Elias Aizenman

Selective modulation of NMDA responses by reduction and oxidation

Electrophysiological responses to the glutamate analog N-methyl-D-aspartate (NMDA) measured in three different central neuronal preparations are subject to a novel modulatory mechanism: they are substantially potentiated after exposure to the disulfide reducing agent dithiothreitol, while oxidation with 5-5-dithiobis-2-nitrobenzoic acid decreases the magnitude of the response. Modification of the NMDA response by either oxidation or reduction does not appear to affect the pharmacological properties of the receptor-channel complex. Since we observe that the redox state of the native receptor-channel complex varies widely among neurons, an in vivo mechanism that can strongly regulate NMDA-activated functions by either reduction or oxidation may exist. In addition, these results suggest that it may be possible to design specific redox agents for characterizing the NMDA receptor-channel complex.

Hundred-fold increase in neuronal vulnerability to glutamate toxicity in astrocyte-poor cultures of rat cerebral cortex

In cultures of rat cerebral cortex in which astrocyte proliferation was stringently suppressed, glutamate neurotoxicity occurred at glutamate concentrations similar to those which are normally found in the extracellular space in the hippocampus. Concentrations of glutamate one hundred-fold higher were required to produce neurotoxicity in the presence of abundant astrocytes. This suggests that the sensitivity of central neurons to glutamate toxicity may be dependent upon astrocyte function.

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.

The Neurophysiology and Pathology of Brain Zinc

Our understanding of the roles played by zinc in the physiological and pathological functioning of the brain is rapidly expanding. The increased availability of genetically modified animal models, selective zinc-sensitive fluorescent probes, and novel chelators is producing a remarkable body of exciting new data that clearly establishes this metal ion as a key modulator of intracellular and intercellular neuronal signaling. In this Mini-Symposium, we will review and discuss the most recent findings that link zinc to synaptic function as well as the injurious effects of zinc dyshomeostasis within the context of neuronal death associated with major human neurological disorders, including stroke, epilepsy, and Alzheimers disease.

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.

Peroxynitrite-Induced Neuronal Apoptosis Is Mediated by Intracellular Zinc Release and 12-Lipoxygenase Activation

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study, we observed that TPEN [N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine], a zinc chelator, protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor, diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition, we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX), p38 mitogen-activated protein kinase (MAPK), and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX, generation of reactive oxygen species (ROS), and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX, ROS accumulation, p38 activation, and caspase-3 activation. Therefore, therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation, stroke, and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death.

Nitric oxide modulates NMDA-induced increases in intracellular Ca2+ in cultured rat forebrain neurons

We studied the effects of nitric oxide (NO) and the NO-releasing agents sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and isosorbide dinitrate (ISDN) on N-methyl-D-aspartate (NMDA)-induced increases in intracellular Ca2+ ([Ca2+]i), whole-cell patch-clamp currents and on glutamate-stimulated [3H]dizocilpine binding. NO and agents that release NO partially inhibit increases in [Ca2+]i at concentrations between 1 microM and 1 mM. These agents also decrease [Ca2+]i changes produced by kainate and potassium, but to a smaller extent. As the effects of NO are still present following alkylation of the redox modulatory site on the NMDA receptor this action of NO is probably not a consequence of oxidation of the redox site. In contrast to SNP, ISDN does not inhibit NMDA-induced whole cell patch-clamp currents suggesting that NO modulates [Ca2+]i via perturbation of a Ca2+ homeostatic process. Furthermore, SNP may have a direct action on the NMDA receptor complex in addition to the generation of NO. 8-Bromo-cGMP does not mimic the inhibitory effect of NO suggesting that this effect is not the result of NO stimulation of neuronal cGMP production. As the production of NO in neurons is dependent on increases in [Ca2+]i associated with NMDA receptor activation, these data suggest that NO-mediated decreases in [Ca2+]i may represent a novel feedback inhibitory mechanism for NO production in the brain.

Responses mediated by excitatory amino acid receptors in solitary retinal ganglion cells from rat.

1. The pharmacological properties of excitatory amino acid responses on ganglion cells dissociated from the rat retina were examined with the use of the whole‐cell voltage‐clamp technique. 2. L‐Glutamate at a concentration of 50 microM produced inward non‐desensitizing currents at negative holding potentials in nearly every cell tested (83%, n = 18) In physiological solutions, L‐glutamate responses reversed at approximately ‐9 mV, and higher concentrations of this agonist introduced a desensitizing component to the response. 3. At negative holding potentials, kainate (25‐125 microM) produced inward currents in all of the cells tested (n = 37). These currents never desensitized, even at high agonist concentrations, and reversed near ‐6 mV. Currents induced by 50 microM‐kainate were reversibly antagonized by kynurenate (100‐300 microM) but not by 100 microM‐2‐amino‐5‐phosphonovalerate (APV). 4. Quisqualate generated smaller, non‐desensitizing currents in only 50% of the cells tested (n = 38). Quisqualate responses reversed in polarity near ‐4 mV and were maximal at an agonist dose of 25 microM, with higher concentrations introducing a rapidly desensitizing component without a detectable increase in amplitude. Currents produced by quisqualate at a concentration of 50 microM were not antagonized by either 750 microM‐kynurenate or 100 microM‐APV. 5. N‐Methyl‐D‐aspartate (NMDA) produced inward currents at negative holding potentials in 68% of the cells tested (n = 31), but only when magnesium was excluded from the extracellular medium. NMDA currents were non‐desensitizing at agonist concentrations of up to 200 microM, with higher concentrations introducing a rapidly desensitizing component. NMDA (200 microM) responses were blocked by APV (100 microM) and kynurenate (300 microM) and reversed near ‐1 mV. 6. Responses generated by kainate (50‐125 microM) were antagonized by quisqualate (30‐250 microM). This antagonism occurred even in cells having no measurable response to quisqualate alone, suggesting the possibility that quisqualate may be acting both as an agonist, in the 50% of the cells that have the quisqualate‐specific receptor, and as an antagonist, at the kainate‐specific site on all cells.(ABSTRACT TRUNCATED AT 400 WORDS)

KCC2 expression in immature rat cortical neurons is sufficient to switch the polarity of GABA responses

During brain development, GABA and glycine switch from being depolarizing to being hyperpolarizing neurotransmitters. This conversion results from a gradual decrease in the chloride electrochemical equilibrium potential (ECl) of developing neurons, which correlates to an increase in the expression or activity of the potassium chloride cotransporter, KCC2. However, evidence as to whether KCC2 expression is sufficient, in and of itself, to induce this switch is lacking. In order to address this question, we used a gain‐of‐function approach by over‐expressing human KCC2 (hKCC2) in immature cortical neurons, before endogenous up‐regulation of KCC2. We found that premature expression of hKCC2 produced a substantial negative shift in the GABA reversal potential and decreased or abolished GABA‐elicited calcium responses in cultured neurons. We conclude that KCC2 expression is not only necessary but is also sufficient for ending the depolarizing period of GABA in developing cortical neurons.