Karen A. Hartnett

Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release.

Abstract: The membrane‐permeant oxidizing agent 2,2′‐dithiodipyridine (DTDP) can induce Zn2+ release from metalloproteins in cell‐free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neurons, detected by the presence of both DNA laddering and asymmetric chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad‐spectrum cysteine protease inhibitor butoxy‐carbonyl‐aspartate‐fluoromethylketone. N,N,N′,N′‐Tetrakis‐(2‐pyridylmethyl)ethylenediamine (TPEN) and other cell‐permeant metal chelators also effectively blocked DTDP‐induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK‐801, by the intracellular calcium release antagonist dantrolene, or by high concentrations of ryanodine. DTDP generated increases in fluorescence signals in cultured neurons loaded with the zinc‐selective dye Newport Green. The fluorescence signals following DTDP treatment also increased in fura‐2‐ and magfura‐2‐loaded neurons. These responses were completely reversed by TPEN, consistent with a DTDP‐mediated increase in intracellular free Zn2+ concentrations. Our studies suggest that under conditions of oxidative stress, Zn2+ released from intracellular stores may contribute to the initiation of neuronal apoptosis.

Caspase 3 activation is essential for neuroprotection in preconditioning

Sublethal insults can induce tolerance to subsequent stressors in neurons. As cell death activators such as ROS generation and decreased ATP can initiate tolerance, we tested whether other cellular elements normally associated with neuronal injury could add to this process. In an in vivo model of ischemic tolerance, we were surprised to observe widespread caspase 3 cleavage, without cell death, in preconditioned tissue. To dissect the preconditioning pathways activating caspases, and the mechanisms by which these proteases are held in check, we developed an in vitro model of excitotoxic tolerance. In this model, antioxidants and caspase inhibitors blocked ischemia-induced protection against N-methyl-d-aspartate toxicity. Moreover, agents that blocked preconditioning also attenuated induction of HSP 70; transient overexpression of a constitutive form of this protein prevented HSP 70 up-regulation and blocked tolerance. We outline a neuroprotective pathway where events normally associated with apoptotic cell death are critical for cell survival.

Oxygen free radicals regulate NMDA receptor function via a redox modulatory site

A novel modulatory site on the N-methyl-D-aspartate (NMDA) receptor that is sensitive to sulfhydryl redox reagents was recently described. Here we report that this redox modulatory site is susceptible to oxidation by reactive oxygen species endogenous to the CNS. Oxygen free radicals generated by xanthine and xanthine oxidase were observed to decrease NMDA-induced changes in intracellular free Ca2+ concentrations and NMDA-evoked cation currents in cortical neurons in culture. Additionally, a sublethal production of free radicals by xanthine and xanthine oxidase reversed a dithiothreitol-induced enhancement of NMDA-mediated neurotoxicity in vitro. These results show that NMDA receptor function is modulated at its redox site by endogenous substances that normally accompany tissue reperfusion following an ischemic event. This novel mechanism for NMDA receptor regulation may have profound implications in the outcome of glutamate neurotoxicity in vivo.

Apoptotic surge of potassium currents is mediated by p38 phosphorylation of Kv2.1

Kv2.1, the primary delayed rectifying potassium channel in neurons, is extensively regulated by phosphorylation. Previous reports have described Kv2.1 phosphorylation events affecting channel gating and the impact of this process on cellular excitability. Kv2.1, however, also provides the critical exit route for potassium ions during neuronal apoptosis via p38 MAPK-dependent membrane insertion, resulting in a pronounced enhancement of K+ currents. Here, electrophysiological and viability studies using Kv2.1 channel mutants identify a p38 phosphorylation site at Ser-800 (S800) that is required for Kv2.1 membrane insertion, K+ current surge, and cell death. In addition, a phospho-specific antibody for S800 detects a p38-dependent increase in Kv2.1 phosphorylation in apoptotic neurons and reveals phosphorylation of S800 in immunopurified channels incubated with active p38. Consequently, phosphorylation of Kv2.1 residue S800 by p38 leads to trafficking and membrane insertion during apoptosis, and remarkably, the absence of S800 phosphorylation is sufficient to prevent completion of the cell death program.

Assessment of Cell Viability in Primary Neuronal Cultures

This unit contains five protocols for assaying cell viability in vitro using primary neuronal cultures, including a novel method for use with transfected neurons. Three of the assays are based on the principle that cell death cascades alter membrane permeability. The lactate dehydrogenase (LDH) release assay measures the amount of the cytoplasmic enzyme released into the bathing medium, while the trypan blue and propidium iodide assays measure the ability of cells to exclude dye from their cytoplasm. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay measures the mitochondrial activity of viable cells by quantifying the conversion of the tetrazolium salt to its formazan product. Finally, the fifth assay details the measurement of luciferase expression as an indication of neuronal viability within a relatively small population of transfected neurons. Curr. Protoc. Neurosci. 44:7.18.1‐7.18.15.

NMDA receptor-mediated neurotoxicity: a paradoxical requirement for extracellular Mg2+ in Na+/Ca2+-free solutions in rat cortical neurons in vitro.

Abstract: Accumulation of intracellular Ca2+ is known to be critically important for the expression of NMDA receptor‐mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg2+ concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg2+ content independently of Ca2+ to investigate the potential role of Mg2+ in excitotoxicity in rat cortical neurons in vitro. In Ca2+‐free solutions in which the Na+ was replaced by N‐methyl‐d‐glucamine or Tris (but not choline), which also contained 9 mM Mg2+, exposure to 100 µM glutamate or 200 µM NMDA for 20 min produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg2+ concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg2+‐dependent NMDA receptor‐mediated toxicity. Thus, the increase in intracellular Mg2+ concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury.

Regulation of apoptotic potassium currents by coordinated zinc‐dependent signalling

Oxidant‐liberated intracellular Zn2+ regulates neuronal apoptosis via an exocytotic membrane insertion of Kv2.1‐encoded ion channels, resulting in an enhancement of voltage‐gated K+ currents and a loss of intracellular K+ that is necessary for caspase‐mediated proteolysis. In the present study we show that an N‐terminal tyrosine of Kv2.1 (Y124), which is a known target of Src kinase, is critical for the apoptotic current surge. Moreover, we demonstrate that Y124 works in concert with a C‐terminal serine (S800) target of p38 mitogen‐activated protein kinase (MAPK) to regulate Kv2.1‐mediated current enhancement. While Zn2+ was previously shown to activate p38, we show here that this metal inhibits cytoplasmic protein tyrosine phosphatase ɛ (Cyt‐PTPɛ), which specifically targets Y124. Importantly, a point mutation of Y124 to a non‐phosphorylatable residue or over‐expression of Cyt‐PTPɛ protects cells from injury. Kv2.1‐encoded channels thus regulate neuronal survival by providing a converging input for two Zn2+‐dependent signal transduction cascades.

Microglia induce neurotoxicity via intraneuronal Zn2+ 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 Zn2+ 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 Zn2+ 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 Zn2+ release and a surge in K+ currents.

Protein kinase C regulation of neuronal zinc signaling mediates survival during preconditioning

Sub‐lethal activation of cell death processes initiate pro‐survival signaling cascades. As intracellular Zn2+ liberation mediates neuronal death pathways, we tested whether a sub‐lethal increase in free Zn2+ could also trigger neuroprotection. Neuronal free Zn2+ transiently increased following preconditioning, and was both necessary and sufficient for conferring excitotoxic tolerance. Lethal exposure to NMDA led to a delayed increase in Zn2+ that contributed significantly to excitotoxicity in non‐preconditioned neurons, but not in tolerant neurons, unless preconditioning‐induced free Zn2+ was chelated. Thus, preconditioning may trigger the expression of Zn2+‐regulating processes, which, in turn, prevent subsequent Zn2+‐mediated toxicity. Indeed, preconditioning increased Zn2+‐regulated gene expression in neurons. Examination of the molecular signaling mechanism leading to this early Zn2+ signal revealed a critical role for protein kinase C (PKC) activity, suggesting that PKC may act directly on the intracellular source of Zn2+. We identified a conserved PKC phosphorylation site at serine‐32 (S32) of metallothionein (MT) that was important in modulating Zn2+‐regulated gene expression and conferring excitotoxic tolerance. Importantly, we observed increased PKC‐induced serine phosphorylation in immunopurified MT1, but not in mutant MT1(S32A). These results indicate that neuronal Zn2+ serves as an important, highly regulated signaling component responsible for the initiation of a neuroprotective pathway.

SNARE-dependent upregulation of potassium chloride co-transporter 2 activity after metabotropic zinc receptor activation in rat cortical neurons in vitro.

The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABA(A) receptor channels. In a recent study, we showed that Zn(2+) regulates GABA(A) reversal potentials in the hippocampus by enhancing the activity of KCC2 through an increase in its surface expression. Zn(2+) initiates this process by activating the Gq-coupled metabotropic Zn(2+) receptor/G protein-linked receptor 39 (mZnR/GPR39). Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture, and then tested the hypothesis that the increase in KCC2 activity is mediated through a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca(2+) indicator and exposing cells to Zn(2+), which triggered consistent Ca(2+) responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist (RS)-α-methyl-4-carboxyphenylglycine (MCPG). Importantly, Zn(2+) treatment under these conditions did not increase the intracellular concentrations of Zn(2+) itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH(4)(+) influx through the co-transporter using an intracellular pH-sensitive fluorescent indicator. We observed that Zn(2+) pretreatment induced a Ca(2+)-dependent increase in KCC2 activity. The effects of Zn(2+) on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39(-/-) mice, suggesting that Zn(2+) acts through mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and synaptosomal-associated protein 25 (SNAP-25). Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn(2+)-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn(2+). These results suggest that SNARE proteins are necessary for the increased activity of KCC2 after Zn(2+) stimulation of mZnR/GPR39.