Lipopolysaccharide sensitizes microglia toward Ca(2+)-induced cell death: mode of cell death shifts from apoptosis to necrosis

Little is known about the effect of microglial activation on cell death. This study examines the effects of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), triggers of microglial activation, on cell death induced by several agents in cultured rat microglia. For comparison, the effect of LPS on cell death is also examined in cultured astrocytes. LPS or IFN-gamma enhanced cell death induced by thapsigargin or ionomycin, an agent that increases intracellular Ca2+ concentration, although LPS or IFN-gamma alone did not affect cell viability. Thapsigargin or ionomycin induced apoptosis in LPS-untreated microglia, while they induced necrosis in LPS-treated microglia, which were partially reversed by O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM, an intracellular Ca2+ chelator). In contrast, LPS treatment did not affect tunicamycin- or staurosporine-induced apoptosis, while it inhibited S-nitroso-N-acetylpenicillamine-induced apoptosis. The effect of LPS on thapsigargin or ionomycin-induced apoptosis was not observed in astrocytes. These results indicate that microglial activation sensitizes the cells toward cell death induced by the change in intracellular Ca2+ concentration and shifts the mode of cell death from apoptosis to necrosis.     

Agmatine induces glutamate release and cell death in cultured rat cerebellar granule neurons

We investigated the effect of agmatine on cell viability of rat cerebellar granule neurons in a high-K+ (27.5 mM) medium. Exposure of cultured rat cerebellar granule neurons to agmatine (200-800 microM) resulted in a significant decrease in cell viability. Agmatine-induced neuronal death began to occur 6-12 h after addition, and gradually progressed. The agmatine neurotoxicity was attenuated by N-methyl-D-aspartate (NMDA) receptor antagonists and by enzymatic degradation of L-glutamate with glutamic pyruvic transaminase. Furthermore, a significant increase in extracellular L-glutamate concentration was detected before cell death occurred. In addition, agmatine-induced glutamate release and cell death were both blocked by pretreatment with botulinum toxin C, which is known to specifically inhibit the exocytosis. The agmatine neurotoxicity was not observed when extracellular K+ concentration was lower (10 mM). These results suggest that agmatine induces glutamate release through the exocytosis and thereby causes NMDA receptor-mediated neuronal death in conditions in which extracellular K+ concentrations are elevated.     

Blockade of NMDA receptor-mediated mobilization of intracellular Ca2+ prevents neurotoxicity.

NMDA receptor activation leads to elevated Ca2+ in cultured rat cortical and retinal ganglion cell neurons. If excessive, this Ca2+ response is associated with delayed neurotoxicity. We used dantrolene and ionomycin to test if the Ca2+ response to NMDA was due to mobilization of intracellular Ca2+ stores rather than merely to Ca2+ influx. In the presence of EGTA, ionomycin resulted in release and subsequent depletion of intracellular Ca2+ stores. Henceforth, despite normal extracellular Ca2+, NMDA elicited only about half of its former Ca2+ response. Similarly, when dantrolene was used to block Ca2+ release from intracellular stores, we observed > 50% smaller NMDA-evoked Ca2+ responses. These results quite surprisingly indicate that at least half the Ca2+ response to NMDA is due to release of intracellular Ca2+, a process triggered by influx of extracellular Ca2+. Dantrolene also protected neurons from NMDA receptor-mediated neurotoxicity. Release of intracellular Ca2+ may therefore be a necessary step in the cascade leading to neuronal damage induced by excessive NMDA receptor stimulation and may be amenable to pharmacological intervention.     

Blockade of NMDA receptor-mediated mobilization of intracellular Ca prevents neurotoxicity

NMDA receptor activation leads to elevated Ca²⁺ in cultured rat cortical and retinal ganglion cell neurons. If excessive, this Ca²⁺ response in associated with delayed neurotoxicity. We used dantrolene and ionomycin to test if the Ca²⁺ response to NMDA was due to mobilization of intracellular Ca²⁺ stores rather than merely to Ca²⁺ influx. In the presence of EGTA, ionomycin resulted in release and subsequent depletion of intracellular Ca²⁺ stores. Henceforth, despite normal extracellular Ca²⁺, NMDA elicited only about half of its former Ca²⁺ response. Similarly, when dantrolene was used to block Ca²⁺ release from intracellular stores, we observed > 50% smaller NMDA-evoked Ca²⁺ responses. These results quite surprisingly indicate that at least half the Ca²⁺ response to NMDA is due to release of intracellular Ca²⁺, a process triggered by influx of extracellular Ca²⁺. Dantrolene also protected neurons from NMDA receptor-mediated neurotoxicity. Release of intracellular Ca²⁺ may therefore be a necessary step in the cascade leading to neuronal damage induced by excessive NMDA receptor stimulation and may be amenable to pharmacological intervention.     

Protective Effect of Memantine Against Doxorubicin Toxicity in Primary Neuronal Cell Cultures: Influence a Development Stage

One of the serious unwanted effects of the anthracycline anticancer drug doxorubicin (Dox, adriamycin) is its neurotoxicity, which can be evoked by the activation of extracellular (FAS/CD95/Apo-1) pathway of apoptosis in cells. Since memantine, a clinically used N-methyl-d-aspartic acid (NMDA) receptor antagonist, shows antiapoptotic action in several models of neuronal cell damage, in this study we evaluated the effect of memantine on the cell death induced by Dox in primary neuronal cell cultures. First, we investigated the effect of different concentrations of Dox (0.1–5 μM) on mouse neocortical, hippocampal, striatal, and cerebellar neurons on 7- and 12-day in vitro (DIV). The 7 DIV neuronal cell cultures were more prone to Dox-induced cell death than 12 DIV cultures. The cerebellar neurons were the most resistant to Dox-induced apoptosis in comparison to neuronal cell cultures derived from the forebrain. Memantine (0.1–2 μM) attenuated the Dox-evoked lactate dehydrogenase release in 7 DIV neuronal cell cultures with no significant effect on 12 DIV cultures. The ameliorating effect of memantine on Dox-mediated cell death was also confirmed by an increase in cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. There was no effect of memantine on Dox-induced caspase-8 and -3 activity and Dox-evoked decrease in mitochondrial potential, although attenuation in the number of cells with apoptotic DNA fragmentation was observed. We also showed that the antiapoptotic effect of memantine in our model was NMDA receptor-independent, since two other antagonists of this receptor, MK-801 and AP-5, did not attenuate Dox-induced cell death. Furthermore, memantine did not influence the Dox-evoked increase in cytoplasmic Ca²⁺ level. The obtained data suggest developmental regulation of both, the Dox-mediated neurotoxicity and efficacy of memantine in alleviating the Dox-induced cell damage in neuronal cell cultures. Moreover, this neuroprotective effect of memantine seems not to be dependent on caspase-3 activity and on the antagonistic action on NMDA receptor.     

Otilonium and pinaverium trigger mitochondrial-mediated apoptosis in rat embryo cortical neurons in vitro

In the frame of a repositioning programme with cholinergic medicines in clinical use searching for neuroprotective properties, we surprisingly found that spasmolytic antimuscarinics otilonium and pinaverium exhibited neurotoxic effects in neuronal cultures. We decided to characterize such unexpected action in primary cultures of rat embryo cortical neurons. Neurotoxicity was time- and concentration-dependent, exhibiting approximate EC₅₀ values of 5 μM for both drugs. Seven antimuscarinic drugs endowed with a quaternary ammonium, and another 10 drugs with different cholinergic activities, carrying in their molecule a ternary ammonium did not exhibit neurotoxicity. Both drugs caused a concentration-dependent blockade of whole-cell inward currents through voltage-activated calcium channels (VACCs). Consistent with this, they also blocked the K⁺-elicited [Ca²⁺]_c transients. Neither antioxidant catalase, glutathione, n-acetylcysteine, nor melatonin protected against neurotoxicity of otilonium or pinaverium. However cyclosporine A, a blocker of the mitochondrial permeability transition pore, prevented the neurotoxic effects of otilonium and pinaverium monitored as the fraction of cells undergoing apoptosis. Furthermore, the caspase-9 and caspase-3 inhibitor Ac-LEHD-CHO mitigated the apoptotic neuronal death of both drugs by around 50%. Data are compatible with the hypothesis that otilonium and pinaverium elicit neuronal death by activating the intrinsic mitochondrial-mediated signaling pathway of apoptosis. This may have its origin in the mitigation of Ca²⁺ entry and the uncoupling of the Ca²⁺-dependent generation of mitochondrial bioenergetics, thus causing the opening of the mitochondrial mPTP to elicit apoptotic neuronal death.     

TGF-β1 prevents gp120-induced impairment of Ca2+ homeostasis and rescues cortical neurons from apoptotic death

HIV-1 infection frequently induces neuronal death responsible for the development of neurological deficits associated with AIDS. Several reports suggest that gp120, the HIV-1 envelope glycoprotein, is the main candidate as mediator of the HIV-1-dependent neurotoxicity. Here we report the effect of gp120 on the survival of cortical neurons in vitro and the possible mechanisms whereby it occurs. Mature cortical neurons, cultured on a feeder layer of astrocytes, were treated with gp120 in a defined culture medium in absence of serum. The treatment with gp120 induced time-dependent neuronal damage displaying apoptotic features, as revealed by in situ labelling of DNA fragmentation. TGF-β1, a cytokine that has been previously shown to exert neuroprotective effects, prevented the cell death induced by exposure of cortical neurons to gp120. The prolonged treatment with gp120 also increased neuronal [Ca²⁺]_i, while the coincubation with TGF-β1 completely prevented the impairment of neuronal Ca²⁺ homeostasis. These data, taken together, demonstrate that gp120 induces apoptosis in cortical neurons, an effect that can be related to the impairment of Ca²⁺ homeostasis, and that TGF-β1 pretreatment reverts both the neuronal death and the alterations in neuronal [Ca²⁺]_i. J. Neurosci. Res. 49:600–607, 1997. © 1997 Wiley-Liss, Inc.     

Porcine pancreatic group IB secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels

Secretory phospholipase A₂ (sPLA₂) exhibits neurotoxicity in the central nervous system. There are high-affinity binding sites of the porcine pancreatic group IB sPLA₂ (sPLA₂-IB) in the brain. sPLA₂-IB causes neuronal cell death via apoptosis in the rat cerebral cortex. Although apoptosis is triggered by an influx of Ca²⁺ into neurons, it has not yet been ascertained whether the Ca²⁺ influx is associated with the neurotoxicity of sPLA₂-IB. We thus examined the possible involvement of Ca²⁺ in the neurotoxicity of sPLA₂-IB in the primary culture of rat cortical neurons. sPLA₂-IB induced neuronal cell death in a concentration- and time-dependent manner. This death was accompanied by condensed chromatin and fragmented DNA, exhibiting apoptotic features. Before apoptosis, sPLA₂-IB markedly enhanced the influx of Ca²⁺ into neurons. A calcium chelator suppressed neurons from sPLA₂-IB-induced neuronal cell death in a concentration-dependent manner. An L-type voltage-sensitive Ca²⁺ channel (L-VSCC) blocker significantly protected the sPLA₂-IB-potentiated influx of Ca²⁺. On the other hand, blockers of N-VSCC and P/Q-VSCC did not. An L-VSCC blocker protected neurons from sPLA₂-IB-induced neuronal cell death. In addition, the L-VSCC blocker ameliorated the apoptotic features of sPLA₂-IB-treated neurons. Neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of the enzyme. In conclusion, these findings demonstrate that the influx of Ca²⁺ into neurons play an important role in the neurotoxicity of sPLA₂-IB. Furthermore, the present study suggests that L-VSCC contribute to the sPLA₂-IB-potentiated influx of Ca²⁺ into neurons.     

Mechanism of neuroprotection by donepezil pretreatment in rat cortical neurons chronically treated with donepezil

Previously, we showed that in rat cortical neurons, chronic donepezil treatment (10 microM, 4 days) up-regulates nicotinic receptors (nAChR) and makes neurons more sensitive to the neuroprotective effect of donepezil. Here we examined the mechanism of donepezil-induced neuroprotection in neurons chronically treated with donepezil. The mechanism of neuroprotection was examined under different conditions of exposure to glutamate, acute and moderate, that induce cell death associated with necrotic and apoptotic cell death, respectively. Concomitant treatment with antagonists of nAChRs but not muscarinic receptors inhibited donepezil pretreatment-induced neuroprotection against acute glutamate treatment-induced death. Donepezil pretreatment prevented acute glutamate- and ionomycin-induced neurotoxicity, but not S-nitrosocysteine-induced neurotoxicity, suggesting that donepezil protects neurons via nAChR at levels before nitric oxide synthase activation against acute glutamate neurotoxicity. Concomitant treatment with antagonists of nAChR or phosphatidylinositol 3-kinase (PI3K) signaling inhibitors significantly inhibited neuroprotection against moderate glutamate neurotoxicity and decreased the phosphorylation level of Akt. Neuroprotection was also inhibited by treatment with inhibitor of mitogen-activated protein kinase (MAPK) kinase. These results suggest that donepezil protects neurons against moderate glutamate neurotoxicity via nAChR-PI3K-Akt and MAPK signaling pathways. This study provides novel insight into the mechanism of donepezil-induced neuroprotection that involves nAChR up-regulation.     

Glutamate mediates cell death and increases the Bax to Bcl-2 ratio in a differentiated neuronal cell line

Excessive stimulation of the NMDA receptor by glutamate induces cell death and has been implicated in the development of several neurodegenerative diseases. While apoptosis plays a role in glutamate-mediated toxicity, the mechanisms underlying this process have yet to be completely determined. Recent evidence has shown that exposure to excitatory amino acids regulates the expression of the antiapoptotic protein, Bcl-2, and the proapoptotic protein, Bax, in neurons. Since it has been suggested that the ratio of Bax to Bcl-2 is an important determinant of neuronal survival, the reciprocal regulation of these Bcl-2 family proteins may play a role in the neurotoxicity mediated by glutamate. Here, we have used a differentiable neuronal cell line, N1E-115, to investigate the molecular properties of glutamate-induced cell death. Annexin V staining was used to determine apoptotic cell death between 0 and 5 days differentiation with DMSO/low serum. Immunoblot analysis was used to determine whether the expression of Bcl-2 or Bax was modulated during the differentiation process. Bcl-2 protein levels were increased during maturation while Bax expression remained unchanged. Maximum Bcl-2 expression was observed following 5 days of differentiation. Examination of Bcl-2 and Bax following glutamate treatment revealed that the expression of these proteins was inversely regulated. Exposure to glutamate (0.001-10 mM) for 20+/-2 h resulted in a dose-dependent decrease in cell survival (as measured by MTT analysis) that was maximal at 10 mM. These results further support the role of apoptosis in glutamate-mediated cell death. Furthermore, a significant decrease in Bcl-2 levels was observed at 1 mM and 10 mM glutamate (32.1%+/-4.8 and 33.7+/-12.8%, respectively) while a significant upregulation of Bax expression (88.2+/-17.9%) was observed at 10 mM glutamate. Interestingly, Bcl-2 and Bax levels in cells treated with glutamate from 12-24 h were not significantly different from those of control. Taken together, these findings provide additional evidence for the reciprocal regulation of Bcl-2 and Bax expression by glutamate and suggest that neuronal excitotoxicity may, in part, result from the inverse regulation of these proteins.     

Time dependency of the action of nitric oxide in lipopolysaccharide-interferon-gamma-induced neuronal cell death in murine primary neuron-glia co-cultures

We investigated the time-dependency of the action of nitric oxide (NO) on glia-mediated neuronal cell death. Cortical neuron–glia co-cultures were treated with lipopolysaccharide and interferon γ (LPS/IFNγ). The production of NO was first detectable 9 h after the exposure to LPS/IFNγ and increased for up to 48 h. A significant neuronal cell death was observed 36–48 h after treatment with LPS/IFNγ. The NO generated at the initial stage of NO synthesis (about 12 h) following exposure to LPS/IFNγ was found to be critical for LPS/IFNγ-induced neurotoxicity. Furthermore, the rate of NO production at the initial stage of NO synthesis was correlated linearly with the extent of neuronal cell death. These findings suggest that the maximal rate of NO synthesis, instead of the accumulated NO₂⁻ level, is a sensitive index for predicting endotoxin-induced cytotoxicity.     

Blockade of adenosine A(2A) receptors prevents staurosporine-induced apoptosis of rat hippocampal neurons

Since adenosine A(2A) receptor (A(2A)Rs) blockade protects against noxious brain insults involving apoptosis, we directly tested if A(2A)R blockade prevents apoptosis induced by staurosporine (STS). Exposure of rat hippocampal neurons to STS (30 nM, 24 h) decreased neuronal viability while increasing the number apoptotic-like neurons and de-localizing mitochondria and cytochrome c immunoreactivities. This was prevented by the selective A(2A)R antagonists, SCH58261 and ZM241385 (50 nM). Shorter incubation periods (6 h) with STS caused no neuronal loss but decreased synaptophysin and MAP-2 immunoreactivities, which was prevented by SCH58261. Furthermore, STS (100 nM) decreased MTT reduction and increased caspase-3 activity in rat hippocampal nerve terminals, which was prevented by SCH58261. These results show that A(2A)R blockade inhibits STS-induced apoptotic-like neuronal cell death. This begins with an apoptotic-like synaptotoxicity, which later evolved into an overt neurotoxicity, and A(2A)Rs effectively control this initial synaptotoxicity, in agreement with their predominant synaptic localization in the hippocampus.     

Apoptosis in the adult striatum after transient forebrain ischemia and the effects of ischemic severity

The mechanisms of neuronal injury after cerebral ischemia have been under active investigation. The medium-size neurons in the dorsal striatum die within 24 h after transient cerebral ischemia. Using electron microscopy, the present study examined the nature of neuronal death in the striatum of adult rats following transient forebrain ischemia and tested the hypothesis that the ischemic severity might influence the nature of cell death. After severe ischemia (approximately 21 min ischemic depolarization), most neurons in the dorsal striatum died with swollen organelles and small irregular chromatin clumps resembling necrosis. The tissue damage in the dorsomedial striatum was less severe than that in the dorsolateral striatum and approximately 5% of the neurons in this region died with large chromatin clumps and relatively intact organelles resembling apoptosis. Some neurons displayed a mixture of necrotic- and apoptotic-like appearance. In contrast, the neurons with large somata only exhibited mild ultrastructural changes. After moderate ischemia (approximately 15 min ischemic depolarization), the tissue damage was less severe and the process of necrosis was temporally prolonged compared with that after severe ischemia. The apoptotic-like neuronal death was observed not only in the dorsomedial (approximately 6%) but also in the dorsolateral striatum (approximately 7%). The neurons in the striatum showed transient reversible changes after mild ischemia (approximately 10 min ischemic depolarization). The present study demonstrates that both apoptosis and necrosis occur in the adult striatum following transient forebrain ischemia and apoptosis occurs in the regions with less severe ischemia. These results suggest that ischemic severity might be one of the contributing factors to necrosis or apoptosis following transient global ischemia.     

Neuronal dysfunction and death in rabies virus infection

Because morphologic changes in natural rabies are usually relatively mild, it is thought that the severe clinical disease with a fatal outcome must be due to neuronal dysfunction of rabies virus-infected neurons. The precise bases of this functional impairment are unknown, and current knowledge on electro-physiological alterations, effects on ion channels and neurotransmission, and neurotoxicity are reviewed. Rabies virus may induce neuronal death, possibly through apoptotic mechanisms. Neuronal apoptosis has been observed in vitro and also in vivo under particular experimental conditions. The relevance of neuronal apoptosis in these situations to natural rabies has not yet been fully elucidated.     

Prostaglandin E2 rescues cortical neurons from amyloid beta protein-induced apoptosis

Cerebrospinal fluid prostaglandin E(2) (PGE(2)) levels are elevated in patients with Alzheimer's disease (AD), suggesting an involvement of PGE(2) in the neurodegeneration. AD is characterized by deposits of amyloid beta protein (Abeta) in various regions of the brain, e.g. the cerebral cortex. In the present study, we investigated the effects of PGE(2) on neuronal survival in primary cultures of rat cortical neurons. PGE(2) had no effect on neuronal cell viability or its morphology. Therefore, we examined the synergistic effects of PGE(2) with Abeta, a neurotoxin. Abeta caused neuronal cell death via apoptosis. PGE(2) significantly suppressed Abeta neurotoxicity, but did not promote the neurotoxicity. Furthermore, PGE(2) ameliorated Abeta-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Abeta increased the influx of Ca(2+) into neurons before cell death. Nimodipine, an inhibitor of the L-type voltage-sensitive calcium channel (L-VSCC), significantly reduced Abeta-potentiated Ca(2+) uptake. On the other hand, there was no effect on the Abeta-induced Ca(2+) influx by an N-VSCC blocker or P/Q-VSCC blockers. Moreover, the inhibitor of L-VSCC suppressed Abeta-induced neuronal cell death, whereas neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of Abeta. PGE(2) also suppressed the Abeta-induced Ca(2+) influx in a concentration-dependent manner. This study demonstrated that PGE(2) rescues cortical neurons from Abeta-induced apoptosis by reducing Ca(2+) influx in the primary culture. Furthermore, the present study suggested that the inhibition of L-VSCC contributes to the neuroprotective effect of PGE(2).     

Apoptotic neurons in brains from paediatric patients with HIV-I encephalitis and progressive encephalopathy

The pathogenesis of human immunodeficiency virus type 1 (HIV-1) associated dementia in adults involves neuronal loss from discrete areas of the neocortex and subcortical regions, but the mechanism for neuronal death is poorly understood. Gene-directed cell death resulting in apoptosis is thought to be a normal feature of neuronal development, but little is known about neuronal apoptosis in disease states. We investigated whether HIV-1 infection of the central nervous system is spatially associated with apoptosis of neurons. Using an in situ technique to identify newly cleaved 3'-OH ends of DNA as a marker for apoptosis, we demonstrate the presence of apoptotic neurons in cerebral cortex and basal ganglia of children that had HIV-1 encephalitis with progressive encephalopathy. Furthermore, an association was observed between the localization of apoptotic neurons and perivascular inflammatory cell infiltrates containing HIV-1 infected macrophages and multinucleated giant cells. Apoptotic neurons and p24–positive macrophages were observed infrequently in cerebral cortex and basal ganglia in children with HIV-1 infection without encephalitis or clinical encephalopathy. In nine control (HIV-1 negative) brains, ranging from the first post-natal month of life to 16.5 years of age, infrequent neuronal apoptosis was observed in three cases. These findings suggest that neuronal apoptosis is unlikely to be associated with post-natal development except in early post-natal germinal matrix, and that it may instead represent the end result of specific pathological processes, such as HIV-1 encephalitis.     

Mechanisms of Dopamine-Induced Cell Death in Cultured Rat Forebrain Neurons: Interactions with and Differences from Glutamate-Induced Cell Death

Injury to the brain, whether by ischemia or trauma, results in the uncontrolled release of many neurotransmitters, including glutamate and dopamine. Both of these neurotransmitters are neurotoxic in high concentrations, and the oxidative stress caused by reactive oxygen species generation has been implicated in the mechanism of neurotoxicity. In this study, we used cultured rat forebrain neurons to characterize cell death caused by exposure to dopamine and/or glutamate and to investigate potential acute mechanisms of toxicity. Dopamine exposure (250 μMfor 2 h) reduced cell viability to 34.3 ± 5.5% of untreated control 20 h later and increased the number of neurons with apoptotic morphology. The antioxidantN-acetylcysteine (100 μM) inhibited dopamine-induced toxicity and prevented the covalent binding of dopamine quinones to protein. In contrast, glutamate toxicity lacked the hallmark characteristics of apoptosis. When neurons were exposed successively to sublethal concentrations of dopamine and glutamate, cell viability at 20 h was reduced to 62.3 ± 5.2% of untreated control. Apoptosis was not evident, andN-acetylcysteine blocked the potentiating effect of dopamine on glutamate-induced toxicity. We used single-cell fluorescence assays to measure changes in intraneuronal glutathione, intraneuronal Ca²⁺, mitochondrial membrane potential, and DNA integrity as potential acute inducers of neuronal injury. While changes in these parameters could be demonstrated, none were identified as the sole acute inducer of cell death caused by dopamine. In summary, we have characterized a number of neuronal responses to lethal dopamine injury. Also, we have demonstrated that dopamine and glutamate can interactin vitroto potentiate cell death and that the potentiation appears to be induced by oxidative stress.     

The neuronal calcium sensor protein VILIP-1 is associated with amyloid plaques and extracellular tangles in Alzheimer's disease and promotes cell death and tau phosphorylation in vitro: a link between calcium sensors and Alzheimer's disease?

To investigate whether the observed association of intracellular neuronal calcium sensor (NCS) proteins with amyloid plaques and neurofibrillar tangles in Alzheimer brains is linked to a possible neuroprotective or neurotoxic activity of the protein, we performed cytotoxicity tests in PC12 cells transfected with the calcium sensor protein VILIP-1 (visinin-like protein) and the calcium buffer protein calbindin-D28K. Whereas VILIP-1 expression enhanced the neurotoxic effect of ionomycin already at low ionophore concentrations, calbindin-D28K protected against ionomycin-induced cytotoxicity only at high ionomycin and therefore calcium concentrations. However, in double-transfected cells calbindin-D28K rescued VILIP-1-mediated cytotoxicity at low ionomycin concentrations. Since VILIP-1 was found to be associated with fibrillar tangles in Alzheimer brains, we tested whether VILIP-1 has an influence on tau hyperphosphorylation. VILIP-1 expression enhanced hyperphosphorylation of tau protein compared to nontransfected or calbindin-D28K-transfected cells. These results raise the possibility that the observed reduction in VILIP-1-expressing cells may indicate a selective vulnerability of these neurons and that the calcium sensor protein is involved in the pathophysiology of Alzheimer's disease. The calcium sensor protein may influence tau phosphorylation and have a role in calcium-mediated neurotoxicity opposed to the previously discovered protective effect of calcium buffer proteins.     

Oxidized proteins in astrocytes generated in a hyperbaric atmosphere induce neuronal apoptosis

In the present study, we investigated the influence of the oxidative damage to astrocytes on neuronal cell survival using cultures of rat cerebral astrocytes and neurons. The exposure of astrocytes to hyperbaric oxygen induced a time-dependent apoptotic cell death, as observed by DNA ladder assessment. When astrocytes damaged by oxidative stress were cocultured with normal neurons from the cerebrum of a newborn rat, neuronal cell death was markedly induced, although normal astrocytes not subjected to hyperoxia cocultured with normal neurons showed no neuronal cell apoptosis. It was found that either the supernatant from the homogenate of astrocytes cultured in hyperbaric oxygen atmosphere or a protein mixture extracted from the supernatant induced neuronal cell death. The level of protein carbonyls, an index of protein oxidation analysis, in cultured astrocytes increased significantly with oxidative stress, and vitamin E inhibited the increase in the level of such oxidized proteins in astrocytes. Furthermore, a two-dimensional (2D) electrophoresis of a protein mixture extracted from the supernatant showed several changes in proteins. These results imply that reactive oxygen species (ROS) induced by oxidative stress attack astrocytes to induce oxidatively denatured proteins in the cells that act as a neurotoxic factor, and that vitamin E protects neurons by inhibiting astrocyte apoptosis caused by oxidative stress.     

Apoptotic cell death progression after photothrombotic focal cerebral ischaemia: effects of the lipophilic iron chelator 2,2'-dipyridyl

Two different forms of cell death have been distinguished morphologically following cerebral ischaemia: necrotic and apoptotic cell death. The aim of this study was to investigate the contribution of apoptosis to ischaemic damage by carefully depicting the temporal and spatial neuronal death following focal ischaemia. For this purpose, rats were subjected to chemical photothrombosis, and histological and biochemical analyses were performed over a period of 24 h after the onset of ischaemia. In addition, the effects of the lipophilic antioxidant iron chelator 2,2'-dipyridyl (DP) were evaluated 24 h after photothrombosis when the lesion volume was maximal. Our results showed two separate waves of neuronal death. In the first wave, shrunken dark neurons were massively present as early as 2 h after photothrombosis in the infarct core. From this initial neuronal abnormal population, progressive and time-dependent changes of both necrotic and apoptotic cell death were observed, leading to ghost neurons and apoptotic bodies after 24 h. The extension of the lesion coincided with a second wave of cell death. Massive and rapid neuronal loss occurred at the infarct border, which appeared as a sharply demarcated pale region. Procaspase and poly(ADP-ribose) polymerase-1 (PARP-1) cleavages were also detected in the infarct core and surrounding damaged tissue. DP treatment markedly blocked the enlargement of the lesion, the infarct border being rescued from infarction. Furthermore, a large decrease of apoptotic bodies was associated with a significant drop of caspase and PARP-1 cleavages, suggesting that the protective effect of DP closely correlates with limitation of apoptosis expansion.