Mitochondrial Inhibitor Models of Huntington's Disease and Parkinson's Disease Induce Zinc Accumulation and Are Attenuated by Inhibition of Zinc Neurotoxicity in vitro or in vivo

Background: Inhibition of mitochondrial function occurs in many neurodegenerative diseases, and inhibitors of mitochondrial complexes I and II are used to model them. The complex II inhibitor, 3-nitroproprionic acid (3-NPA), kills the striatal neurons susceptible in Huntington's disease. The complex I inhibitor N-methyl-4-phenylpyridium (MPP(+)) and 6-hydroxydopamine (6-OHDA) are used to model Parkinson's disease. Zinc (Zn(2+)) accumulates after 3-NPA, 6-OHDA and MPP(+) in situ or in vivo. Objective: We will investigate the role of Zn(2+) neurotoxicity in 3-NPA, 6-OHDA and MPP(+). Methods: Murine striatal/midbrain tyrosine hydroxylase positive, or near-pure cortical neuronal cultures, or animals were exposed to 3-NPA or MPP(+) and 6-OHDA with or without neuroprotective compounds. Intracellular zinc ([Zn(2+)](i)), nicotinamide adenine dinucleotide (NAD(+)), NADH, glycolytic intermediates and neurotoxicity were measured. Results: We showed that compounds or genetics which restore NAD(+) and attenuate Zn(2+) neurotoxicity (pyruvate, nicotinamide, NAD(+), increased NAD(+) synthesis, sirtuin inhibition or Zn(2+) chelation) attenuated the neuronal death induced by these toxins. The increase in [Zn(2+)](i) preceded a reduction in the NAD(+)/NADH ratio that caused a reversible glycolytic inhibition. Pyruvate, nicotinamide and NAD(+) reversed the reductions in the NAD(+)/NADH ratio, glycolysis and neuronal death after challenge with 3-NPA, 6-OHDA or MPP(+), as was previously shown for exogenous Zn(2+). To test efficacy in vivo, we injected 3-NPA into the striatum of rats and systemically into mice, with or without pyruvate. We observed early striatal Zn(2+) fluorescence, and pyruvate significantly attenuated the 3-NPA-induced lesion and restored behavioral scores. Conclusions: Together, these studies suggest that Zn(2+) accumulation caused by MPP(+) and 3-NPA is a novel preventable mechanism of the resultant neurotoxicity.     

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.     

Serum or target deprivation‐induced neuronal death causes oxidative neuronal accumulation of Zn2+ and loss of NAD+

Trophic deprivation‐mediated neuronal death is important during development, after acute brain or nerve trauma, and in neurodegeneration. Serum deprivation (SD) approximates trophic deprivation in vitro, and an in vivo model is provided by neuronal death in the mouse dorsal lateral geniculate nucleus (LGNd) after ablation of the visual cortex (VCA). Oxidant‐induced intracellular Zn²⁺ release ([Zn²⁺]_i) from metallothionein‐3 (MT‐III), mitochondria or ‘protein Zn²⁺’, was implicated in trophic deprivation neurotoxicity. We have previously shown that neurotoxicity of extracellular Zn²⁺ required entry, increased [Zn²⁺]_i, and reduction of NAD⁺ and ATP levels causing inhibition of glycolysis and cellular metabolism. Exogenous NAD⁺ and sirtuin inhibition attenuated Zn²⁺ neurotoxicity. Here we show that: (1) Zn²⁺ is released intracellularly after oxidant and SD injuries, and that sensitivity to these injuries is proportional to neuronal Zn²⁺ content; (2) NAD⁺ loss is involved – restoration of NAD⁺ using exogenous NAD⁺, pyruvate or nicotinamide attenuated these injuries, and potentiation of NAD⁺ loss potentiated injury; (3) neurons from genetically modified mouse strains which reduce intracellular Zn²⁺ content (MT‐III knockout), reduce NAD⁺ catabolism (PARP‐1 knockout) or increase expression of an NAD⁺ synthetic enzyme (Wld^s) each had attenuated SD and oxidant neurotoxicities; (4) sirtuin inhibitors attenuated and sirtuin activators potentiated these neurotoxicities; (5) visual cortex ablation (VCA) induces Zn²⁺ staining and death only in ipsilateral LGNd neurons, and a 1 mg/kg Zn²⁺ diet attenuated injury; and finally (6) NAD⁺ synthesis and levels are involved given that LGNd neuronal death after VCA was dramatically reduced in Wld^s animals, and by intraperitoneal pyruvate or nicotinamide. Zn²⁺ toxicity is involved in serum and trophic deprivation‐induced neuronal death.     

Involvement of poly ADP ribosyl polymerase-1 in acute but not chronic zinc toxicity

We have previously suggested that zinc-induced neuronal death may be mediated in part by inhibition of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), secondary to depletion of the essential cosubstrate NAD+. Given convergent evidence implicating the NAD+-catabolizing enzyme, poly ADP ribosyl polymerase (PARP) in mediating ATP depletion and neuronal death after excitotoxic and ischemic insults, we tested the specific hypothesis that the neuronal death induced by exposure to toxic levels of extracellular zinc might be partly mediated by PARP. PARP was activated in cultured mouse cortical astrocytes after a toxic acute Zn2+ exposure (350 microm Zn2+ for 15 min), but not in cortical neurons or glia after exposure to a toxic chronic Zn2+ exposure (40 microm Zn2+ for 1-4 h), an exposure sufficient to deplete NAD+ and ATP levels. Furthermore, the neurotoxicity induced by acute, but not chronic, Zn2+ exposure was reduced in mixed neuronal-glial cultures prepared from mutant mice lacking the PARP gene. These data suggest PARP activation may contribute to more fulminant forms of Zn2+-induced neuronal death.     

Alterations of intracellular calcium homeostasis and mitochondrial function are involved in ruthenium red neurotoxicity in primary cortical cultures

Ruthenium red (RR) is a polycationic dye that induces neuronal death in vivo and in primary cultures. To characterize this neurotoxic action and to determine the mechanisms involved, we have analyzed the ultrastructural alterations induced by RR in rat cortical neuronal cultures and measured its effect on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and on mitochondrial function. RR produced a dose-dependent, progressive disruption of neurites and plasma membrane of neuronal somata after 8-24 hr of incubation. RR caused also an elevation of both the basal [Ca(2+)](i) and its maximal levels after K(+) depolarization. Mitochondrial oxidative function, assessed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and by changes in dihydrorhodamine-123 fluorescence, was significantly diminished after treatment with RR, both in cultured neurons and in isolated brain mitochondria. La(3+) did not prevent but rather potentiated RR-induced cell death. Glutamate receptor antagonists also failed to prevent RR neurotoxicity. Apoptotic electron microscope images were not observed, and protein synthesis inhibitors did not show any protective effect. It is concluded that RR penetrates neurons and that its neurotoxic damage probably is due to intracellular Ca(2+) dishomeostasis and disruption of mitochondrial oxidative function. These results enhance our understanding of the intracellular mechanisms underlying neuronal death.     

Depolarization-induced 65zinc influx into cultured cortical neurons

Toxic Zn(2+) influx may be a key mechanism underlying selective neuronal death after transient global ischemia in rats. To identify routes responsible for neuronal Zn(2+) influx, we measured the accumulation of (65)Zn(2+) into cultured murine cortical cells under depolarizing conditions (60 mM K(+)) associated with severe hypoxia-ischemia in brain tissue. Addition of 60 mM K(+) or 300 microM kainate substantially increased (65)Zn(2+) accumulation into mixed cultures of neurons and glia, but not glia alone. (65)Zn(2+) accumulation was attenuated by increasing concentrations of extracellular Ca(2+) or trypsin pretreatment, but not by late trypsinization, and corresponded to an increase in atomic Zn(2+). Confirming predominantly neuronal entry, K(+)-induced (65)Zn(2+) accumulation was reduced by prior selective destruction of neurons with NMDA. K(+)-induced (65)Zn(2+) influx was not sensitive to glutamate receptor antagonists, but was attenuated by Gd(3+) and Cd(2+) as well as 1 microM nimodipine; it was partially sensitive to 1 microM omega-conotoxin-GVIA, and insensitive to 1 microM omega-agatoxin-IVA. K(+)-induced, Gd(3+)-sensitive (45)Ca(2+) accumulation but not (65)Zn(2+) accumulation was sharply attenuated by lowering extracellular pH to 6.6.     

Sodium overload through voltage-dependent Na(+) channels induces necrosis and apoptosis of rat superior cervical ganglion cells in vitro

Using the failure to exclude trypan blue as a criterion for cell death, we found that veratridine, the voltage-dependent Na(+) channel activator, exerted its toxicity to cultured sympathetic neurons in a dose-dependent manner (half-maximal toxicity occurred at 2 microM). The co-presence of tetrodotoxin completely reversed the toxicity only at concentrations of veratridine < 20 microM. Veratridine neurotoxicity was due to the influx of Na(+); a medium low in Na(+) (36 mM) completely abolished its neurotoxicity, whereas a Ca(2+)-free medium did not attenuate its neurotoxicity. Furthermore, the buffering action of 1, 2-Bis-(2-aminophenoxy)ethane-N,N,N',N',-tetraacetate (BAPTA) on veratridine-induced increase in intracellular Ca(2+) levels neither blocked veratridine neurotoxicity in normal medium, nor attenuated the low Na(+) effect. Elevated K(+) effectively blocked veratridine neurotoxicity in a Ca(2+)-dependent manner. Cytoplasmic pH measurements using a fluorescent pH indicator demonstrated that cellular acidification (from pH 7.0 to pH 6.5) occurred upon treatment with veratridine. Both veratridine-induced acidification and cell death were ameliorated by 5-(N-ethyl-N-isopropyl)amiloride, the specific inhibitor of the Na(+)/H(+) exchanger (IC(50) = 0.5 microM). Finally, necrosis occurred predominantly in veratridine neurotoxicity, but both staining with bis-benzimide and TUNEL analysis showed nuclear features of apoptosis in sympathetic neurons undergoing cell death.     

L-type Ca(2+) channel-mediated Zn(2+) toxicity and modulation by ZnT-1 in PC12 cells

In view of evidence that Zn(2+) neurotoxicity contributes to some forms of pathological neuronal death, we developed a model of Zn(2+) neurotoxicity in a cell line amenable to genetic manipulations. Exposure to 500 microM ZnCl(2) for 15 min under depolarizing conditions resulted in modest levels of PC12 cell death, that was reduced by the L-type Ca(2+) channel antagonist, nimodipine, and increased by the L-type Ca(2+) channel opener, S(-)-Bay K 8644. At lower insult levels (200 micrometer Zn(2+)+Bay K 8644), Zn(2+)-induced death appeared apoptotic under electron microscopy and was sensitive to the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-CH(2)F (Z-VAD); at higher insult levels (1000 microM+Bay K 8644), cells underwent necrosis insensitive to Z-VAD. To test the hypothesis that the plasma membrane transporter, ZnT-1, modulates Zn(2+) neurotoxicity, we generated stable PC12 cell lines overexpressing wild type or dominant negative forms of rat ZnT-1 (rZnT-1). Clones T9 and T23 overexpressing wild type rZnT-1 exhibited enhanced Zn(2+) efflux and reduced vulnerability to Zn(2+)-induced death compared to the parental line, whereas clones D5 and D16 expressing dominant negative rZnT-1 exhibited the opposite characteristics.     

Microglia induce neurotoxicity via intraneuronal Zn(2+) release and a K(+) current surge

Microglial cells are critical components of the injurious cascade in a large number of neurodegenerative diseases. However, the precise molecular mechanisms by which microglia mediate neuronal cell death have not been fully delineated. We report here that reactive species released from activated microglia induce the liberation of Zn(2+) from intracellular stores in cultured cortical neurons, with a subsequent enhancement in neuronal voltage-gated K(+) currents, two events that have been intimately linked to apoptosis. Both the intraneuronal Zn(2+) release and the K(+) current surge could be prevented by the NADPH oxidase inhibitor apocynin, the free radical scavenging mixture of superoxide dismutase and catalase, as well as by 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride. The enhancement of K(+) currents was prevented by neuronal overexpression of metallothionein III or by expression of a dominant negative (DN) vector for the upstream mitogen-activated protein kinase apoptosis signal regulating kinase-1 (ASK-1). Importantly, neurons overexpressing metallothionein-III or transfected with DN vectors for ASK-1 or Kv2.1-encoded K(+) channels were resistant to microglial-induced toxicity. These results establish a direct link between microglial-generated oxygen and nitrogen reactive products and neuronal cell death mediated by intracellular Zn(2+) release and a surge in K(+) currents.     

Neurosteroids block the increase in intracellular calcium level induced by Alzheimer’s β-amyloid protein in long-term cultured rat hippocampal neurons

The neurotoxicity of beta-amyloid protein (AbetaP) is implicated in the etiology of Alzheimer's disease. We previously have demonstrated that AbetaP forms Ca(2+)-permeable pores on neuronal membranes, causes a marked increase in intracellular calcium level, and leads to neuronal death. Here, we investigated in detail the features of AbetaP-induced changes in intracellular Ca(2+) level in primary cultured rat hippocampal neurons using a multisite Ca(2+)-imaging system with fura-2 as a fluorescent probe. Only a small fraction of short-term cultured hippocampal neurons (ca 1 week in vitro) exhibited changes in intracellular Ca(2+) level after AbetaP exposure. However, AbetaP caused an acute increase in intracellular Ca(2+) level in long-term cultured neurons (ca 1 month in vitro). The responses to AbetaP were highly heterogeneous, and immunohistochemical analysis using an antibody to AbetaP revealed that AbetaP is deposited on some but not all neurons. Considering that the disruption of Ca(2+) homeostasis is the primary event in AbetaP neurotoxicity, substances that protect neurons from an AbetaP-induced intracellular Ca(2+) level increase may be candidates as therapeutic drugs for Alzheimer's disease. In line with the search for such protective substances, we found that the preadministration of neurosteroids including dehydroepiandrosterone, dehydroepiandrosterone sulfate, and pregnenolone significantly inhibits the increase in intracellular calcium level induced by AbetaP. Our results suggest the possible significance of neurosteroids, whose levels are reduced in the elderly, in preventing AbetaP neurotoxicity.     

Astrocytes are more resistant than neurons to the cytotoxic effects of increased [Zn(2+)](i)

Increased intracellular free Zn(2+) ([Zn(2+)](i)) is toxic to neurons. Glia are more resistant to Zn(2+)-mediated toxicity; however, it is not known if this is because glia are less permeable to Zn(2+) or if glia possess intrinsic mechanisms that serve to buffer or extrude excess [Zn(2+)](i). We used the Zn(2+)-selective ionophore pyrithione to directly increase [Zn(2+)](i) in both neurons and astrocytes. In neurons, a 5-min exposure to 1 microM extracellular Zn(2+) in combination with pyrithione produced widespread toxicity, whereas extensive astrocyte injury was not observed until extracellular Zn(2+) was increased to 10 microM. Measurements with magfura-2 demonstrated that pyrithione increased [Zn(2+)](i) to similar levels in both cell types. We also measured how increased [Zn(2+)](i) affects mitochondrial membrane potential (Deltapsi(m)). In astrocytes, but not in neurons, toxic [Zn(2+)](i) resulted in an acute loss of Deltapsi(m), suggesting that mitochondrial dysregulation may be an early event in [Zn(2+)](i)-induced astrocyte but not neuronal death.     

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).     

The effect of tertiary butylhydroperoxide and nicotinamide on human cortical neurons

It is well known that the generation of oxygen radicals can cause neuronal death by both apoptosis and necrosis, which may lead to the onset of neurodegenerative diseases. In previous in vivo studies, nicotinamide was found to prevent both DNA fragmentation and apoptosis that were induced by free radical generating toxins like tertiary butylhydroperoxide (t-BuOOH). Nicotinamide is a precursor for NAD and is an inhibitor of the enzyme poly(ADP-ribose) polymerase (PARP). However, the effect of nicotinamide on the regulation of pro- and anti-apoptotic proteins in neurons is not clear. In our study, the human cortical neuronal cell line HCN1-A has been used to determine the mechanism of action of nicotinamide at the cellular level. Cell viability studies showed that t-BuOOH treatment (both 100 microM and 1mM) caused significant cell death at 24 and 48h compared to control cells. Pretreatment with 1mM nicotinamide before t-BuOOH administration caused significant reduction in cell death. Moreover, the morphology of HCN1-A cells that were treated with both nicotinamide and t-BuOOH appeared to be closer to control cells when compared to HCN1-A cells treated with only t-BuOOH. Also, t-BuOOH treatment caused an elevation in the levels of the pro-apoptotic proteins p53 and p21/WAF-1 and a reduction in the levels of the anti-apoptotic protein bcl-2 compared to their levels in control HCN1-A cells, while pretreatment with nicotinamide reduced p53 and p21/WAF-1 levels even in the presence of t-BuOOH. However nicotinamide did not seem to alter bcl-2 levels. These results indicate that nicotinamide treatment can protect human neuronal cells from the toxic effects of t-BuOOH.     

Prevention of acute/severe hypoglycemia-induced neuron death by lactate administration

Hypoglycemia-induced cerebral neuropathy can occur in patients with diabetes who attempt tight control of blood glucose and may lead to cognitive dysfunction. Accumulating evidence from animal models suggests that hypoglycemia-induced neuronal death is not a simple result of glucose deprivation, but is instead the end result of a multifactorial process. In particular, the excessive activation of poly (ADP-ribose) polymerase-1 (PARP-1) consumes cytosolic nicotinamide adenine dinucleotide (NAD(+)), resulting in energy failure. In this study, we investigate whether lactate administration in the absence of cytosolic NAD(+) affords neuroprotection against hypoglycemia-induced neuronal death. Intraperitoneal injection of sodium L-lactate corrected arterial blood pH and blood lactate concentration after hypoglycemia. Lactate administered without glucose was not sufficient to promote electroencephalogram recovery from an isoelectric state during hypoglycemia. However, supplementation of glucose with lactate reduced neuronal death by ～80% in the hippocampus. Hypoglycemia-induced superoxide production and microglia activation was also substantially reduced by administration of lactate. Taken together, these results suggest an intriguing possibility: that increasing brain lactate following hypoglycemia offsets the decrease in NAD(+) due to overactivation of PARP-1 by acting as an alternative energy substrate that can effectively bypass glycolysis and be fed directly to the citric acid cycle to maintain cellular ATP levels.     

Protective effects of extracellular glutathione against Zn2+-induced cell death in vitro and in vivo

The central nervous system reserves high concentrations of free Zn(2+) in certain excitatory synaptic vesicles. In pathological conditions such as transient cerebral ischemia, traumatic brain injury, and kainic acid (KA)-induced seizure, free Zn(2+) is released in excess at synapses, which causes neuronal and glial death. We report here that glutathione (GSH) can be used as an effective means for protection of neural cells from Zn(2+)-induced cell death in vitro and in vivo. Chronic treatment with 35 microM Zn(2+) led to death of primary cortical neurons and primary astrocytes. The Zn(2+) toxicity of cortical neurons was partially protected by 1 mM of GSH, whereas the Zn(2+) toxicity of primary astrocyte cultures was blocked completely by 100 microM of GSH. To evaluate the beneficial effects of GSH in vivo, an excitotoxin-induced neural cell death model was established by intracerebroventricular (i.c.v.) injection of 0.94 nmol (0.2 microg) KA, which produced selective neuronal death, especially in CA1 and CA3 hippocampal regions. The i.c.v. co-injection of 200 pmol of GSH significantly attenuated KA-induced neuronal cell death and reactive gliosis in hippocampus. The results of this study suggest the contribution of Zn(2+) in the excitotoxin-induced neural cell death model and a potential value of GSH as a therapeutic means against Zn(2+)-induced pathogenesis in brain.     

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.     

Involvement of calmodulin in neuronal cell death

A large body of evidence indicates that disturbances of Ca(2+) homeostasis may be a causative factor in the neurotoxicity following cerebral ischemia. However, the mechanisms by which Ca(2+) overload leads to neuronal cell death have not been fully elucidated. Calmodulin, a major intracellular Ca(2+)-binding protein found mainly in the central nervous system, mediates many physiological functions in response to changes in the intracellular Ca(2+) concentration, whereas Ca(2+) overload in neurons after excitotoxic insult may induce excessive activation of calmodulin signaling pathways, leading to neuronal cell death. To determine the role of calmodulin in the induction of neuronal cell death, we generated primary rat cortical neurons that express a mutant calmodulin with a defect in Ca(2+)-binding affinity. Neurons expressing the mutant had low responses of calmodulin-dependent signaling to membrane depolarization by high KCl and became resistant to glutamate-triggered excitotoxic neuronal cell death compared with the vector or wild-type calmodulin-transfected cells, indicating that blocking calmodulin function is protective against excitotoxic insult. These results suggest that calmodulin plays a crucial role in the processes of Ca(2+)-induced neuronal cell death and the possibility that the blockage of calmodulin attenuates brain injury after cerebral ischemia.     

High activity of K+-dependent plasmalemmal Na+/Ca2+ exchangers in hippocampal CA1 neurons

Ca(2+) influx via reversed K(+)-dependent (NCKX) and/or K(+)-independent (NCX) plasmalemmal Na(+)/Ca(2+) exchangers may play a role in neuronal death following global brain ischemia to which CA1 neurons are particularly vulnerable. Therefore, this work tested whether the rates of Ca(2+) influx via reversed NCKX or NCX in cultured rat CA1 neurons differ from those in forebrain neurons (FNs) or cerebellar granule cells (CGCs). The NCKX-mediated Ca(2+) influx was several times more rapid in CA1 neurons than in FNs or CGCs and was not affected by Na(+)/Ca(2+) exchange inhibitors, KB-R7943 or bepridil. NCKX reversal inhibitors are not yet available. Their development would greatly facilitate further testing the role of NCKX in ischemic death of CA1 neurons.     

Attenuation of Zn2+ neurotoxicity by aspirin: role of N-type Ca2+ channel and the carboxyl acid group

Synaptically released Zn2+ ions enter into neurons primarily through voltage-gated Ca2+ channels (VGCC) or N-methyl-d-aspartate (NMDA) receptors, which can mediate pathological neuronal death. We studied the possibility (and underlying mechanisms) that aspirin, known to prevent NMDA neurotoxicity, would also attenuate Zn2+ neurotoxicity. Administration of 3 to 10 mM aspirin, in cortical cell cultures, attenuated the evolution of neuronal death following exposure to 300 microM Zn2+ for 30 min. This neuroprotective effect of aspirin was attributable to the prevention of Zn2+ ion entry. Aspirin interfered with inward currents and an increase in [Ca2+]i through VGCC and selective binding of omega-conotoxin, sensitive to N-type Ca2+ channel. The omega-conotoxins GVIA or MVIIC, the selective inhibitors of N-type Ca2+ channels, attenuated Zn2+ neurotoxicity. Aspirin derivatives lacking the carboxyl acid group did not reduce Zn2+ neurotoxicity. The present findings suggest that aspirin prevents Zn2+-mediated neuronal death by interfering with VGCC, and its action specifically requires the carboxyl acid group.     

Pyruvate and oxaloacetate limit zinc-induced oxidative HT-22 neuronal cell injury

During CNS ischaemia, accumulating evidence suggests that raised intracellular Zn(2+) levels may play a significant role in inducing neuronal cell death. Several mechanisms mediating Zn(2+)-induced cell death have been suggested, however the precise molecular mechanisms remain uncertain. Employing the HT-22 murine hippocampal neuronal cell line, we have evaluated possible mechanisms of cytotoxic extracellular Zn(2+) insults. Increased extracellular Zn(2+) levels was found to induce concentration-dependent cytotoxicity. When tested at 200muM, Zn(2+) increased intracellular Zn(2+) levels (determined via FluoZin-3 fluorescence) and rapidly induced cell death. However, neither L-type (nimodipine) nor T-type (mibefradil) voltage-activated Ca(2+) channel inhibitors limited Zn(2+)-induced cytotoxicity. Furthermore, and in contrast with staurosporine, Zn(2+) cytotoxic insults failed to induce significant caspase-3 activation and were insensitive to the poly-caspase inhibitor, zVAD-fmk. Antioxidant co-application (Trolox and N,N'-diphenyl-1,4-phenylenediamine (DPPD)) was neuroprotective versus 6h Zn(2+) insults. Additionally, despite inducing significant mitochondrial membrane potential loss, Zn(2+) failed to induce detectable increased superoxide production. However, both pyruvate and oxaloacetate were found to afford significant neuroprotection versus Zn(2+) cytotoxic insults, without significantly influencing intracellular Zn(2+) accumulation. We conclude that cultured HT-22 neurones are vulnerable to Zn(2+) cytotoxic insults via a non-caspase-3 mediated mechanism, which involves glycolytic inhibition.