Zn2+ entry produces oxidative neuronal necrosis in cortical cell cultures

Evidence has accumulated that Zn²⁺ plays a central role in neurodegenerative processes following brain injuries including ischaemia or epilepsy. In the present study, we examined patterns and possible mechanisms of Zn²⁺ neurotoxicity. Inclusion of 30–300 μm Zn²⁺ for 30 min caused neuronal necrosis apparent by cell body and mitochondrial swelling in cortical cell cultures. This Zn²⁺ neurotoxicity was not attenuated by antiapoptosis agents, inhibitors of protein synthesis or caspase. Blockade of glutamate receptors or nitric oxide synthase showed no beneficial effect against Zn²⁺ neurotoxicity. Interestingly, antioxidants, trolox or SKF38393, attenuated Zn²⁺-induced neuronal necrosis. Pretreatment with insulin or brain-derived neurotrophic factor increased the Zn²⁺-induced free radical injury. Kainate or AMPA facilitated Zn²⁺ entry and potentiated Zn²⁺ neurotoxicity in a way sensitive to trolox. Reactive oxygen species and lipid peroxidation were generated in the early phase of Zn²⁺ neurotoxicity. These findings indicate that entry and accumulation of Zn²⁺ result in generation of toxic free radicals and then cause necrotic neuronal degeneration under certain pathological conditions in the brain.     

Zn2+ regulates Kv2.1 voltage‐dependent gating and localization following ischemia

The delayed‐rectifier K⁺ channel Kv2.1 exists in highly phosphorylated somatodendritic clusters. Ischemia induces rapid Kv2.1 dephosphorylation and a dispersal of these clusters, accompanied by a hyperpolarizing shift in their voltage‐dependent activation kinetics. Transient modulation of Kv2.1 activity and localization following ischemia is dependent on a rise in intracellular Ca²⁺and the protein phosphatase calcineurin. Here, we show that neuronal free Zn²⁺also plays a critical role in the ischemic modulation of Kv2.1. We found that sub‐lethal ischemia in cultured rat cortical neurons led to characteristic hyperpolarizing shifts in K⁺ current voltage dependency and pronounced dephosphorylation of Kv2.1. Zn²⁺chelation, similar to calcineurin inhibition, attenuated ischemic induced changes in K⁺ channel activation kinetics. Zn²⁺chelation during ischemia also blocked Kv2.1 declustering. Surprisingly, we found that the Zn²⁺rise following ischemia occurred in spite of calcineurin inhibition. Therefore, a calcineurin‐independent rise in neuronal free Zn²⁺ is critical in altering Kv2.1 channel activity and localization following ischemia. The identification of Zn²⁺ in mediating ischemic modulation of Kv2.1 may lead to a better understanding of cellular adaptive responses to injury.     

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

In view of evidence that Zn²⁺ neurotoxicity contributes to some forms of pathological neuronal death, we developed a model of Zn²⁺ neurotoxicity in a cell line amenable to genetic manipulations. Exposure to 500 μM ZnCl₂ for 15 min under depolarizing conditions resulted in modest levels of PC12 cell death, that was reduced by the L-type Ca²⁺ channel antagonist, nimodipine, and increased by the L-type Ca²⁺ channel opener, S(−)-Bay K 8644. At lower insult levels (200 μM Zn²⁺+Bay K 8644), Zn²⁺-induced death appeared apoptotic under electron microscopy and was sensitive to the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-CH₂F (Z-VAD); at higher insult levels (1000 μM+Bay K 8644), cells underwent necrosis insensitive to Z-VAD. To test the hypothesis that the plasma membrane transporter, ZnT-1, modulates Zn²⁺ 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 Zn²⁺ efflux and reduced vulnerability to Zn²⁺-induced death compared to the parental line, whereas clones D5 and D16 expressing dominant negative rZnT-1 exhibited the opposite characteristics.     

Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca²⁺ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca²⁺ media (0–1.0 mM), but was completely abolished in a low Na⁺ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na⁺, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na⁺ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca²⁺ was detected under these conditions. Though Na⁺ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na⁺-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na⁺ influx via voltage-dependent Na⁺ channels, and suggests the presence of Na⁺-dependent regulatory mechanism(s) in neuronal survival.     

Nicotinamide Prevents NAD+ Depletion and Protects Neurons Against Excitotoxicity and Cerebral Ischemia: NAD+ Consumption by SIRT1 may Endanger Energetically Compromised Neurons

Neurons require large amounts of energy to support their survival and function, and are therefore susceptible to excitotoxicity, a form of cell death involving bioenergetic stress that may occur in several neurological disorders including stroke and Alzheimer’s disease. Here we studied the roles of NAD⁺ bioenergetic state, and the NAD⁺-dependent enzymes SIRT1 and PARP-1, in excitotoxic neuronal death in cultured neurons and in a mouse model of focal ischemic stroke. Excitotoxic activation of NMDA receptors induced a rapid decrease of cellular NAD(P)H levels and mitochondrial membrane potential. Decreased NAD⁺ levels and poly (ADP-ribose) polymer (PAR) accumulation in nuclei were relatively early events (<4 h) that preceded the appearance of propidium iodide- and TUNEL-positive cells (markers of necrotic cell death and DNA strand breakage, respectively) which became evident by 6 h. Nicotinamide, an NAD⁺ precursor and an inhibitor of SIRT1 and PARP1, inhibited SIRT1 deacetylase activity without affecting SIRT1 protein levels. NAD⁺ levels were preserved and PAR accumulation and neuronal death induced by excitotoxic insults were attenuated in nicotinamide-treated cells. Treatment of neurons with the SIRT1 activator resveratrol did not protect them from glutamate/NMDA-induced NAD⁺ depletion and death. In a mouse model of focal cerebral ischemic stroke, NAD⁺ levels were decreased in both the contralateral and ipsilateral cortex 6 h after the onset of ischemia. Stroke resulted in dynamic changes of SIRT1 protein and activity levels which varied among brain regions. Administration of nicotinamide (200 mg/kg, i.p.) up to 1 h after the onset of ischemia elevated brain NAD⁺ levels and reduced ischemic infarct size. Our findings demonstrate that the NAD⁺ bioenergetic state is critical in determining whether neurons live or die in excitotoxic and ischemic conditions, and suggest a potential therapeutic benefit in stroke of agents that preserve cellular NAD⁺ levels. Our data further suggest that, SIRT1 is linked to bioenergetic state and stress responses in neurons, and that under conditions of reduced cellular energy levels SIRT1 enzyme activity may consume sufficient NAD⁺ to nullify any cell survival-promoting effects of its deacetylase action on protein substrates.     

Zinc neurotoxicity is dependent on intracellular NAD levels and the sirtuin pathway

Zinc neurotoxicity has been demonstrated in ischemic, seizure, hypoglycemic, and trauma-induced neuronal death where Zn(2+) is thought to be synaptically released and taken up in neighbouring neurons, reaching toxic concentrations. We previously demonstrated that toxicity of extracellular Zn(2+) depended on entry, elevation in intracellular free Zn(2+) ([Zn(2+)](i)), a reduction in NAD(+) and ATP levels, and dysfunction of glycolysis and cellular metabolism. We suggested that PARP-1 activation alone can not explain this loss of neuronal NAD(+). NAD(+) was recently demonstrated to permeate neurons and glia, and we have now shown that exogenous NAD(+) can reduce Zn(2+) neurotoxicity, and 3-acetylpyridine, which generates inactive NAD(+), potentiated Zn(2+) neurotoxicity. Sirtinol and 2-hydroxynaphthaldehyde, inhibitors of the sirtuin pathway (SIRT proteins are NAD(+)-catabolic protein deacetylases), attenuated both acute and chronic Zn(2+) neurotoxicity. Resveratrol and fisetin (sirtuin activators) potentiated NAD(+) loss and Zn(2+) neurotoxicities. Furthermore, neuronal cultures derived from the Wld(s) mouse, which overexpress the NAD(+) synthetic enzyme nicotinamide mononucleotide adenyl transferase (NMNAT-1), had reduced sensitivity to Zn(2+) neurotoxicity. Finally, nicotinamide was demonstrated to attenuate CA1 neuronal death after 10 min of global ischemia in rat even if administered 1 h after the insult. Together with previous data, these results further implicate NAD(+) levels in Zn(2+) neurotoxicity.     

Release, neuronal effects and removal of extracellular β-nicotinamide adenine dinucleotide (β-NAD⁺) in the rat brain

Recent evidence supports an emerging role of β‐nicotinamide adenine dinucleotide (β‐NAD⁺) as a novel neurotransmitter and neuromodulator in the peripheral nervous system –β‐NAD⁺ is released in nerve‐smooth muscle preparations and adrenal chromaffin cells in a manner characteristic of a neurotransmitter. It is currently unclear whether this holds true for the CNS. Using a small‐chamber superfusion assay and high‐sensitivity high‐pressure liquid chromatography techniques, we demonstrate that high‐K⁺ stimulation of rat forebrain synaptosomes evokes overflow of β‐NAD⁺, adenosine 5′‐triphosphate, and their metabolites adenosine 5′‐diphosphate (ADP), adenosine 5′‐monophosphate, adenosine, ADP‐ribose (ADPR) and cyclic ADPR. The high‐K⁺‐evoked overflow of β‐NAD⁺ is attenuated by cleavage of SNAP‐25 with botulinum neurotoxin A, by inhibition of N‐type voltage‐dependent Ca²⁺ channels with ω‐conotoxin GVIA, and by inhibition of the proton gradient of synaptic vesicles with bafilomycin A1, suggesting that β‐NAD⁺ is likely released via vesicle exocytosis. Western analysis demonstrates that CD38, a multifunctional protein that metabolizes β‐NAD⁺, is present on synaptosomal membranes and in the cytosol. Intact synaptosomes degrade β‐NAD⁺. 1,N ⁶‐etheno‐NAD, a fluorescent analog of β‐NAD⁺, is taken by synaptosomes and this uptake is attenuated by authentic β‐NAD⁺, but not by the connexin 43 inhibitor Gap 27. In cortical neurons local applications of β‐NAD⁺ cause rapid Ca²⁺ transients, likely due to influx of extracellular Ca²⁺. Therefore, rat brain synaptosomes can actively release, degrade and uptake β‐NAD⁺, and β‐NAD⁺ can stimulate postsynaptic neurons, all criteria needed for a substance to be considered a candidate neurotransmitter in the brain.     

Zn2+‐induced ERK activation mediates PARP‐1‐dependent ischemic‐reoxygenation damage to oligodendrocytes

Much of the cell death following episodes of anoxia and ischemia in the mammalian central nervous system has been attributed to extracellular accumulation of glutamate and ATP, which causes a rise in [Ca²⁺]_i, loss of mitochondrial potential, and cell death. However, restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury (the oxygen paradox). Herein we describe a novel signaling pathway that is activated during ischemia‐like conditions (oxygen and glucose deprivation; OGD) and contributes to ischemia‐induced oligodendroglial cell death. OGD induced a retarded and sustained increase in extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation after restoring glucose and O₂ (reperfusion‐like conditions). Blocking the ERK1/2 pathway with the MEK inhibitor UO126 largely protected oligodendrocytes against ischemic insults. ERK1/2 activation was blocked by the high‐affinity Zn²⁺ chelator TPEN, but not by antagonists of AMPA/kainate or P2X7 receptors that were previously shown to be involved in ischemic oligodendroglial cell death. Using a high‐affinity Zn²⁺ probe, we showed that ischemia induced an intracellular Zn²⁺ rise in oligodendrocytes, and that incubation with TPEN prevented mitochondrial depolarization and ROS generation after ischemia. Accordingly, exposure to TPEN and the antioxidant Trolox reduced ischemia‐induced oligodendrocyte death. Moreover, UO126 blocked the ischemia‐induced increase in poly‐[ADP]‐ribosylation of proteins, and the poly[ADP]‐ribose polymerase 1 (PARP‐1) inhibitor DPQ significantly inhibited ischemia‐induced oligodendroglial cell death—demonstrating that PARP‐1 was required downstream in the Zn²⁺‐ERK oligodendrocyte cell death pathway. Chelation of cytosolic Zn²⁺, blocking ERK signaling, and antioxidants may be beneficial for treating CNS white matter ischemia‐reperfusion injury. Importantly, all the inhibitors of this pathway protected oligodendrocytes when applied after the ischemic insult. © 2012 Wiley Periodicals, Inc.     

Modification of hippocampal excitability in brain slices pretreated with a low nanomolar concentration of Zn2+

Synaptic Zn²⁺ homeostasis may be changed during brain slice preparation. However, much less attention has been paid to Zn²⁺ in artificial cerebrospinal fluid (ACSF) used for slice experiments than has been paid to Ca²⁺. The present study assesses addition of Zn²⁺ to ACSF, focused on hippocampal excitability after acute brain slice preparation. When the static levels of intracellular Zn²⁺ and Ca²⁺ were compared between brain slices prepared with conventional ACSF without Zn²⁺ and those pretreated with ACSF containing 20 nM ZnCl₂ for 1 hr, both levels were almost the same. On the other hand, intracellular Ca²⁺ levels were significantly increased in the stratum lucidum of the control brain slices after stimulation with high K⁺, although the increase was significantly suppressed by the pretreatment with ACSF containing Zn²⁺, suggesting that neuronal excitation is enhanced in brain slices prepared with ACSF without Zn²⁺. The increase in extracellular Zn²⁺ level, an index of glutamate release, after stimulation with high K⁺ was also significantly suppressed by pretreatment with ACSF containing Zn²⁺. When mossy fiber excitation was assessed in brain slices with FM4‐64, an indicator of presynaptic activity, attenuation of FM 4‐64 fluorescence based on presynaptic activity was suppressed in the stratum lucidum of brain slices pretreated with ACSF containing Zn²⁺. The present study indicates that hippocampal excitability is enhanced in brain slices prepared with ACSF without Zn²⁺. It is likely that a low nanomolar concentration of Zn²⁺ is necessary for ACSF. © 2015 Wiley Periodicals, Inc.     

Delayed application of aurintricarboxylic acid reduces glutamate-induced cortical neuronal injury

The non-specific endonuclease inhibitor, aurintricarboxylic acid (ATA), attenuated glutamate-induced destruction of cultured cortical neurons. In part, this protective effect likely reflected the ability of ATA to produce a slowly developing block of N-methyl-D-aspartate receptor-mediated inward whole cell current or increase in intracellular free Ca²⁺. However, ATA also attenuated a high K⁺-induced increase in intracellular free Ca²⁺ in the presence of D-aminophosphonovalerate, suggesting that ATA may have a more general effect on Ca²⁺ homeostasis. In addition, ATA attenuated glutamate neurotoxicity even if added up to 2 hr after completion of glutamate exposure, a time when glutamate antagonists or lipid peroxidation inhibitors are no longer neuroprotective. Involvement of apoptosis in this excitotoxic death is unlikely, as Southern blotting of genomic DNA revealed no evidence of fragmentation, and death was not prevented by inhibitors of RNA or protein synthesis. Most likely, ATA interferes with some key downstream consequences of excitotoxic glutamate receptor overactivation. © 1994 Wiley-Liss, Inc.     

Involvement of intracellular Zn2+ signaling in LTP at perforant pathway–CA1 pyramidal cell synapse

Physiological significance of synaptic Zn²⁺ signaling was examined at perforant pathway–CA1 pyramidal cell synapses. In vivo long‐term potentiation (LTP) at perforant pathway–CA1 pyramidal cell synapses was induced using a recording electrode attached to a microdialysis probe and the recording region was locally perfused with artificial cerebrospinal fluid (ACSF) via the microdialysis probe. Perforant pathway LTP was not attenuated under perfusion with CaEDTA (10 mM), an extracellular Zn²⁺ chelator, but attenuated under perfusion with ZnAF‐2DA (50 μM), an intracellular Zn²⁺ chelator, suggesting that intracellular Zn²⁺ signaling is required for perforant pathway LTP. Even in rat brain slices bathed in CaEDTA in ACSF, intracellular Zn²⁺ level, which was measured with intracellular ZnAF‐2, was increased in the stratum lacunosum‐moleculare where perforant pathway–CA1 pyramidal cell synapses were contained after tetanic stimulation. These results suggest that intracellular Zn²⁺ signaling, which originates in internal stores/proteins, is involved in LTP at perforant pathway–CA1 pyramidal cell synapses. Because the influx of extracellular Zn²⁺, which originates in presynaptic Zn²⁺ release, is involved in LTP at Schaffer collateral‐CA1 pyramidal cell synapses, synapse‐dependent Zn²⁺ dynamics may be involved in plasticity of postsynaptic CA1 pyramidal cells.     

Comparative effects of metal chelating agents on the neuronal cytotoxicity induced by copper (Cu), iron (Fe) and zinc in the hippocampus

The ability of metal chelating agents to prevent neuronal death caused by intra-hippocampal injections of cupric sulphate, ferric citrate and zinc chloride was investigated. Ammonium tetrathiomolybdate was itself toxic after injection into the hippocampus, but this toxicity was reduced by formation of a metal ion/tetrathiomolybdate complex with Cu⁺². Disodium bathocuproine disulphonate (BCDS) prevented neuronal death caused by Cu⁺², but not that induced by Fe⁺³ or Zn⁺². Desferrioxamine prevented death caused by Fe⁺³, had no significant effect of the toxicity of Zn⁺², and increased that caused by Cu⁺². Even though N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) has a higher affinity for Cu⁺² than for Zn⁺², TPEN had no effect on the toxicity of Cu⁺² while totally preventing damage caused by Fe⁺³ or Zn⁺². Ethylenediaminetetra-acetic acid (EDTA) prevented the toxicity of all three metal ions. Motor seizure activity occurred in most rats after injections of Fe⁺³; or combinations of Cu⁺² plus TPEN, or 4 nmol Fe⁺³ plus 0.1 nmol desferrioxamine. However, apart from the low dose desferrioxamine/Fe⁺³ combination, only the occasional brain contained seizure-induced neuronal loss in limbic regions outside the injected hippocampus, and these brains were not used for analysis. Seizure activity was found even with very low levels of Cu⁺² with a fixed amount of TPEN (a ratio of Cu⁺²/TPEN of 1:100), but the extent of hippocampal damage in these brains was not significantly different to that caused by injections of saline. These studies demonstrate that idiosyncratic interactions can occur between metal ions and chelating agents. Thus further investigations are needed before chelating agents can be examined for their protective properties in various neurodegenerative diseases.     

The effects of innervation on the developmental expression of Ca2+-activated K+ currents in embryonic parasympathetic neurons are not activity-dependent

Chronic blockade of synaptic transmission in ovo using mecamylamine, a neuronal nicotinic receptor antagonist, caused a large increase in naturally occurring cell death in the embryonic chick ciliary ganglion. However, the Ca²⁺-activated K⁺ currents in embryonic day 13 mecamylamine-treated ciliary ganglion neurons were indistinguishable from those of saline-treated controls. Therefore, the trophic effect of preganglionic innervation on the developmental expression of Ca ²⁺-activated K⁺ current is not dependent upon intact nicotinic cholinergic synaptic transmission and may instead be mediated by a nerve terminal-derived differentiation factor.     

Prevention of hypoglycemia-induced neuronal death by hypothermia

Hypothermia reduces neuronal damage after cerebral ischemia and traumatic brain injury, while hyperthermia exacerbates damage from these insults. Previously we have shown that temperature-dependent modulation of excitotoxic neuronal death is mediated in part by temperature-dependent changes in the synaptic release/translocation of Zn²⁺. In this study, we hypothesize that brain temperature also affects hypoglycemia-induced neuronal death by modulation of vesicular Zn²⁺ release from presynaptic terminals. To test our hypothesis, we used a rat model of insulin-induced hypoglycemia. Here we found that hypoglycemia-induced neuronal injury was significantly affected by brain temperature, that is, hypothermia inhibited while hyperthermia aggravated neuronal death. To investigate the mechanism of temperature-dependent neuronal death after hypoglycemia, we measured zinc release/translocation, reactive oxygen species (ROS) production, and microglia activation. Here we found that hypoglycemia-induced Zn²⁺ release/translocation, ROS production, and microglia activation were inhibited by hypothermia but aggravated by hyperthermia. Even when the insult was accompanied by hyperthermic conditions, zinc chelation inhibited ROS production and microglia activation. Zinc chelation during hyperthermia reduced neuronal death, superoxide production, and microglia activation, which was comparable to the protective effects of hypothermia. We conclude that neuronal death after hypoglycemia is temperature-dependent and is mediated by increased Zn²⁺ release, superoxide production, and microglia activation.     

Neuroprotective activity of stiripentol with a possible involvement of voltage‐dependent calcium and sodium channels

A growing body of data has shown that recurrent epileptic seizures may be caused by an excessive release of the excitatory neurotransmitter glutamate in the brain. Glutamatergic overstimulation results in massive neuronal influxes of calcium and sodium through N‐methyl‐D‐aspartate (NMDA), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, and kainic acid glutamate subtype receptors and also through voltage‐gated calcium and sodium channels. These persistent and abnormal sodium and calcium entry points have deleterious consequences (neurotoxicity) for neuronal function. The therapeutic value of an antiepileptic drug would include not only control of seizure activity but also protection of neuronal tissue. The present study examines the in vitro neuroprotective effects of stiripentol, an antiepileptic compound with γ‐aminobutyric acidergic properties, on neuronal–astroglial cultures from rat cerebral cortex exposed to oxygen–glucose deprivation (OGD) or to glutamate (40 µM for 20 min), two in vitro models of brain injury. In addition, the affinity of stiripentol for the different glutamate receptor subtypes and the interaction with the cell influx of Na⁺ and of Ca²⁺ enhanced by veratridine and NMDA, respectively, are assessed. Stiripentol (10–100 µM) included in the culture medium during OGD or with glutamate significantly increased the number of surviving neurons relative to controls. Stiripentol displayed no binding affinity for different subtypes of glutamate receptors (IC₅₀ > 100 µM) but significantly blocked the entry of Na⁺ and Ca²⁺ activated by veratridine and NMDA, respectively. These results suggest that Na⁺ and Ca²⁺ channels could contribute to the neuroprotective properties of sitiripentol. © 2015 Wiley Periodicals, Inc.     

Gray and white matter astrocytes differ in basal metabolism but respond similarly to neuronal activity

Astrocytes are a heterogeneous population of glial cells in the brain, which adapt their properties to the requirements of the local environment. Two major groups of astrocytes are protoplasmic astrocytes residing in gray matter as well as fibrous astrocytes of white matter. Here, we compared the energy metabolism of astrocytes in the cortex and corpus callosum as representative gray matter and white matter regions, in acute brain slices taking advantage of genetically encoded fluorescent nanosensors for the NADH/NAD⁺ redox ratio and for ATP. Astrocytes of the corpus callosum presented a more reduced basal NADH/NAD⁺ redox ratio, and a lower cytosolic concentration of ATP compared to cortical astrocytes. In cortical astrocytes, the neurotransmitter glutamate and increased extracellular concentrations of K⁺, typical correlates of neuronal activity, induced a more reduced NADH/NAD⁺ redox ratio. While application of glutamate decreased [ATP], K⁺ as well as the combination of glutamate and K⁺ resulted in an increase of ATP levels. Strikingly, a very similar regulation of metabolism by K⁺ and glutamate was observed in astrocytes in the corpus callosum. Finally, strong intrinsic neuronal activity provoked by application of bicuculline and withdrawal of Mg²⁺ caused a shift of the NADH/NAD⁺ redox ratio to a more reduced state as well as a slight reduction of [ATP] in gray and white matter astrocytes. In summary, the metabolism of astrocytes in cortex and corpus callosum shows distinct basal properties, but qualitatively similar responses to neuronal activity, probably reflecting the different environment and requirements of these brain regions.     

Nicotinamide mononucleotide alters mitochondrial dynamics by SIRT3-dependent mechanism in male mice

Nicotinamide adenine dinucleotide (NAD⁺) is a central signaling molecule and enzyme cofactor that is involved in a variety of fundamental biological processes. NAD⁺ levels decline with age, neurodegenerative conditions, acute brain injury, and in obesity or diabetes. Loss of NAD⁺ results in impaired mitochondrial and cellular functions. Administration of NAD⁺ precursor, nicotinamide mononucleotide (NMN), has shown to improve mitochondrial bioenergetics, reverse age-associated physiological decline, and inhibit postischemic NAD⁺ degradation and cellular death. In this study, we identified a novel link between NAD⁺ metabolism and mitochondrial dynamics. A single dose (62.5 mg/kg) of NMN, administered to male mice, increases hippocampal mitochondria NAD⁺ pools for up to 24 hr posttreatment and drives a sirtuin 3 (SIRT3)-mediated global decrease in mitochondrial protein acetylation. This results in a reduction of hippocampal reactive oxygen species levels via SIRT3-driven deacetylation of mitochondrial manganese superoxide dismutase. Consequently, mitochondria in neurons become less fragmented due to lower interaction of phosphorylated fission protein, dynamin-related protein 1 (pDrp1 [S616]), with mitochondria. In conclusion, manipulation of mitochondrial NAD⁺ levels by NMN results in metabolic changes that protect mitochondria against reactive oxygen species and excessive fragmentation, offering therapeutic approaches for pathophysiologic stress conditions.     

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.     

Calpain activation and Na+/Ca2+ exchanger degradation occur downstream of calcium deregulation in hippocampal neurons exposed to excitotoxic glutamate

Delayed calcium deregulation (DCD) plays an essential role in glutamate excitotoxicity, a major detrimental factor in stroke, traumatic brain injury, and various neurodegenerations. In the present study, we examined the role of calpain activation and Na⁺/Ca²⁺ exchanger (NCX) degradation in DCD and excitotoxic cell death in cultured hippocampal neurons. Exposure of neurons to glutamate caused DCD accompanied by secondary mitochondrial depolarization. Activation of calpain was evidenced by detecting NCX isoform 3 (NCX3) degradation products. Degradation of NCX isoform 1 (NCX1) was below the detection limit of Western blotting. Degradation of NCX3 was detected only after 1 hr of incubation with glutamate, whereas DCD occurred on average within 15 min after glutamate application. Calpeptin, an inhibitor of calpain, significantly attenuated NCX3 degradation but failed to inhibit DCD and excitotoxic neuronal death. Calpain inhibitors I, III, and VI also failed to influence DCD and glutamate‐induced neuronal death. On the other hand, MK801, an inhibitor of the NMDA subtype of glutamate receptors, added shortly after the initial glutamate‐induced jump in cytosolic Ca²⁺, completely prevented DCD and activation of calpain and strongly protected neurons against excitotoxicity. Taken together, our results suggest that, in glutamate‐treated hippocampal neurons, the initial increase in cytosolic Ca²⁺ that precedes DCD is insufficient for sustained calpain activation, which most likely occurs downstream of DCD. © 2009 Wiley‐Liss, Inc.