Congeners of nitrogen monoxide (NO) are neuroprotective and neurodestructive. To address this apparent paradox, we considered the effects on neurons of compounds characterized by alternative redox states of NO: nitric oxide (NO.) and nitrosonium ion (NO+). Nitric oxide, generated from NO. donors or synthesized endogenously after NMDA (N-methyl-D-aspartate) receptor activation, can lead to neurotoxicity. Here, we report that NO.- mediated neurotoxicity is engendered, at least in part, by reaction with superoxide anion (O2.-), apparently leading to formation of peroxynitrite (ONOO-), and not by NO. alone. In contrast, the neuroprotective effects of NO result from downregulation of NMDA-receptor activity by reaction with thiol group(s) of the receptor's redox modulatory site. This reaction is not mediated by NO. itself, but occurs under conditions supporting S-nitrosylation of NMDA receptor thiol (reaction or transfer of NO+). Moreover, the redox versatility of NO allows for its interconversion from neuroprotective to neurotoxic species by a change in the ambient redox milieu. The details of this complex redox chemistry of NO may provide a mechanism for harnessing neuroprotective effects and avoiding neurotoxicity in the central nervous system.

Severely elevated levels of total homocysteine (approximately millimolar) in the blood typify the childhood disease homocystinuria, whereas modest levels (tens of micromolar) are commonly found in adults who are at increased risk for vascular disease and stroke. Activation of the coagulation system and adverse effects of homocysteine on the endothelium and vessel wall are believed to underlie disease pathogenesis. Here we show that homocysteine acts as an agonist at the glutamate binding site of the N-methyl-Daspartate receptor, but also as a partial antagonist of the glycine coagonist site. With physiological levels of glycine, neurotoxic concentrations of homocysteine are on the order of millimolar. However, under pathological conditions in which glycine levels in the nervous system are elevated, such as stroke and head trauma, homocysteine's neurotoxic (agonist) attributes at 10-100 M levels outweigh its neuroprotective (antagonist) activity. Under these conditions neuronal damage derives from excessive Ca 2؉ inf lux and reactive oxygen generation. Accordingly, homocysteine neurotoxicity through overstimulation of N-methyl-D-aspartate receptors may contribute to the pathogenesis of both homocystinuria and modest hyperhomocysteinemia.Elevated levels of homocysteine in the blood predispose to arteriosclerosis and stroke. In children with the relatively rare condition of homocystinuria, levels of total homocysteine approach millimolar concentrations. However, more modest levels (Ϸ15-50 M) are found very commonly in the general population (a condition known as hyperhomocysteinemia) (1, 2), and a concentration of up to 10 M has been measured in brain (3). Indeed, it has been recently estimated that as many as 47% of patients with arterial occlusions manifest these modest elevations in plasma homocysteine (1, 2). Included among the many causes are genetic alterations in enzymes such as cystathionine ␤-synthase, a defect found in 1-2% of the general population, and deficiencies in vitamins B 6 , B 12 , and folate, whose intake is suboptimal in perhaps 40% of the population (4). The strength of the association between homocysteine and cerebrovascular disease appears to be greater than that between homocysteine and coronary heart disease or peripheral vascular disease (1, 5). Current theories on homocysteine arteriosclerosis do not explain this predilection, nor do they give insight into the cognitive deficits seen in some patients. In the present study, we show that homocysteine causes direct neurotoxicity by activating the N-methyl-Daspartate (NMDA) subtype of glutamate receptor. Excessive stimulation of these receptors is known to mediate brain damage in focal ischemia (6, 7). Thus homocysteine may not only be associated with the vascular injury leading to stroke but may also participate in the ensuing neurotoxic response in the brain. MATERIALS AND METHODSHomocysteine and Derivatives. D,L-Homocysteine was used here because the L-form was not commercially available. However, based upon previous data, it is ...

Prions are proteins that can assume at least two distinct conformational states, one of which is dominant and self-perpetuating. Previously we found that a translation regulator CPEB from Aplysia, ApCPEB, that stabilizes activity-dependent changes in synaptic efficacy can display prion-like properties in yeast. Here we find that, when exogenously expressed in sensory neurons, ApCPEB can form an amyloidogenic self-sustaining multimer, consistent with it being a prion-like protein. In addition, we find that conversion of both the exogenous and the endogenous ApCPEB to the multimeric state is enhanced by the neurotransmitter serotonin and that an antibody that recognizes preferentially the multimeric ApCPEB blocks persistence of synaptic facilitation. These results are consistent with the idea that ApCPEB can act as a self-sustaining prion-like protein in the nervous system and thereby might allow the activity-dependent change in synaptic efficacy to persist for long periods of time.

Several ion channels are thought to be directly modulated by nitric oxide (NO), but the molecular basis of this regulation is unclear. Here we show that the NMDA receptor (NMDAR)-associated ion channel was modulated not only by exogenous NO but also by endogenous NO. Site-directed mutagenesis identified a critical cysteine residue (Cys 399) on the NR2A subunit whose S-nitrosylation (NO+ transfer) under physiological conditions underlies this modulation. In cell systems expressing NMDARs with mutant NR2A subunits in which this single cysteine was replaced by an alanine, the effect of endogenous NO was lost. Thus endogenous S-nitrosylation can regulate ion channel activity.

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Zinc (Zn2+) inhibition of N-methyl-D-aspartate receptor (NMDAR) activity involves both voltage-independent and voltage-dependent components. Recombinant NR1/NR2A and NR1/NR2B receptors exhibit similar voltage-dependent block, but voltage-independent Zn2+ inhibition occurs with much higher affinity for NR1/NR2A than NR1/NR2B receptors (nanomolar versus micromolar IC50, respectively). Here, we show that two neighboring histidine residues on NR2A represent the critical determinant (termed the "short spacer") for high-affinity, voltage-independent Zn2+ inhibition using the Xenopus oocyte expression system and site-directed mutagenesis. Mutation of either one of these two histidine residues (H42 and H44) in the extracellular N-terminal domain of NR2A shifted the IC50 for high-affinity Zn2+ inhibition approximately 200-fold without affecting the EC50 of the coagonists NMDA and glycine. We suggest that the mechanism of high-affinity Zn2+ inhibition on the NMDAR involves enhancement of proton inhibition.

Neuroligin-1 is a potent trigger for the de novo formation of synaptic connections, and it has recently been suggested that it is required for the maturation of functionally competent excitatory synapses. Despite evidence for the role of neuroligin-1 in specifying excitatory synapses, the underlying molecular mechanisms and physiological consequences that neuroligin-1 may have at mature synapses of normal adult animals remain unknown. By silencing endogenous neuroligin-1 acutely in the amygdala of live behaving animals, we have found that neuroligin-1 is required for the storage of associative fear memory. Subsequent cellular physiological studies showed that suppression of neuroligin-1 reduces NMDA receptor-mediated currents and prevents the expression of long-term potentiation without affecting basal synaptic connectivity at the thalamo-amygdala pathway. These results indicate that persistent expression of neuroligin-1 is required for the maintenance of NMDAR-mediated synaptic transmission, which enables normal development of synaptic plasticity and long-term memory in the amygdala of adult animals.synaptic plasticity ͉ neuroligin ͉ autism S everal studies have found that synaptically localized cell adhesion molecules not only trigger synapse formation but also play a major role in regulating both basal synaptic transmission and synaptic plasticity (1, 2). Among them, neurexins and neuroligins (NLs), which undergo a heterophilic interaction with each other, have emerged as important organizers of de novo synapse formation (3). Moreover, modifying the interaction of neuroligin-1 and PSD-95 alters the balance of neuronal excitation and inhibition required for normal brain function (4). The indispensable role of neuroligins for proper neuronal connectivity is further supported by the genetic linkage of neuroligin mutations with autism, a disease that is thought to be a disorder in social cognition that critically involves the amygdala (5, 6).Because neuroligins are present both during development and throughout adulthood (7,8), it is likely that neuroligins play roles other than that of an inducer of synaptogenesis in the adult brain. Indeed, a recent study of knockout (KO) mice deficient in neuroligin-1 demonstrated that neuroligin-1 regulates excitatory synaptic responses (9). Although neuroligin-1 has been suggested to be essential for maintaining normal N-methyl-D-aspartate (NMDA)-type glutamate receptor-mediated currents (9), the underlying mechanism and its physiological consequence remain to be identified. Furthermore, because the regulation of NMDA receptor (NMDAR) is critical for long-term synaptic modification (10), alterations of NMDAR-dependent currents regulated by neuroligin-1 are likely to have effects on synaptic plasticity and long-term memory in adult animals.To address the functional role of neuroligin-1 at existing mature synapses, we used virus-mediated RNA interference to deplete endogenous neuroligin-1 in the lateral nucleus of the amygdala (LA) of adult animals. We investigated the actions...

Recent evidence indicates that the NO-related species, nitroxyl anion (NO), is produced in physiological systems by several redox metal-containing proteins, including hemoglobin, nitric oxide synthase (NOS), superoxide dismutase, and S-nitrosothiols (SNOs), which have recently been identified in brain. However, the chemical biology of NO- remains largely unknown. Here, we show that NO- -unlike NO*, but reminiscent of NO+ transfer (or S-nitrosylation)- -reacts mainly with Cys-399 in the NR2A subunit of the N-methyl-D-aspartate (NMDA) receptor to curtail excessive Ca2+ influx and thus provide neuroprotection from excitotoxic insults. This effect of NO- closely resembles that of NOS, which also downregulates NMDA receptor activity under similar conditions in culture.