Nicotinamide mononucleotide protects against ß-amyloid oligomer-induced cognitive impairment and neuronal death.

Amyloid-ß (Aß) oligomers are recognized as the primary neurotoxic agents in Alzheimer's disease (AD). Impaired brain energy metabolism and oxidative stress are implicated in cognitive decline in AD. Nicotinamide adenine dinucleotide (NAD(+)), a coenzyme involved in redox activities in the mitochondrial electron transport chain, has been identified as a key regulator of the lifespan-extending effects, and the activation of NAD(+) expression has been linked with a decrease in Aß toxicity in AD. One of the key precursors of NAD(+) is nicotinamide mononucleotide (NMN), a product of the nicotinamide phosphoribosyltransferase reaction. To determine whether improving brain energy metabolism will forestall disease progress in AD, the impact of the NAD(+) precursor NMN on Aß oligomer-induced neuronal death and cognitive impairment were studied in organotypic hippocampal slice cultures (OHCs) and in a rat model of AD. Treatment of intracerebroventricular Aß oligomer infusion AD model rats with NMN (500mg/kg, intraperitoneally) sustained improvement in cognitive function as assessed by the Morris water maze. In OHCs, Aß oligomer-treated culture media with NMN attenuated neuronal cell death. NMN treatment also significantly prevented the Aß oligomer-induced inhibition of LTP. Furthermore, NMN restored levels of NAD(+) and ATP, eliminated accumulation of reactive oxygen species (ROS) in the Aß oligomer-treated hippocampal slices. All these protective effects were reversed by 3-acetylpyridine, which generates inactive NAD(+). The present study indicates that NMN could restore cognition in AD model rats. The beneficial effect of NMN is produced by ameliorating neuron survival, improving energy metabolism and reducing ROS accumulation. These results suggest that NMN may become a promising therapeutic drug for AD.

[Causes and background of death in elderly patients with advanced dementia].

AIM: To examine the causes of death in elderly patients with advanced dementia, we retrospectively investigated the medical records for death discharge cases hospitalized in the Department of Geriatric Medicine at Saiseikai-Nakatsu Hospital and examined death certificates issued throughout the hospital. METHODS: (1) From 2010 to 2013, 31 patients with advanced dementia died in the hospital or were discharged to receive terminal care at home. We evaluated their medical records to examine the pathological background and disease with which they were diagnosed when admitted to and discharged from the hospital. (2) In order to assess the relationship between disease and dementia, we examined death certificates with "senility" or " (aspiration) pneumonia" recorded as the direct death cause issued throughout the hospital in the one-year period of 2013. RESULTS: (1) There were many cases in which eating problems and dysphagia influenced the clinical course. A total of 21 patients died from eating problems and/or dysphagia. (2) All 13 cases with "senility" recorded as the direct death cause on the death certificate involved severe dysphagia. Investigating the medical records, 11 patients had advanced dementia and two patients had end-stage Parkinson's disease. In total, 46 cases were diagnosed as involving " (aspiration) pneumonia", whereas there were no cases in which the records mentioned dementia or dysphagia in another column on the death certification. CONCLUSIONS: Advanced dementia is a mortal illness, and most patients with advanced dementia have dysphagia. Clinicians should be aware of the fact that dysphagia may lead to aspiration pneumonia and is a significant cause of death. Understanding the clinical course of dementia is important for determining the cause of death.

Systemic pyruvate administration markedly reduces neuronal death and cognitive impairment in a rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a major neurodegenerative disease of old age, characterized by progressive cognitive impairment, dementia and atrophy of the central nervous system. Amyloid-ß (Aß) oligomers are derived from proteolytic cleavage of amyloid precursor protein (APP) and recognized as the primary neurotoxic agents in AD. Pyruvate has a protective effect against Aß oligomer-induced neuronal cell death and inhibition of long-term potentiation (LTP) in hippocampal slice cultures, leading us to investigate the effect of systemic pyruvate administration in an intracerebroventricular Aß oligomer infusion model. We found that sodium pyruvate (500 mg/kg, intraperitoneally) improved neuron survival and sustained improvement in cognitive function as assessed by the Morris water maze. Pyuvate prevented the Aß oligomer-induced inhibition of LTP and protein phosphatase 2A (PP2A) activation. Pyruvate suppressed the Aß oligomer-induced poly[adenosine diphosphate (ADP) ribose] polymerase-1 (PARP-1) activity and ameliorated Aß oligomer-induced decrease of NAD(+) level. Moreover, pyuvate, but not lactate, decreased reactive oxygen species (ROS) accumulation in hippocampus of Aß1-42 oligomer-injection rat model. These results suggest that systemic pyruvate administration could significantly ameliorate Aß oligomer-induced spatial learning and memory impairment by the improvement of neuron survival and prevention of LTP inhibition, and the beneficial effect of pyruvate could be linked, at least in part, to the elimination of ROS accumulation, prevention of PP2A activation, amelioration of NAD(+) level and suppression of PARP-1 activity.

A warning index used in prescreening for Alzheimer's disease, based on self-reported cognitive deficits and vascular risk factors for dementia in elderly patients with type 2 diabetes.

Background/Aims. Diabetes might increase the risk of Alzheimer's disease (AD). For detecting dementia, it is typical to obtain informants' perceptions of cognitive deficits, but such interviews are usually difficult in routine care. We aimed to develop a model for predicting mild to moderate AD using a self-reported questionnaire and by evaluating vascular risk factors for dementia in elderly subjects with diabetes. Methods. We recruited 286 diabetic and 155 nondiabetic elderly subjects. There were 25 patients with AD and 261 cognitively normal individuals versus 30 with AD and 125 normal subjects, respectively. Each participant answered subjective questions on memory deficits and daily functioning. Information on vascular risk factors was obtained from clinical charts, and multivariate logistic regression was used to develop a model for predicting AD. Results. The predicted probabilities used in screening for AD in diabetic subjects constituted age, education, lower diastolic blood pressure, subjective complaints of memory dysfunction noticeable by others, and impaired medication, shopping, and travel outside a familiar locality. Receiver operating characteristic analysis revealed a satisfactory discrimination for AD specific for diabetic elderly subjects, with 95.2% sensitivity and 90.6% specificity. Conclusion. This is the first useful index that can prescreen for AD in elderly subjects with diabetes.

Dietary zinc reduction, pyruvate supplementation, or zinc transporter 5 knockout attenuates ß-cell death in nonobese diabetic mice, islets, and insulinoma cells.

Pancreatic zinc (Zn(2+)) concentrations are linked to diabetes and pancreatic dysfunction, but Zn(2+) is also required for insulin processing and packaging. Zn(2+) released with insulin increases ß-cell pancreatic death after streptozotocin toxin exposure in vitro and in vivo. Triosephosphate accumulation, caused by NAD(+) loss and glycolytic enzyme dysfunction, occur in type-1 diabetics (T1DM) and animal models. We previously showed these mechanisms are also involved in Zn(2+) neurotoxicity and are attenuated by nicotinamide- or pyruvate-induced restoration of NAD(+) concentrations, Zn(2+) restriction, or inhibition of Sir2 proteins. We tested the hypothesis that similar Zn(2+)- and NAD(+)-mediated mechanisms are involved in ß-cell toxicity in models of ongoing T1DM using mouse insulinoma cells, islets, and nonobese diabetic (NOD) mice. Zn(2+), streptozotocin, and cytokines caused NAD(+) loss and death in insulinoma cells and islets, which were attenuated by Zn(2+) restriction, pyruvate, nicotinamide, NAD(+), and inhibitors of Sir2 proteins. We measured diabetes incidence and mortality in NOD mice and demonstrated that pyruvate supplementation, or genetic or dietary Zn(2+) reduction, attenuated these measures. T-lymphocyte infiltration, punctate Zn(2+) staining, and ß-cell loss increased with time in islets of NOD mice. Dietary Zn(2+) restriction or Zn(2+) transporter 5 knockout reduced pancreatic Zn(2+) staining and increased ß-cell mass, glucose homeostasis, and survival in NOD mice, whereas Zn(2+) supplementation had the opposite effects. Pancreatic Zn(2+) reduction or NAD(+) restoration (pyruvate or nicotinamide supplementation) are suggested as novel targets for attenuating T1DM.

Pyruvate prevents the inhibition of the long-term potentiation induced by amyloid-ß through protein phosphatase 2A inactivation.

Amyloid-ß (Aß) oligomers are derived from proteolytic cleavage of amyloid-ß protein precursor and can impair memory and hippocampal long-term potentiation (LTP) in vivo and in vitro. They are recognized as the primary neurotoxic agents in Alzheimer's disease. Pyruvate has a protective effect against Aß-induced neuronal cell death in hippocampal slice cultures. However, whether pyruvate also has a protective effect against the inhibition of neuronal plasticity induced by Aß remains to be elucidated. This study examined the effect of pyruvate on the Aß-induced inhibition of LTP in the rat hippocampus. We found that pyruvate prevented the Aß-induced inhibition of LTP as strong as fostriecin, a specific protein phosphatase 2A (PP2A) inhibitor. Pyruvate prevented the Aß block of Ca(2+)/calmodulin dependent protein kinase 2 (CaMK2) autophosphorylation and the Aß-induced PP2A activation. Pyruvate, but not lactate, decreased reactive oxygen species levels in CA1 slices exposed to Aß. We propose that pyruvate could prevent the Aß-induced inhibition of LTP by the re-autophosphorylation of CaMK2 through PP2A inactivation. The reduction of reactive oxygen species production is considered to be the upstream mechanism of this observed pyruvate protection.

Amyloid-ß neurotoxicity restricts glucose window for neuronal survival in rat hippocampal slice cultures.

Diabetes may increase the risk of Alzheimer's disease (AD). However, a preventive strategy to combat cognitive decline in diabetic elderly with preexisting AD has remained unknown. The aim of this study was to determine the effects of metabolic perturbation on amyloid-ß (Aß) neurotoxicity and the optimal glucose range for improved neuronal survival, which is referred to as the "glucose window". Organotypic hippocampal slice cultures were incubated in either normoglycemic or hyperglycemic medium for 48 h, and subsequently treated in experimental media containing 0-30 mM glucose, with and without Aß(25-35). Neuronal survival was evaluated by the propidium iodide method. Aß neurotoxicity was exacerbated during hypoglycemia/hyperglycemia (≦2 mM/≧30 mM) without Aß and ≦3 mM/≧20 mM with Aß. ROS elevated in the respective glucose ranges and supplementation of ROS scavengers effectively improved neuronal survival. Interestingly, a sharp and sudden drop in glucose levels from preceding hyperglycemia further increased Aß neurotoxicity. Supplementation of pyruvate protected exacerbated Aß neurotoxicity. These results indicate that increased oxidative stress during severe hypoglycemia, hyperglycemia and fluctuation of blood glucose enhances neuronal cell death, resulting in the extremely limited glucose window, and therefore suggest that careful management of glucose avoiding hypoglycemia is needed to prevent brain degeneration in diabetic patients with AD.

The double-edged effect of insulin on the neuronal cell death associated with hypoglycemia on the hippocampal slice culture.

It is well known that the central nervous system (CNS) is vulnerable to hypoglycemia and hyperglycemia. Insulin is indispensable for serum glucose control and diabetes patients are on the relative or absolute deficient state of insulin. The role of insulin on the CNS, however, has not been fully elucidated, yet. To reveal the role of insulin on the neuronal survival, we have used in vitro system of an organotypic hippocampal slice culture from rat, and examine the neuronal cell death at the various glucose concentrations in the presence or absence of insulin. When glucose concentrations is varied to 0, 1, 3, 5 and 30 mM in the incubation medium, the neuronal cell death was minimum at 5mM, and no neuronal survival was observed under 1mM on the CA1. On the dentate gyrus granule cells (DG), on the other hand, the significant neuronal survival was observed even as low as 1mM. In the presence of 1 nM concentration of insulin, the neuronal cell death curve showed the U-shape, and the minimum death point was 3-5mM glucose concentrations at the CA1. At the DG, insulin did not show the protective effect up to 48 hours culture regardless of glucose concentration. In the absence of glucose, insulin accelerated the neuronal cell death both in the CA1 and DG. We concluded that insulin has a double-edged effect on the neuronal cell death dependent on glucose concentration, and that the CA1 and the DG have a different sensitivity to insulin in terms of cell survival.

[A case of elderly onset loss of consciousness due to bacterial meningitis and subsequent vasculitis].

We reported an 83-year-old woman, who suffered from bacterial meningitis and subsequent vasculitis. She experienced episodes of loss of consciousness several times in July, 2006. She also had recurrent fever and was admitted to a local hospital. Routine examinations, including brain MRI and electroencephalogram, were negative and urinary tract infection was diagnosed. After successful antibiotic therapy, she was transferred to a rehabilitation hospital. After transfer, she had no headache, but presented fever again, and a reduced level of consciousness. Cerebrospinal fluid test showed that cell counts were high with a predominance of neutrophils, and her glucose level was low. She received antibiotic therapy on her suspicion of bacterial meningitis. Bacterial cultures of CSF and blood were negative, probably due to the previous antibiotic therapy. Repeated CSF analysis showed a decrease in cell counts, but her lower consciousness did not improve. Moreover, neurological symptoms such as left pyramidal tract sign appeared. She was transferred to our hospital on the suspicion of vasculitis. Diffusion MRI showed high intensity in the right middle cerebral artery (MCA) area and CT angiography showed the stenosis of the right MCA at the M3 portion. Two courses of steroid pulse therapy were performed. Her consciousness gradually improved and eventually could talk although cognitive decline remained as a residual deficit. Our patient failed to be diagnosed early because of atypical symptoms of meningitis. Caution seems necessary for elderly cases presenting with atypical initial symptoms of meningitis. Steroid pulse therapy was effective for the subsequent vasculitis, as reported previously.

Different effects of monocarboxylates on neuronal survival and beta-amyloid toxicity.

Glucose is a principal metabolic fuel in the central nervous system, but, when glucose is unavailable, the brain can utilize alternative metabolic substrates such as monocarboxylates to sustain brain functions. This study examined whether the replacement of glucose with monocarboxylates (particularly pyruvate and lactate) had an equivalent effect of glucose on neuronal survival in rat hippocampal organotypic slice cultures, or ameliorate the neurotoxicity induced by amyloid beta-peptide (Abeta). The possible mechanism was also explored. We found that pyruvate and lactate alone increased cell death in the hippocampal slice cultures at 24 and 48 h. Supplementation of glucose-containing culture media and Abeta-treated culture media with pyruvate, but not lactate, attenuated cell death as strong as with trolox, known as a reactive oxygen species scavenger, and niacinamide, an NAD(+) precursor, and this protective effect was reversed by alpha-cyano-4-hydroxycinnamic acid. Pyruvate significantly increased the aconitase activity and the NAD(+) levels in the hippocampal slices in the presence of Abeta, but did not maintain the ATP levels. Our results indicate that pyruvate and lactate alone cannot replace glucose as an alternative energy source to preserve the neuronal viability in the hippocampal slice cultures. Pyruvate, in the presence of glucose, improves neuronal survival in the hippocampal slice cultures and also protects neurons against Abeta-induced cell death in which mitochondrial NAD(P) redox status may play a central role.

Voltage-dependent and Src-mediated regulation of NMDA receptor single channel outward currents in cortical neurons.

A voltage-dependent but Ca2+-independent regulation of N-methyl-D-aspartate (NMDA) receptor outward activity was studied at the single channel level using outside-out patches of cultured mouse cortical neurons. Unlike the inward activity associated with Ca2+ and Na+ influx, the NMDA receptor outward K+ conductance was unaffected by changes in Ca2+ concentration. Following a depolarizing pre-pulse, the single channel open probability (NP o), amplitude, and open duration of the NMDA inward current decreased, whereas the same pre-depolarization increased those parameters of the NMDA outward current (pre-pulse facilitation). The outward NP o was increased by the pre-pulse facilitation, disregarding Ca2+ changes. The voltage-current relationships of the inward and outward currents were shifted by the pre-depolarization toward opposite directions. The Src family kinase inhibitor, PP1, and the Src kinase antibody, but not the anti-Fyn antibody, blocked the pre-pulse facilitation of the NMDA outward activity. On the other hand, a hyperpolarizing pre-pulse showed no effect on NMDA inward currents but inhibited outward currents (pre-pulse depression). Application of Src kinase, but not Fyn kinase, prevented the pre-pulse depression. We additionally showed that a depolarization pre-pulse potentiated miniature excitatory synaptic currents (mEPSCs). The effect was blocked by application of the NMDA receptor antagonist AP-5 during depolarization. These data suggest a voltage-sensitive regulation of NMDA receptor channels mediated by Src kinase. The selective changes in the NMDA receptor-mediated K+ efflux may represent a physiological and pathophysiological plasticity at the receptor level in response to dynamic changes in the membrane potential of central neurons.

Impaired response of perforating arteries to hypercapnia in chronic hyperglycemia.

Diabetes mellitus increases the risk of cerebrovascular disease, the effects of hypercapnia on CBF (cerebral blood flow) and cerebrovascular reactivity during diabetes are still inconsistent. Here, we have established a new microangiographic technique using synchrotron radiation (SPring-8, Japan), which enabled us to visualize rat cerebral vessels with high spatial resolution in real time. The goal of the study presented here was to identify the effects of chronic hyperglycemia on hypercapnia-induced vascular responses (endothelium-dependent vasodilatation) and nitric oxide (NO) donor- induced vascular responses (endothelium-independent) of perforating arteries and of the deeply located large cerebral arteries. We found a significant vasodilatation of rat perforating arteries after hypercapnia with a maximum diameter of approximately 140% of baseline in normal Wistar rats. Chronic hyperglycemia impaired vasodilatation of perforating arteries in genetically diabetic GK rats. SNP (sodium nitroprusside) caused a similar vasodilatation of perforating vessels in normal and chronic hyperglycemia, indicating that endothelium-dependent vasodilatation of perforating arteries may be specifically impaired in chronic hyperglycemia. Possible impairment of endothelium-dependent vasodilatation in perforating vessels during chronic hyperglycemia may cause decreased vascular reserve capacity of perforating artery, resulting in the increased ischemic insults and cerebrovascular diseases in diabetes.

Cognitive dysfunction associates with white matter hyperintensities and subcortical atrophy on magnetic resonance imaging of the elderly diabetes mellitus Japanese elderly diabetes intervention trial (J-EDIT).

BACKGROUND: Type 2 diabetes is associated with cognitive dysfunction and increases the risk of dementia in the elderly. The aim of this study was to explore, by means of magnetic resonance (MR) imaging, possible relationships among clinical profiles of diabetes, cognitive function, white matter hyperintensities (WMHs) and subcortical brain atrophy. METHODS: Data were obtained from 95 nondemented type 2 diabetic participants aged 65 years or over, enrolled in an intervention trial for Japanese elderly diabetic patients. Cognitive function was measured with neuropsychiatric tests, including mini-mental state examination (MMSE), verbal memory, digit symbol substitution and Stroop tests. Hyperintensity was classified into periventricular, deep white matter, thalamic and basal ganglia. Four ventricle-to-brain ratios were used to measure subcortical atrophy. To identify clinical features of diabetes, indices of glycemic control, lipid metabolism, blood pressure and complications were examined. Canonical correlation analysis and regression analysis were used to assess correlation. RESULTS: Scores for digit symbol substitution and MMSE negatively correlated with WMHs in the parietal lobe and hyperintensities in the thalamus, respectively. Lower scores for memory and digit symbol substitution showed positive association with enlarged subcortical atrophy adjacent to lateral ventricles. There was no association between clinical pictures of diabetic patients with cognitive dysfunction and of those with morphological changes in the brain. CONCLUSIONS: Impaired cognitive domains of the speed of mental processes and memory were associated with WMHs and subcortical atrophy. Degenerative changes in the cerebral small vessels may constitute predictive factors for the rate of cognitive dysfunction in elderly diabetic patients.

Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells.

Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.

Neuroprotective effect of D-fructose-1,6-bisphosphate against beta-amyloid induced neurotoxicity in rat hippocampal organotypic slice culture: involvement of PLC and MEK/ERK signaling pathways.

D-fructose-1,6-bisphosphate (FBP) is an endogenous intermediate of glycolytic pathway which has potent neuroprotective effect against various neurotoxic insults. This study examined whether FBP could antagonize the neurotoxicity induced by amyloid beta-peptide (Abeta) in rat hippocampal organotypic slice cultures, and the possible mechanism was also explored. Treatment with FBP (concentration ranges from 1.7 mM to 10 mM) significantly decreased the cell death in hippocampal slices in the presence of Abeta at 24h, 48 h and 72 h, and this neuroprotective effect of FBP against Abeta was not in a dose-dependent manner, FBP 3.5 mM has better neuroprotective effect than that of other FBP concentration groups. Treatment with FBP slightly but significantly increases the ATP levels in hippocampal slices in the presence of Abeta. However, the increment of ATP levels was similar among various FBP concentration groups. Neuroprotective effect of FBP 3.5 mM against Abeta induced neurotoxicity in hippocampal slices was attenuated by addition of phospholipase C (PLC) inhibitor, U73122, mitogen activated extracellular signal protein kinase (MEK) inhibitor, U0126, or extracellular signal activated protein kinase (ERK) inhibitor, PD98059 at 24 h, 48 h and 72 h. However, co-treatment with these three kinds of inhibitors did not change the FBP's effect on ATP levels. Our results suggested FBP has neuroprotective effect against Abeta induced neurotoxicity in hippocampal slice cultures, and FBP plays role not only as an alternative energy source, but also a modulator of PLC and MEK/ERK pathways to regulate the cellular response and survival.

Glycolysis regulates the induction of lactate utilization for synaptic potentials after hypoxia in the granule cell of guinea pig hippocampus.

Lactate is considered an alternative substrate that is capable of replacing glucose in maintaining synaptic function in adult neurons. But, we found recently that lactate could be utilized for maintenance of synaptic potentials only after the activation of NMDA and voltage-dependent-calcium channel during glucose deprivation. To clarify more on the relationship between glycolysis and induction of lactate utilization, we tested lower concentration of glucose with hypoxia to induce a relative shortage of anaerobic energy production. Population spikes are not maintained with lactate following hypoxia in 10 mM glucose medium, but are maintained at their original levels with lactate after exposure to hypoxia in lower concentration (5 mM) of glucose. Hypothermia during low glucose-hypoxia, bath application of the NMDA channel blocker and the voltage-sensitive calcium channel blocker, as well as the omission of extracellular calcium prevented the induction of the lactate-supported population spikes. ATP levels in the tissue slices are relatively preserved in the conditions that block the induction of lactate-supported population spikes. From these observations, we propose that the energy source for maintenance of synaptic function in adult neuron changes from adult form (glucose alone) to immature one (glucose and/or lactate) after short of glucose supply.

Potassium attenuates zinc-induced death of cultured cortical astrocytes.

Transient global ischemia induces CA1 hippocampal neuronal death without astrocyte death, perhaps mediated in part by the toxic translocation of zinc from presynaptic terminals to postsynaptic neurons. We tested the hypothesis that cellular depolarization, which occurs in the ischemic brain due to increased extracellular potassium and energy failure, might contribute to astrocyte resistance to zinc-induced death. We previously reported that neurons in mixed cortical neuronal-astrocyte cultures were more vulnerable to a 5-15-min exposure to Zn(2+) than astrocytes in the same cultures. In the present report, we show that (1) neurons in isolation or in conjunction with astrocytes were 2-3-fold more sensitive to a 15-min nondepolarizing Zn(2+) exposure than are glia; (2) KCl-induced depolarization attenuated glial vulnerability to zinc toxicity but potentiated neuronal vulnerability to zinc toxicity; (3) Zn(2+)-induced glial death was attenuated by T-type Ca(2+) channel blockade, as well as compounds that increase NAD(+) levels; and (4) both astrocytic (65)Zn(2+) accumulation and the increase in astrocytic [Zn(2+)](i) induced by Zn(2+) exposure were also attenuated by depolarization or T-type Ca(2+) channel blockers. Zn(2+)-induced cell death in astrocytes was at least in part apoptotic, as caspase-3 was activated, and the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone partially attenuated Zn(2+)-induced death. The levels of peak [Zn(2+)](i) achieved in astrocytes during this toxic nondepolarizing Zn(2+) exposure (250 nM) were substantially greater than those achieved in neurons (40 nM). In glia, exposure to 400 microM Zn(2+) induced a 13-mV depolarization, which can activate T-type Ca(2+) channels. This Zn(2+)-induced astrocyte death, like neuronal death, was attenuated by the addition of pyruvate or niacinamide to the exposure medium.