Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina

Excitotoxic studies using isolated chick embryo retina indicated that such an in vitro model provides a valid tool to characterize the effect of different agonists for subtypes of glutamate ionotropic receptors. In retinas maintained for 24 h in a Krebs medium, after a brief exposure (30 min) to glutamate agonists, we compared the effects produced by NMDA and non-NMDA-agonists, such as kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Delayed retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium after exposure to the previously named agonists. Although at high concentrations, both KA and AMPA produced more relevant release than NMDA, 7-8% of total retinal LDH was released after exposure to a 50 microM concentration of non-NMDA agonists. These values were similar to those obtained after 100 microM NMDA. In this regard, retinal tissue appeared to be less sensitive to excitotoxicity based on the activation of NMDA receptor subtype. All three agents produced histopathological lesions typical for excitotoxic damage. A delayed form of excitotoxicity observed in retina segments was predominated by necrotic features. However, the activation of apoptotic machinery early during the incubation period subsequent to brief exposure to NMDA (100 microM) was also present. The activation of caspase enzymes was studied by a fluorometric protease activity assay as well as by western blot analysis. Caspase-3-like activity reached the highest value within 3 h of incubation after exposure to excitotoxin, then the level of enzyme activity declined to lower values. As confirmed by a time-related appearance of TUNEL-positive nuclei, apoptotic features appeared to be specific for retina response to NMDA. In contrast, the exposure to a 50 microM concentration of KA or AMPA induced necrotic cell damage which was evident through the incubation, leading to a delayed mechanism of excitotoxicity. These observations provide evidence that in the retinal model, with regard to agonist concentrations and subtype of glutamate receptors, the cascade of events leading to excitotoxicity may result in either apoptotic or necrotic neuronal cell damage.     

Contributing mechanisms for cysteine excitotoxicity in cultured cerebellar granule cells

Several possible mechanisms for cysteine toxicity on rat cerebellar granule cells were studied and compared with the excitotoxic effect of glutamate. It was shown that the excitotoxic potency of both cysteine and glutamate increased in the presence of elevated concentrations, of bicarbonate or increased pH. Pharmacological studies showed that the cysteine toxicity was specifically coupled to the NMDA receptor, whereas the glutamate toxicity was mediated to a smaller extent also by non-NMDA receptors. Treatment of cerebellar granule cells with cysteine led to an increased extracellular level of glutamate. In addition, cysteine sensitized NMDA receptors by reducing disulfide bonds in the receptor to sulfhydryl groups. A mechanism for cysteine excitotoxicity may therefore be formation of cysteine-sensitized NMDA receptors that are stimulated either by cysteine and/or by endogenous glutamate. This mechanism may also be important for the effects observed during regulated physiological release of cysteine.     

Developmental and cell-selective variations in N-methyl-D-aspartate receptor degradation by calpain

NMDA receptors play critical roles in synaptic modulation and neurological disorders. In this study, we investigated the developmental changes in NR2 cleavage by NMDA receptor-activated calpain in cultured cortical and hippocampal neurons. Calpain activity increased with development, associated with increased expression of NMDA receptors but not of calpain I. The activation of calpain in immature and mature cortical cultures was inhibited by antagonists of NR1/2B and NR1/2A/2B receptors, whereas the inhibition of NR1/2B receptors did not alter calpain activation in mature hippocampal cultures. The degradation of NR2 subunits by calpain differed with developmental age. NR2A was not a substrate of calpain in mature hippocampal cultures, but was cleaved in immature cortical and hippocampal cultures. NR2B degradation by calpain in cortical cultures decreased with development, but the level of degradation of NR2B in hippocampal cultures did not change. The kinetics of NMDA receptor-gated whole cell currents were also modulated by calpain activation in a manner that varied with developmental stage in vitro. In early (but not later) developmental stages, calpain activation altered the NMDA-evoked current rise time and time constants for both desensitization and deactivation. Our data suggest that the susceptibility of the NMDA receptor to cleavage by calpain varies with neuronal maturity in a manner that may alter its electrophysiological properties.     

Reduction of functional N-methyl-D-aspartate receptors in neurons by RNase P-mediated cleavage of the NR1 mRNA

One approach to studying the functional role of individual NMDA receptor subunits involves the reduction in the abundance of the protein subunit in neurons. We have pursued a strategy to achieve this goal that involves the use of a small guide RNA which can lead to the destruction of the mRNA for a specific receptor subunit. We designed a small RNA molecule, termed 'external guide sequence' (EGS), which binds to the NR1 mRNA and directs the endonuclease RNase P to cleave the target message. This EGS has exquisite specificity and directed the RNase P-dependent cleavage at the targeted location within the NR1 mRNA. To improve the efficiency of this EGS, an in vitro evolution strategy was employed which led to a second generation EGS that was 10 times more potent than the parent molecule. We constructed an expression cassette by flanking the EGS with self-cleaving ribozymes and this permitted generation of the specified EGS RNA sequence from any promoter. Using a recombinant Herpes simplex virus (HSV), we expressed the EGS in neurons and showed the potency of the EGS to reduce NR1 protein within neurons. In an excitotoxicity assay, we showed that expression of the EGS in cortical neurons is neuroprotective. Our results demonstrate the utility of EGSs to reduce the expression of any gene (and potentially any splice variant) in neurons.     

GluR2-free alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors intensify demyelination in experimental autoimmune encephalomyelitis

We adopted a genetic approach to test the importance of edited GluR2-free, Ca(2+)-permeable, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in the pathophysiology of experimental autoimmune encephalomyelitis, an inflammatory demyelinative disorder resembling multiple sclerosis. Initial studies showed that oligodendroglial lineage cells from mice lacking functional copies of the gene encoding the GluR3 AMPA receptor subunit (Gria3) had a diminished capacity to assemble edited GluR2-free AMPA receptors, and were resistant to excitotoxicity in vitro. Neurological deficits and spinal cord demyelination elicited by immunization with myelin oligodendrocyte glycoprotein peptide were substantially milder in these Gria3 mutant mice than in their wild-type littermates. These results support the hypothesis that oligodendroglial excitotoxicity mediated by AMPA receptors that do not contain edited GluR2 subunits contributes to demyelination in experimental autoimmune encephalomyelitis, and suggest that inhibiting these Ca(2+)-permeable AMPA receptors would be therapeutic in multiple sclerosis.     

GABAC receptors in the vertebrate retina

In the central nervous system (CNS), the inhibitory transmitter GABA interacts with three subtypes of GABA receptors, type A, type B, and type C. Historically, GABA receptors have been classified as either the inotropic GABA_A receptors or the metabotropic GABA_B receptors. Over the past 10 yr, studies have shown that a third class, called the GABA_C receptor, also exists. GABA_C receptors are found primarily in the vertebrate retina and to some extent in other parts of the CNS. Although GABA_A and GABA_C receptors both gate chloride channels, they are pharmacologically, molecularly, and functionally distinct. The ρ subunit of the GABA_C receptor, which has about 35% amino acid homology to GABA_A receptor subunits, was cloned from the retina and, when expressed inXenopus oocytes, has properties similar to retinal GABA_C receptors. There are probably distinct roles for GABA_C receptors in the retina, because they are found on only a subset of neurons, whereas GABA_A receptors are ubiquitous. This article reviews recent electrophysiological and molecular studies that have characterized the unique properties of GABA_C receptors and describes the roles that these receptors may play in visual information processing in the retina.     

Development of glutamate-induced intracellular Ca2+ rise in the embryonic chick retina

Neurotransmitters affect neuronal development by regulating intracellular Ca²⁺ concentrations. We studied spatiotemporal pattern of the development of glutamate-induced intracellular Ca²⁺ rise in the embryonic chick retina, where developmental changes in mitotic activity, cell death, and synapse formation have been well established. Glutamate was bath-applied to the central part of the retina dissected at embryonic day 3 (E3) to E13, and changes in intracellular Ca²⁺ concentration were measured with Fura-2 fluorescence. The Ca²⁺ rise to glutamate first appeared at E6, reached a maximum at E9–10, and then declined before the appearance of synaptic structures (E12). Ca²⁺ rises to kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) appeared earlier and were larger in amplitude than those to N-methyl-D -aspartic acid. The KA/AMPA receptor of the E9 chick retina was permeable for Ca²⁺, suggesting the functional expression of Ca²⁺-permeable KA/AMPA receptors at the stage of retinal cell death. The Ca²⁺rise to glutamate and KA occurred intensely at the inner plexiform layer, the inner part of inner nuclear layer, and the ganglion cell layer, where the cell death occurs. The Ca²⁺ rise to high K²⁺, in contrast, occurred intensely at the nerve fiber layer and the ganglion cell layer, developing continuously from E3 until E11. Our study shows that the Ca²⁺ rise to glutamate develops with the decline of the mitotic activity of the retinal cells and is transiently enhanced during the period of cell death in the embryonic chick retina. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 113–125, 1998     

A role for polyamines in retinal ganglion cell excitotoxic death

Neuronal death due to excessive activation of N -methyl- d -aspartate (NMDA) receptors is a hallmark of neurodegenerative diseases. The polyamines: putrescine, spermine, and spermidine, bind to specific sites on the NMDA receptor and promote its activation, but their role in NMDA-induced neuronal death is ill defined. In this study, we characterized the role of polyamines in excitotoxic death of retinal ganglion cells (RGCs), a population of central neurons susceptible to NMDA-induced damage. Our data show that endogenous arginase I, the rate limiting enzyme for polyamine biosynthesis, is expressed in the intact, adult retina. Intraocular injection of NMDA visibly increased arginase I expression in Müller cells, the predominant glial cell-type in the mammalian retina. Inhibition of polyamine synthesis using di-fluoro-methyl-ornithine (DFMO) was markedly neuroprotective, while injection of exogenous polyamines in conjunction with NMDA exacerbated RGC death. Blockade of the polyamine binding sites on NMDA receptors using the non-competitive antagonist ifenprodil was neuroprotective, suggesting that polyamines contribute to excitotoxic death, at least partly, by binding to NMDA receptors. Importantly, we also demonstrate that NMDA leads to activation of both the Erk1/2 and PI3 K/Akt pathways, but only the PI3 K/Akt kinase was required for di-fluoro-methyl-ornithine-induced RGC survival. In summary, our study reveals that polyamines modulate neuronal death in the retina via different mechanisms that potentiate NMDA-triggered excitotoxicity.     

Subcellular segregation of distinct heteromeric NMDA glutamate receptors in the striatum

Functional N-methyl-d-aspartate (NMDA) glutamate receptors are composed of heteromeric complexes of NR1, the obligatory subunit for channel activity, and NR2 or NR3 family members, which confer variability in the properties of the receptors. Recent studies have provided evidence for the existence of both binary (containing NR1 and either NR2A or NR2B) and ternary (containing NR1, NR2A, and NR2B) receptor complexes in the adult mammalian brain. However, the mechanisms regulating subunit assembly and receptor localization are not well understood. In the CNS, NMDA subunits are present both at intracellular sites and the post-synaptic membrane of neurons. Using biochemical protein fractionation and co-immunoprecipitation approaches we have found that in rat striatum binary NMDA receptors are widely distributed, and can be identified in the light membrane, synaptosomal membrane, and synaptic vesicle-enriched subcellular compartments. In contrast, ternary receptors are found exclusively in the synaptosomal membranes. When striatal proteins are chemically cross-linked prior to subcellular fractionation, ternary NMDA receptors can be precipitated from the light membrane and synaptic vesicle-enriched fractions where this type of receptor complex is not detectable under normal conditions. These findings suggest differential targeting of distinct types of NMDA receptor assemblies between intracellular and post-synaptic sites based on subunit composition. This targeting may underlie important differences in the regulation of the transport pathways involved in both normal as well as pathological receptor functions.     

NMDA excitotoxicity and free radical generation in rat brain homogenates: Application of a chemiluminescence assay

NMDA, the specific agonist of glutamate gated ion channels permeable to calcium, is implicated as a causal factor in the pathogenesis of several neurobiological disorders such as stroke, seizures, ischemia, and chronic neurodegenerative disease. On the other hand, evidence on the roles of oxidative mechanisms involved in NMDA-induced neurotoxicity is accumulating. In this study, we have used chemiluminescence measurements as an easy, rapid and sensitive assay to investigate the effects of NMDA and oxidative stress on brain cell vulnerability. Rat brain homogenates were incubated with increasing concentrations of glutamate and NMDA. Production of reactive oxygen species was followed by single photon emission measurements using the specific enhancers luminol and lucigenin. Increases in emission were observed at excitotoxic concentrations of glutamate and NMDA. Other parameters of oxidative stress such as diene conjugates, TBARS and carbonyl groups were also investigated. Our results indicated that chemiluminescence measurements may be used to study involvement of oxidative stress in neurotoxicity.     

Calcium channels and GABA receptors in the early embryonic chick retina

The properties of calcium channels were studied at the period of neurogenesis in the early embryonic chick retina. The whole neural retina was isolated from embryonic day 3 (E3) chick and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). The retinal cells were depolarized by puff application of high-K⁺ solutions. Increases in intracellular Ca²⁺ concentrations were evoked by the depolarization through calcium channels. The type of calcium channel was identified as l-type by the sensitivity to dihydropyridines. The Ca²⁺ response was completely blocked by 10 μM nifedipine, whereas it was remarkably enhanced by 5 μM Bay K 8644. Then we sought a factor to activate the calcium channel and found that GABA could activate it by membrane depolarization at the E3 chick retina. Puff application of 100 μM GABA raised intracellular Ca²⁺ concentrations, and this Ca²⁺ response to GABA was also sensitive to the two dihydropyridines. Intracellular potential recordings verified clear depolarization by bath-applied 100 μM GABA. The Ca²⁺ response to GABA was mediated by GABA_A receptors, since the GABA response was blocked by 10 μgM bicuculline or 50 μM picrotoxin, and mimicked by muscimol but not by baclofen. Neither glutamate, kainate, nor glycine evoked any Ca²⁺ response. We conclude that l-type calcium channels and GABA_A receptors are already are already expressed before differentiation of retinal cells and synapse formation in the chick retina. A possibility is proposed that GABA might act as a trophic factor by activating l-type calcium channels via GABA_A receptors during the early period of retinal neurogenesis. © 1993 John Wiley & Sons, Inc.     

Cytoprotection by endogenous zinc in the vertebrate retina

Our recent studies have shown that endogenous zinc, co‐released with glutamate from the synaptic terminals of vertebrate retinal photoreceptors, provides a feedback mechanism that reduces calcium entry and the concomitant vesicular release of glutamate. We hypothesized that zinc feedback may serve to protect the retina from glutamate excitotoxicity, and conducted in vivo experiments on the retina of the skate (Raja erinacea) to determine the effects of removing endogenous zinc by chelation. These studies showed that removal of zinc by injecting the zinc chelator histidine results in inner retinal damage similar to that induced by the glutamate receptor agonist kainic acid. In contrast, when an equimolar quantity of zinc followed the injection of histidine, the retinal cells were unaffected. Our results are a good indication that zinc, co‐released with glutamate by photoreceptors, provides an auto‐feedback system that plays an important cytoprotective role in the retina.

     

Molecular pharmacology of human NMDA receptors

N-methyl-d-aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission. NMDA receptors are also important drug targets that are implicated in a number of pathophysiological conditions. To facilitate the transition from lead compounds in pre-clinical animal models to drug candidates for human use, it is important to establish whether NMDA receptor ligands have similar properties at rodent and human NMDA receptors. Here, we compare amino acid sequences for human and rat NMDA receptor subunits and discuss inter-species variation in the context of our current knowledge of the relationship between NMDA receptor structure and function. We summarize studies on the biophysical properties of human NMDA receptors and compare these properties to those of rat orthologs. Finally, we provide a comprehensive pharmacological characterization that allows side-by-side comparison of agonists, un-competitive antagonists, GluN2B-selective non-competitive antagonists, and GluN2C/D-selective modulators at recombinant human and rat NMDA receptors. The evaluation of biophysical properties and pharmacological probes acting at different sites on the receptor suggest that the binding sites and conformational changes leading to channel gating in response to agonist binding are highly conserved between human and rat NMDA receptors. In summary, the results of this study suggest that no major detectable differences exist in the pharmacological and functional properties of human and rat NMDA receptors.     

Group I metabotropic glutamate receptors are expressed in the chicken retina and by cultured retinal amacrine cells

Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double-labeling experiments, most metabotropic glutamate receptor 1-positive cell bodies in the inner nuclear layer also label with anti-GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca(2+) elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca(2+) elevations were enhanced by metabotropic glutamate receptor 1-selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca(2+) signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells.     

NMDA receptor signaling: death or survival?

Glutamate-induced neuronal damage is mainly caused by overactivation of N-methyl-D-aspartate (NMDA) receptors.Conversely,normal physiological brain function and neuronal survival require adequate activ...     

Localization of glutamate in the human retina during early prenatal development

In this study we have localized glutamate (GLU) in fetal (14–25 weeks gestation, Wg) human retinas by immunohistochemistry. At 14 Wg, GLU-immunoreactivity (IR) was localized only in the central part of retina, showing a prominently labelled nerve fiber layero A few ganglion cells and displaced amacrine cells were very weakly labelled. At 17 Wg, GLU was localized conspicuously in many ganglion cells, displaced amacrine cells, some amacrine cells and the prospective photoreceptor cell bodies in the neuroepithelial layero With progressive development at 20 and 25 Wg, the IR for GLU was found additionally in the Müller cell endfeet, some bipolar cells as well as in the horizontal cells that were aligned in a row along the outer border of the inner nuclear layer of the central retinao The photoreceptor cell bodies in the outer nuclear layer were also prominently immunopositive for GLU. The developmental distribution of GLU in the human retina tends to indicate that it plays an important role in the differentiation and maturation of retinal neurons.     

Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.     

NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury

Excitotoxic neuronal damage via over-activation of the NMDA receptor has been implicated in many neurodegenerative diseases. In vitro modeling of excitotoxic injury has shown that activation of G-protein coupled receptors (GPCRs) counteracts such injury through modulation of neuronal pro-survival pathways and/or NMDA receptor signaling. We have previously demonstrated that the GPCR APJ and its endogenous neuropeptide ligand apelin can protect neurons against excitotoxicity, but the mechanism(s) of this neuroprotection remain incompletely understood. We hypothesized that apelin can promote neuronal survival by activating pro-survival signaling as well as inhibiting NMDA receptor-mediated excitotoxic signaling cascades. Our results demonstrate that (i) apelin activates pro-survival signaling via inositol trisphosphate (IP(3) ), protein kinase C (PKC), mitogen-activated protein kinase kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase-1/2 (ERK1/2) to protect against excitotoxicity, and (ii) apelin inhibits excitotoxic signaling by attenuating NMDA receptor and calpain activity, and by modulating NMDA receptor subunit NR2B phosphorylation at serine 1480. These studies delineate a novel apelinergic signaling pathway that concurrently promotes survival and limits NMDA receptor-mediated injury to protect neurons against excitotoxicity. Defining apelin-mediated neuroprotection advances our understanding of neuroprotective pathways and will potentially improve our ability to develop therapeutics for excitotoxicity-associated neurodegenerative disorders.     

High levels of extracellular glutamate are present in retina during neonatal development

The three major classes of neurons which comprise the primary visual pathway in retina are glutamatergic. These cells are generated in two separate developmental stages, with one subclass of photoreceptors (cones) and ganglion cells generated before birth; and the other subclass of photoreceptors (rods) and bipolar cells generated during the first week after birth. Gas chromatography/mass spectroscopy analysis coupled with a new method for collecting small samples of extracellular fluids from retina were used to determine the levels of endogenous glutamate present during differentiation and synaptogenesis of these different cell types. As expected the total retinal content of glutamate increased during the postnatal period in synchrony with the generation and maturation of glutamatergic cells. However, a significant proportion of the endogenous pool was found extracellularly at birth. Intracellular glutamate is localized within cell bodies and growing processes of cones and ganglion cells at this time but few glutamatergic synapses are present. The extracellular concentration of glutamate actually declined during the most active period of synaptogenesis, reaching very low levels in the adult. The high concentrations of extracellular glutamate in neonatal retina could play an important role in a variety of developmental events such as dendritic pruning, programmed cell death and neurite sprouting. Special issue dedicated to Dr. Kinya Kuriyama.