classified neurodegeneration in each of these models with respect to whether it resembles apoptosis, necrosis, or an intermediate form of cell death falling along an apoptosis-necrosis continuum. We found that N-methyl-D-aspartate (NMDA) receptor- and non-NMDA receptor-mediated excitotoxic injury results in neurodegeneration along an apoptosis-necrosis continuum, in which neuronal death (appearing as apoptotic, necrotic, or intermediate between the two extremes) is influenced by the degree of brain maturity and the subtype of glutamate receptor that is stimulated. Global cerebral ischemia produces neuronal death that has commonalities with excitotoxicity and target deprivation. Degeneration of selectively vulnerable populations of neurons after ischemia is morphologically nonapoptotic and is indistinguishable from NMDA receptor-mediated excitotoxic death of mature neurons. However, prominent apoptotic cell death occurs following global ischemia in neuronal groups that are interconnected with selectively vulnerable populations of neurons and also in nonneuronal cells. This apoptotic neuronal death is similar to some forms of retrograde neuronal apoptosis that occur following target deprivation. We conclude that cell death in the CNS following injury can coexist as apoptosis, necrosis, and hybrid forms along an apoptosis-necrosis continuum. These different forms of cell death have varying contributions to the neuropathology resulting from excitotoxicity, cerebral ischemia, and target deprivation/axotomy. Degeneration of different populations of cells (neurons and nonneuronal cells) may be mediated by distinct or common causal mechanisms that can temporally overlap and perhaps differ mechanistically in the rate of progression of cell death.     

Central infusion of glucagon-like peptide-1-(7-36) amide (GLP-1) receptor antagonist attenuates lithium chloride-induced c-Fos induction in rat brainstem

Studies of neuronal injury and death after cerebral ischemia and various neurodegenerative diseases have increasingly focused on the interactions between mitochondrial function, reactive oxygen species (ROS) production and glutamate neurotoxicity. Recent findings suggest that increased mitochondrial ROS production precedes neuronal death after glutamate treatment. It is hypothesized that under pathological conditions when mitochondrial function is compromised, extracellular glutamate may exacerbate neuronal injury. In the present study, we focus on the relationship between mitochondrial superoxide production and glutamate neurotoxicity in cultured cortical neurons with normal or reduced levels of manganese-superoxide dismutase (MnSOD) activity. Our results demonstrate that neurons with reduced MnSOD activity are significantly more sensitive to transient exposure to extracellular glutamate. The increased sensitivity of cultured cortical neurons with reduced MnSOD activity is characteristically subject only to treatment by glutamate but not to other glutamate receptor agonists, such as N-methyl-d-aspartate, kainate and quisqualate. We suggest that the reduced MnSOD activity in neurons may exacerbate glutamate neurotoxicity via a mechanism independent of receptor activation.     

NMDA-induced superoxide production and neurotoxicity in cultured rat hippocampal neurons: role of mitochondria

Excitotoxic mechanisms are believed to be involved in the death of neurons after trauma, epileptic seizures and cerebral ischaemia. We investigated the role of mitochondrial superoxide production in excitotoxic cell death of cultured rat hippocampal neurons. Brief exposure to the selective glutamate agonist N-methyl-D-aspartate (NMDA; 100-300 microM, 10 min) induced significant neuronal death, which was sensitive to cycloheximide (1 microM) and the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-chloromethylketone (10 microM). Intracellular superoxide production was monitored semiquantitatively on sister cultures from the same platings using the oxidation-sensitive probe, hydroethidine. Brief exposures to toxic NMDA concentrations induced significant increases in superoxide production which correlated with the degree of neuronal injury. However, subtoxic NMDA exposures also produced moderate, yet statistically significant increases in superoxide production. Both NMDA-induced superoxide production and neurotoxicity were reduced by inhibition of mitochondrial electron transport using either sodium cyanide (1 mM), or a combination of rotenone (2 microM) and oligomycin (2 microM). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 1 microM) mimicked the effect of NMDA on mitochondrial superoxide production. Both NMDA-induced superoxide production and neurotoxicity were potentiated by FCCP (1 microM). Exposure to FCCP alone (1-10 microM, 10 min), however, failed to produce any toxicity. Our data suggest that mitochondrial superoxide production per se is not sufficient to trigger the degeneration of cultured hippocampal neurons, but that manipulation of mitochondrial activity alters NMDA-induced superoxide production and neurotoxicity.     

Uric acid protects neurons against excitotoxic and metabolic insults in cell culture, and against focal ischemic brain injury in vivo

Uric acid is a well-known natural antioxidant present in fluids and tissues throughout the body. Oxyradical production and cellular calcium overload are believed to contribute to the damage and death of neurons that occurs following cerebral ischemia in victims of stroke. We now report that uric acid protects cultured rat hippocampal neurons against cell death induced by insults relevant to the pathogenesis of cerebral ischemia, including exposure to the excitatory amino acid glutamate and the metabolic poison cyanide. Confocal laser scanning microscope analyses showed that uric acid suppresses the accumulation of reactive oxygen species (hydrogen peroxide and peroxynitrite), and lipid peroxidation, associated with each insult. Mitochondrial function was compromised by the excitotoxic and metabolic insults, and was preserved in neurons treated with uric acid. Delayed elevations of intracellular free calcium levels induced by glutamate and cyanide were significantly attenuated in neurons treated with uric acid. These data demonstrate a neuroprotective action of uric acid that involves suppression of oxyradical accumulation, stabilization of calcium homeostasis, and preservation of mitochondrial function. Administration of uric acid to adult rats either 24 hr prior to middle cerebral artery occlusion (62.5 mg uric acid/kg, intraperitoneally) or 1 hr following reperfusion (16 mg uric acid/kg, intravenously) resulted in a highly significant reduction in ischemic damage to cerebral cortex and striatum, and improved behavioral outcome. These findings support a central role for oxyradicals in excitotoxic and ischemic neuronal injury, and suggest a potential therapeutic use for uric acid in ischemic stroke and related neurodegenerative conditions.     

Self-adaptation in evolving systems

While a high rate of cell loss is tolerated and even required to model the developing nervous system, an increased rate of cell death in the adult nervous system underlies neurodegenerative disease. Evolutionarily conserved mechanisms involving proteases, Bcl-2-related proteins, p53, and mitochondrial factors participate in the modulation and execution of cell death. In addition, specific death mechanisms, based on specific neuronal characteristics such as excitability and the presence of specific channels or enzymes, have been unraveled in the brain. Particularly important for various human diseases are excessive nitric oxide (NO) production and excitotoxicity. These two pathological mechanisms are closely linked, since excitotoxic stimulation of neurons may trigger enhanced NO production and exposure of neurons to NO may trigger the release of excitotoxins. Depending on the experimental situation and cell type, excitotoxic neuronal death may either be apoptotic or necrotic.     

Progressive neurodegeneration after intracerebroventricular kainic acid administration in rats: implications for schizophrenia?

BACKGROUND: Intracerebroventricular (ICV) administration of kainic acid to rats produces limbic-cortical neuronal damage that has been compared to the neuropathology of schizophrenia. METHODS: Groups of adult rats were administered ICV kainic acid and then assessed for neuronal loss and the expression of proteins relevant to mechanisms of neuronal damage after one and fourteen days. Neuronal loss was assessed by two-dimensional cell counting and protein expression was assessed by immunohistochemistry. RESULTS: ICV kainic acid administration was associated with both immediate (day 1) and delayed (day 14) neuronal loss in the dorsal hippocampus. The immediate injury was largely limited to the CA3 hippocampal subfield, while the delayed injury included the CA1 subfield. Multiple mechanisms of cell death appeared to be involved in the delayed neuronal loss, as evidenced by changes in the expression of glutamate receptor subunits, heat shock protein and jun protein. CONCLUSIONS: ICV kainic acid administration to adult rats produces progressive damage to limbic-cortical neurons, involving both fast and slow mechanisms of cell death. Given the evidence for clinical deterioration, cognitive deficits and hippocampal neuropathy in some cases of schizophrenia, this animal model may be relevant for hypotheses regarding mechanisms of neurodegeneration in that disorder.     

Oxidative metabolism, apoptosis and perinatal brain injury

Perinatal hypoxic-ischaemic injury (HII) is a significant cause of neurodevelopmental impairment and disability. Studies employing 31P magnetic resonance spectroscopy to measure phosphorus metabolites in situ in the brains of newborn infants and animals have demonstrated that transient hypoxia-ischaemia leads to a delayed disruption in cerebral energy metabolism, the magnitude of which correlates with the subsequent neurodevelopmental impairment. Prominent among the biochemical features of HII is the loss of cellular ATP, resulting in increased intracellular Na+ and Ca2+, and decreased intracellular K+. These ionic imbalances, together with a breakdown in cellular defence systems following HII, can contribute to oxidative stress with a net increase in reactive oxygen species. Subsequent damage to lipids, proteins, and DNA and inactivation of key cellular enzymes leads ultimately to cell death. Although the precise mechanisms of neuronal loss are unclear, it is now clear both apoptosis and necrosis are the significant components of cell death following HII. A number of different factors influence whether a cell will undergo apoptosis or necrosis, including the stage of development, cell type, severity of mitochondrial injury and the availability of ATP for apoptotic execution. This review will focus on some pathological mechanisms of cell death in which there is a disruption to oxidative metabolism. The first sections will discuss the process of damage to oxidative metabolism, covering the data collected both from human infants and from animal models. Following sections will deal with the molecular mechanisms that may underlie cerebral energy failure and cell death in this form of brain injury, with a particular emphasis on the role of apoptosis and mitochondria.     

Lateralization of temporal lobe epilepsy based on regional metabolic abnormalities in proton magnetic resonance spectroscopic images

Magnetic resonance spectroscopic imaging (MRSI) is capable of determining the spatial distribution in vivo of cerebral metabolites, including N-acetylaspartate (NAA), a compound found only in neurons. We used this technique in 10 patients with temporal lobe epilepsy (TLE) to determine the location of maximal neuronal/axonal loss or damage and to evaluate the potential of MRSI for presurgical lateralization. Asymmetry of the relative resonance intensity of NAA to creatine was determined for mid and posterior regions of the temporal lobes defined anatomically and also for "metabolic lesions" defined as the regions of maximal abnormality on MRSI. MRSI revealed decreased relative signal intensity in at least one temporal lobe of all patients. Two patients had a widespread reduction in NAA in both temporal lobes. The region of maximal abnormality was usually in the posterior temporal lobe but sometimes in the mid temporal lobe. The side of lowest NAA was ipsilateral to the clinical electroencephalographic lateralization in all patients. Lateralization based on NAA to creatine correlated with the atrophy of amygdala and hippocampus in 8 patients who showed this on magnetic resonance imaging volumetric measurements. MRSI can demonstrate regional neuronal loss or damage that correlates with clinical electroencephalographic and structural lateralization in temporal lobe epilepsy. The ability to identify a region of maximal metabolic abnormality on spectroscopic images may confer greater sensitivity than that available from single voxel methods. The maximal metabolic abnormality may not be located in a voxel defined a priori, and based on anatomical considerations, without knowledge of the distribution of the metabolic abnormality.     

Nitric oxide acutely inhibits neuronal energy production. The Committees on Neurobiology and Cell Physiology

Disruption of mitochondrial respiration has been proposed as an action of nitric oxide (NO) responsible for its toxicity, but the effects of NO on the energetics of intact central neurons have not been reported. We examined the effects of NO on mitochondrial function and energy metabolism in cultured hippocampal neurons. The application of NO from NO donors or from dissolved gas produced a rapid, reversible depolarization of mitochondrial membrane potential, as detected by rhodamine-123 fluorescence. NO also produced a progressive concentration-dependent depletion of cellular ATP over 20 min exposures. The energy depletion produced by higher levels of NO (2 microM or more) was profound and irreversible and proceeded to subsequent neuronal death. In contrast to the effects of NO, mitochondrial protonophores produced complete depolarizations of mitochondrial membrane potential but depleted the neuronal ATP stores only partially. Inhibitors of mitochondrial oxidative phosphorylation (rotenone or 3-nitropropionic acid) or of glycolysis (iodoacetate plus pyruvate) also produced only partial ATP depletion, suggesting that either process alone could partially maintain ATP stores. Only by combining the inhibition of glycolytic energy production with the inhibition of mitochondria could the effects of NO in depleting energy and inducing delayed toxicity be duplicated. These results show that NO has rapid inhibitory actions on mitochondrial metabolism in living neurons. However, the severe ATP-depleting effects of high concentrations of NO are not fully explained by the direct effects on mitochondrial activity alone but must involve the inhibition of glycolysis as well. These inhibitory effects on energy production may contribute to the delayed toxicity of NO in vitro and in ischemic stroke.     

Glutamate in neurologic diseases

Excitotoxicity has been implicated as a mechanism of neuronal death in acute and chronic neurologic diseases. Cerebral ischemia, head and spinal cord injury, and prolonged seizure activity are associated with excessive release of glutamate into the extracellular space and subsequent neurotoxicity. Accumulating evidence suggests that impairment of intracellular energy metabolism increases neuronal vulnerability to glutamate which, even when present at physiologic concentrations, can damage neurons. This mechanism of slow excitotoxicity may be involved in neuronal death in chronic neurodegenerative diseases such as the mitochondrial encephalomyopathies, Huntington's disease, spinocerebellar degeneration syndromes, and motor neuron diseases. If so, glutamate antagonists in combination with agents that selectively inhibit the multiple steps downstream of the excitotoxic cascade or help improve intracellular energy metabolism may slow the neurodegenerative process and offer a therapeutic approach to treat these disorders.     

Differential use of immunoglobulin light chain genes and B lymphocyte expansion at sites of disease in rheumatoid arthritis (RA) compared with circulating B lymphocytes

Patients infected with HIV-1 often exhibit cognitive deficits that are related to progressive neuronal degeneration and cell death. The protein Tat, which is released from HIV-1-infected cells, was recently shown to be toxic toward cultured neurons. We now report that Tat induces apoptosis in cultured embryonic rat hippocampal neurons. Tat induced caspase activation, and the caspase inhibitor zVAD-fmk prevented Tat-induced neuronal death. Tat induced a progressive elevation of cytoplasmic-free calcium levels, which was followed by mitochondrial calcium uptake and generation of mitochondrial-reactive oxygen species (ROS). The intracellular calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red protected neurons against Tat-induced apoptosis. zVAD-fmk suppressed Tat-induced increases of cytoplasmic calcium levels and mitochondrial ROS accumulation, indicating roles for caspases in the perturbed calcium homeostasis and oxidative stress induced by Tat. An inhibitor of nitric oxide synthase, and the peroxynitrite scavenger uric acid, protected neurons against Tat-induced apoptosis, indicating requirements for nitric oxide production and peroxynitrite formation in the cell death process. Finally, Tat caused a delayed and progressive mitochondrial membrane depolarization, and cyclosporin A prevented Tat-induced apoptosis, suggesting an important role for mitochondrial membrane permeability transition in Tat-induced apoptosis. Collectively, our data demonstrate that Tat can induce neuronal apoptosis by a mechanism involving disruption of calcium homeostasis, caspase activation, and mitochondrial calcium uptake and ROS accumulation. Agents that interupt this apoptotic cascade may prove beneficial in preventing neuronal degeneration and associated dementia in AIDS patients.     

S100beta induces neuronal cell death through nitric oxide release from astrocytes

The glial-derived neurotrophic protein S100beta has been implicated in the development and maintenance of the nervous system. S100beta has also been postulated to play a role in mechanisms of neuropathology because of its specific localization and selective overexpression in Alzheimer's disease. However, the exact relationship between S100beta overexpression and neurodegeneration is unclear. Recent data have demonstrated that treatment of cultured rat astrocytes with high concentrations of S100beta results in a potent activation of inducible nitric oxide synthase (iNOS) and a subsequent generation of nitric oxide (NO), which can lead to astrocytic cell death. To investigate whether S100beta-induced NO release from astroctyes might influence neurons, we studied S100beta effects on neuroblastoma B104 cells or primary hippocampal neurons co-cultured with astrocytes. We found that S100beta treatment of astrocyte-neuron co-cultures resulted in neuronal cell death by both necrosis and apoptosis. Neuronal cell death induced by S100beta required the presence of astrocytes and depended on activation of iNOS. Cell death correlated with the levels of NO and was blocked by a specific NOS inhibitor. Our data support the idea that overexpression of S100beta may be an exacerbating factor in the neurodegeneration of Alzheimer's disease.     

Glutamate, glutamine, and GABA as substrates for the neuronal and glial compartments after focal cerebral ischemia in rats

BACKGROUND AND PURPOSE: Even though the utilization of substrates alternative to glucose may play an important role in the survival of brain cells under ischemic conditions, evidence on changes in substrate selection by the adult brain in vivo during ischemic episodes remains very limited. This study investigates the utilization of glutamate, glutamine, and GABA as fuel by the neuronal and glial tricarboxylic acid cycles of both cerebral hemispheres after partially reversible focal cerebral ischemia (FCI). METHODS: Right hemisphere infarct was induced in adult Long-Evans rats by permanent occlusion of the right middle cerebral artery and transitory occlusion of both common carotid arteries. (1,2-13C2) acetate was infused for 60 minutes in the right carotid artery immediately after carotid recirculation had been re-established (1-hour group) or 23 hours later (24-hour group). Extracts from both cerebral hemispheres were prepared and analyzed separately by 13C nuclear magnetic resonance and computer-assisted metabolic modeling. RESULTS: FCI decreased the oxidative metabolism of glucose in the brain in a time-dependent manner. Reduced glucose oxidation was compensated for by increased oxidations of (13C) glutamate and (13C) GABA in the astrocytes of the ipsilateral hemispheres of both groups. Increased oxidative metabolism of (13C) glutamine in the neurons was favored by increased activity of the neuronal pyruvate recycling system in the 24-hour group. CONCLUSIONS: Data were obtained consistent with time-dependent changes in the utilization of glutamate and GABA or glutamine as metabolic substrates for the glial or neuronal compartments of rat brain after FCI.     

Effects of extracellular ATP on hair cells isolated from the guinea-pig semicircular canals

Using stereological methods, two cerebral cortical areas from AIDS brains were investigated. Neuronal density, profile area of neurons, and perikaryon volume fraction were measured and compared to age-matched control brains. In the fronto-orbital cortex (area 11) of AIDS brains, a significant loss of neurons was seen. The perikaryon volume fraction was likewise decreased. The size of neurons did not differ between control and AIDS brains. In patients with clinical signs of progressive dementia and in brains with human immunodeficiency virus (HIV)-specific neuropathology (HIV-leukoencephalopathy and/or HIV-encephalitis) as compared to patients lacking these features, a small decrease in neuronal density was noted but this difference did not reach the level of statistical significance (P = 0.16). In the superior parietal lobule (area 7) of AIDS brains, no loss of nerve cells was noted. AIDS patients with progressive dementia and brains with HIV-specific neuropathology showed no difference in neuronal densities as compared to those without such features. We conclude that the fronto-orbital cortex, in contrast to the parietal cortex, is mainly damaged in AIDS brains. Neuronal loss was not significantly correlated with development of dementing symptoms and of HIV-specific neuropathology.     

Cellular mechanisms involved in brain ischemia

Cellular mechanisms, both destructive and protective, that are associated with cerebral ischemia are reviewed in this paper. Central to understanding the evolution of stroke are the concepts of ischemic core and surrounding penumbral region damage, delayed neuronal death, and neuronal rescue. The role of spreading depression in the evolution of subsequent ATP depletion, ion shifts, glutamate release, activation of glutamate receptors, intracellular Ca2+ changes, and generation of reactive oxygen species in the penumbra in relationship to neuronal and glial cell damage are discussed. We conclude that the most fruitful areas for future stroke research include traditional approaches as well as novel approaches. Traditional approaches include stroke prevention and examination of the effects of combinations of proven and promising effective therapeutic interventions. Novel approaches include delineating mechanisms whereby growth factors and compounds such as deprenyl and staurosporine afford neuroprotection, ultimately leading to direct manipulation of the signal transduction pathways that lead to neuronal dysfunction and death. This includes determining which genes are activated and repressed in specific response to hypoxia-ischemia and determining how such alterations in gene expression affect survival and function of neurons. We also suggest that advantage be taken of the blood-brain barrier compromise during stroke in designing neuroprotective therapies.     

Catecholamines potentiate amyloid beta-peptide neurotoxicity: involvement of oxidative stress, mitochondrial dysfunction, and perturbed calcium homeostasis

Oxidative stress and mitochondrial dysfunction are implicated in the neuronal cell death that occurs in physiological settings and in neurodegenerative disorders. In Alzheimer's disease (AD) degenerating neurons are associated with deposits of amyloid beta-peptide (A beta), and there is evidence for increased membrane lipid peroxidation and protein oxidation in the degenerating neurons. Cell culture studies have shown that A beta can disrupt calcium homeostasis and induce apoptosis in neurons by a mechanism involving oxidative stress. We now report that catecholamines (norepinephrine, epinephrine, and dopamine) increase the vulnerability of cultured hippocampal neurons to A beta toxicity. The catecholamines were effective in potentiating A beta toxicity at concentrations of 10-200 microM, with the higher concentrations (100-200 microM) themselves inducing cell death. Serotonin and acetylcholine were not neurotoxic and did not modify A beta toxicity. Levels of membrane lipid peroxidation, and cytoplasmic and mitochondrial reactive oxygen species, were increased following exposure to neurons to A beta, and catecholamines exacerbated the oxidative stress. Subtoxic concentrations of catecholamines exacerbated decreases in mitochondrial energy charge and transmembrane potential caused by A beta, and higher concentrations of catecholamines alone induced mitochondrial dysfunction. Antioxidants (vitamin E, glutathione, and propyl gallate) protected neurons against the damaging effects of A beta and catecholamines, whereas the beta-adrenergic receptor antagonist propanolol and the dopamine (D1) receptor antagonist SCH23390 were ineffective. Measurements of intracellular free Ca2+ ([Ca2+]i) showed that A beta induced a slow elevation of [Ca2+]i which was greatly enhanced in cultures cotreated with catecholamines. Collectively, these data indicate a role for catecholamines in exacerbating A beta-mediated neuronal degeneration in AD and, when taken together with previous findings, suggest roles for oxidative stress induced by catecholamines in several different neurodegenerative conditions.     

Metabolic inhibition enhances selective toxicity of L-DOPA toward mesencephalic dopamine neurons in vitro

Recent in vitro studies have described the toxicity of levodopa (L-DOPA) to dopamine (DA) neurons. We investigated whether metabolic inhibition with rotenone, an inhibitor of complex I of the mitochondrial respiratory chain, may enhance the toxicity of L-DOPA toward DA neurons in mesencephalic cultures. The uptakes of DA and GABA were determined to evaluate the functional and morphological integrity of DA and non-DA neurons, respectively. Pretreatment with rotenone significantly augmented the toxic effect of L-DOPA on DA neurons. Interestingly, prior metabolic inhibition with rotenone rendered DA cells susceptible to a dose (5 microM) of L-DOPA that otherwise exhibited no toxic effect. DA uptake was more intensely attenuated than GABA uptake after the combined exposure to rotenone and L-DOPA. This was confirmed by cell survival estimation showing that tyrosine hydroxylase-positive DA cells are more vulnerable to the sequential exposure to the drugs than total cells. The selective toxic effect of L-DOPA on rotenone-pretreated DA neurons was significantly blocked by antioxidants, but not antagonists of NMDA or non-NMDA glutamate receptors. This indicates that oxidative stress play a central role in mediating the selective damage of DA cells in the present experimental paradigm. Our results raise the possibility that long-term L-DOPA treatment could accelerate the progression of degeneration of DA neurons in patients with Parkinson's disease where potential energy failure due to mitochondrial defects has been demonstrated to take place.     

Glucose transporter expression in brain: relationship to cerebral glucose utilization

Glucose is the principle energy source for mammalian brain. Delivery of glucose from the blood to the brain requires its transport across the endothelial cells of the blood-brain barrier and across the plasma membranes of neurons and glia, which is mediated by the facilitative glucose transporter proteins. The two primary glucose transporter isoforms which function in cerebral glucose metabolism are GLUT1 and GLUT3. GLUT1 is the primary transporter in the blood-brain barrier, choroid plexus, ependyma, and glia; GLUT3 is the neuronal glucose transporter. The levels of expression of both transporters are regulated in concert with metabolic demand and regional rates of cerebral glucose utilization. We present several experimental paradigms in which alterations in energetic demand and/or substrate supply affect glucose transporter expression. These include normal cerebral development in the rat, Alzheimer's disease, neuronal differentiation in vitro, and dehydration in the rat.     

Metabolic and genetic analyses of apoptosis in potassium/serum-deprived rat cerebellar granule cells

Cerebellar granule cells maintained in medium containing serum and 25 mM potassium undergo an apoptotic death within 96 hr when switched to serum-free medium with 5 mM potassium. Because large numbers of apparently homogeneous neurons can be obtained, this represents a potentially useful model of neuronal programmed cell death (PCD). Analysis of the time course and extent of death after removal of either serum or K+ alone demonstrated that a fast-dying (T(1/2) = 4 hr) population (20%) responded to serum deprivation, whereas a slow-dying (T(1/2) = 25 hr) population (80%) died in response to K+ deprivation. Taking advantage of the complete death after removing both K+ and serum, changes in metabolic events and mRNA levels were analyzed in this model. Glucose uptake, protein synthesis, and RNA synthesis fell to <35% of control by 9 hr after potassium/serum deprivation, a time when 85% of the cells were still viable. The pattern of the fall in these metabolic parameters was similar to that reported for trophic factor-deprived sympathetic neurons. Most mRNAs decreased markedly after K+/serum deprivation. Levels of c-jun mRNA increased fivefold in potassium/serum-deprived granule cells; c-jun is required for cell death of sympathetic neurons. mRNA levels of cyclin D1, c-myb, collagenase, and transin remained relatively constant in potassium/serum-deprived granule cells. These data demonstrate the existence of two populations of granule cells with respect to cell death and define common metabolic and genetic events involved in neuronal PCD.     

Astrocytic gap junctional communication decreases neuronal vulnerability to oxidative stress-induced disruption of Ca2+ homeostasis and cell death

We investigated the effect of uncoupling astrocytic gap junctions on neuronal vulnerability to oxidative injury in embryonic rat hippocampal cell cultures. Mixed cultures (neurons growing on an astrocyte monolayer) treated with 18-alpha-glycyrrhetinic acid (GA), an uncoupler of gap junctions, showed markedly enhanced generation of intracellular peroxides (2,7-dichlorofluorescein fluorescence), impairment of mitochondrial function [(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and cell death (lactate dehydrogenase release) following exposure to oxidative insults (FeSO4 and 4-hydroxynonenal). GA alone had little or no effect on basal levels of peroxides, mitochondrial function, or neuronal survival. Intercellular dye transfer analyses revealed extensive astrocyte-astrocyte coupling but no astrocyte-neuron or neuron-neuron coupling in the mixed cultures. Studies of pure astrocyte cultures and microscope analyses of neurons in mixed cultures showed that the increased oxidative stress and cell death in GA-treated cultures occurred only in neurons and not in astrocytes. Antioxidants (propyl gallate and glutathione) blocked the death of neurons exposed to FeSO4/GA. Elevations of neuronal intracellular calcium levels ([Ca2+]i) induced by FeSO4 were enhanced in neurons in mixed cultures exposed to GA. Removal of extracellular Ca2+ and the L-type Ca2+ channel blocker nimodipine prevented impairment of mitochondrial function and cell death induced by FeSO4 and GA, whereas glutamate receptor antagonists were ineffective. Finally, GA exacerbated kainate- and FeSO4-induced injury to pyramidal neurons in organotypic hippocampal slice cultures. The data suggest that interastrocytic gap junctional communication decreases neuronal vulnerability to oxidative injury by a mechanism involving stabilization of cellular calcium homeostasis and dissipation of oxidative stress.