Remodeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca(2+) permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca(2+) influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca(2+)-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.     

Status epilepticus decreases glutamate receptor 2 mRNA and protein expression in hippocampal pyramidal cells before neuronal death

Kainic acid (KA)-induced status epilepticus in adult rats leads to delayed, selective death of pyramidal neurons in the hippocampal CA1 and CA3. Death is preceded by down-regulation of glutamate receptor 2 (GluR2) mRNA and protein [the subunit that limits Ca(2+) permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors] in CA1 and CA3, as indicated by in situ hybridization, immunolabeling, and quantitative Western blotting. GluR1 mRNA and protein are unchanged or slightly increased before cell death. These changes could lead to formation of GluR2-lacking, Ca(2+)-permeable AMPA receptors and increased toxicity of endogenous glutamate. GluR2 immunolabeling is unchanged in granule cells of the dentate gyrus, which are resistant to seizure-induced death. Thus, formation of Ca(2+)-permeable AMPA receptors may be a critical mediator of delayed neurodegeneration after status epilepticus.     

Blockade of calcium-permeable AMPA receptors protects hippocampal neurons against global ischemia-induced death

Transient global or forebrain ischemia induced experimentally in animals can cause selective, delayed neuronal death of hippocampal CA1 pyramidal neurons. A striking feature is a delayed rise in intracellular free Zn(2+) in CA1 neurons just before the onset of histologically detectable cell death. Here we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) at Schaffer collateral to CA1 synapses in postischemic hippocampus exhibit properties of Ca(2+)/Zn(2+)-permeable, Glu receptor 2 (GluR2)-lacking AMPARs before the rise in Zn(2+) and cell death. At 42 h after ischemia, AMPA excitatory postsynaptic currents exhibited pronounced inward rectification and marked sensitivity to 1-naphthyl acetyl spermine (Naspm), a selective channel blocker of GluR2-lacking AMPARs. In control hippocampus, AMPA excitatory postsynaptic currents were electrically linear and relatively insensitive to Naspm. Naspm injected intrahippocampally at 9-40 h after insult greatly reduced the late rise in intracellular free Zn(2+) in postischemic CA1 neurons and afforded partial protection against ischemia-induced cell death. These results implicate GluR2-lacking AMPA receptors in the ischemia-induced rise in free Zn(2+) and death of CA1 neurons, although a direct action at the time of the rise in Zn(2+) is unproven. This receptor subtype appears to be an important therapeutic target for intervention in ischemia-induced neuronal death in humans.     

Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.

Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeable to Ca2+; heteromeric channels containing GluR2 subunits are Ca(2+)-impermeable. Thus, altered expression of GluR2 in development or following a neurological insult or injury to the brain can act as a switch to modify Ca2+ permeability. To investigate the molecular mechanism underlying delayed postischemic cell death, GluR1, GluR2, and GluR3 gene expression was examined by in situ hybridization in postischemic rats. Following severe, transient forebrain ischemia GluR2 gene expression was preferentially reduced in CA1 hippocampal neurons at a time point that preceded their degeneration. The switch in expression of kainate/AMPA receptor subunits coincided with the previously reported increase in Ca2+ influx into CA1 cells. Timing of the switch indicates that it may play a causal role in postischemic cell death.     

Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity

Influx of Ca(2+) ions through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors contributes to neuronal damage in stroke, epilepsy, and neurodegenerative disorders such as ALS. The Ca(2+) permeability of AMPA receptors is largely determined by the glutamate receptor 2 (GluR2) subunit, receptors lacking GluR2 being permeable to Ca(2+) ions. We identified a difference in GluR2 expression in motor neurons from two rat strains, resulting in a difference in vulnerability to AMPA receptor-mediated excitotoxicity both in vitro and in vivo. Astrocytes from the ventral spinal cord were found to mediate this difference in GluR2 expression in motor neurons. The presence of ALS-causing mutant superoxide dismutase 1 in astrocytes abolished their GluR2-regulating capacity and thus affected motor neuron vulnerability to AMPA receptor-mediated excitotoxicity. These results reveal a mechanism through which astrocytes influence neuronal functioning in health and disease.     

Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the glutamate receptor GluR2 subunit are Ca(2+)-permeable and exhibit inwardly rectifying current responses to kainate and AMPA. A proportion of cultured rat hippocampal neurons show similar Ca(2+)-permeable inwardly rectifying AMPA receptor currents. Inward rectification in these neurons was lost with intracellular dialysis and was not present in excised outside-out patches but was maintained in perforated-patch whole-cell recordings, suggesting that a diffusible cytoplasmic factor may be responsible for rectification. Inclusion of the naturally occurring polyamines spermine and spermidine in the recording pipette prevented loss of rectification in both whole-cell and excised-patch recordings; Mg2+ and putrescine were without effect. Inward rectification of Ca(2+)-permeable AMPA receptors may reflect voltage-dependent channel block by intracellular polyamines.     

Astrocyte-mediated activation of neuronal kainate receptors

Exogenous kainate receptor agonists have been shown to modulate inhibitory synaptic transmission in the hippocampus, but the pathways involved in physiological activation of the receptors remain largely unknown. Accumulating evidence indicates that astrocytes can release glutamate in a Ca(2+)-dependent manner and signal to neighboring neurons. We tested the hypothesis that astrocyte-derived glutamate activates kainate receptors on hippocampal interneurons. We report here that elevation of intracellular Ca(2+) in astrocytes, induced by uncaging Ca(2+), o-nitrophenyl-EGTA, increased action potential-driven spontaneous inhibitory postsynaptic currents in nearby interneurons in rat hippocampal slices. This effect was blocked by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptor antagonists, but not by selective AMPA receptor or N-methyl-d-aspartate receptor antagonists. This pharmacological profile indicates that kainate receptors were activated during Ca(2+) elevation in astrocytes. Kainate receptors containing the GluR5 subunit seemed to mediate the observed effect because a selective GluR5-containing kainate receptor antagonist blocked the changes in sIPSCs induced by Ca(2+) uncaging, and bath application of a selective GluR5-containing receptor agonist robustly potentiated sIPSCs. When tetrodotoxin was included to block action potentials, Ca(2+) uncaging induced a small decrease in the frequency of miniature inhibitory postsynaptic currents, which was not affected by AMPA/kainate receptor antagonists. Our data suggest that an astrocyte-derived, nonsynaptic source of glutamate represents a signaling pathway that can activate neuronal kainate receptors. By modulating the activity of interneurons, astrocytes may play a critical role in circuit function of hippocampus.     

Estrogen can act via estrogen receptor alpha and beta to protect hippocampal neurons against global ischemia-induced cell death

Estradiol at physiological concentrations intervenes in apoptotic death cascades and ameliorates neuronal death in experimental models of focal and global ischemia. The cellular targets that mediate estradiol protection of hippocampal neurons in global ischemia are, however, unclear. The present study examined the hypothesis that estradiol protects hippocampal neurons in ovariectomized rats via estrogen receptor (ER)alpha and/or beta. Estradiol (14 d pretreatment) afforded robust protection of CA1 neurons against global ischemia-induced death. The broad-spectrum ER antagonist ICI 182,780 (intracerebroventricularly, 0 and 12 h after ischemia) abolished estrogen protection, consistent with a role for ERs. To evaluate the potential roles of ERalpha vs. ERbeta in estrogen protection, we administered subtype-selective agonists for 14 d before and 7 d after ischemia. The ERalpha-selective agonist propyl pyrazole triol (PPT, 10 mg/kg) and ERbeta-selective agonist WAY 200070-3 (1 mg/kg) produced nearly complete protection of CA1 neurons in approximately 50% of the animals. PPT, but not WAY 200070-3, at doses used for protection, elicited lordosis, induced negative feedback inhibition of LH release, and reduced weight gain. These findings establish the efficacy of the PPT dose in neuroendocrine assays and specificity of WAY 200070-3 for ERbeta. We also examined the ability of estradiol and neuronal injury to regulate ERalpha and ERbeta expression. Both estradiol and global ischemia markedly increased ERalpha, but not ERbeta, protein in CA1. These data indicate that estradiol can act via ERalpha and ERbeta to protect CA1 neurons from global ischemia-induced death and that both estradiol and global ischemia modulate ERalpha expression in hippocampal CA1.     

The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations

We have considered the extracellular serine protease thrombin and its receptor as endogenous mediators of neuronal protection against brain ischemia. Exposure of gerbils to prior mild ischemic insults, here two relatively short-lasting occlusions (2 min) of both common carotid arteries applied at 1-day intervals 2 days before a severe occlusion (6 min), caused a robust ischemic tolerance of hippocampal CA1 neurons. This resistance was impaired if the specific thrombin inhibitor hirudin was injected intracerebroventricularly before each short-lasting insult. Thus, efficient native neuroprotective mechanisms exist and endogenous thrombin seems to be involved therein. In vitro experiments using organotypic slice cultures of rat hippocampus revealed that thrombin can have protective but also deleterious effects on hippocampal CA1 neurons. Low concentrations of thrombin (50 pM, 0.01 unit/ml) or of a synthetic thrombin receptor agonist (10 microM) induced significant neuroprotection against experimental ischemia. In contrast, 50 nM (10 units/ml) thrombin decreased further the reduced neuronal survival that follows the deprivation of oxygen and glucose, and 500 nM even caused neuronal cell death by itself. Degenerative thrombin actions also might be relevant in vivo, because hirudin increased the number of surviving neurons when applied before a 6-min occlusion. Among the thrombin concentrations tested, 50 pM induced intracellular Ca(2+) spikes in fura-2-loaded CA1 neurons whereas higher concentrations caused a sustained Ca(2+) elevation. Thus, distinct Ca(2+) signals may define whether or not thrombin initiates protection. Taken together, in vivo and in vitro data suggest that thrombin can determine neuronal cell death or survival after brain ischemia.     

TRPM7 channels in hippocampal neurons detect levels of extracellular divalent cations

Exposure to low Ca(2+) and/or Mg(2+) is tolerated by cardiac myocytes, astrocytes, and neurons, but restoration to normal divalent cation levels paradoxically causes Ca(2+) overload and cell death. This phenomenon has been called the "Ca(2+) paradox" of ischemia-reperfusion. The mechanism by which a decrease in extracellular Ca(2+) and Mg(2+) is "detected" and triggers subsequent cell death is unknown. Transient periods of brain ischemia are characterized by substantial decreases in extracellular Ca(2+) and Mg(2+) that mimic the initial condition of the Ca(2+) paradox. In CA1 hippocampal neurons, lowering extracellular divalents stimulates a nonselective cation current. We show that this current resembles TRPM7 currents in several ways. Both (i) respond to transient decreases in extracellular divalents with inward currents and cell excitation, (ii) demonstrate outward rectification that depends on the presence of extracellular divalents, (iii) are inhibited by physiological concentrations of intracellular Mg(2+), (iv) are enhanced by intracellular phosphatidylinositol 4,5-bisphosphate (PIP(2)), and (v) can be inhibited by Galphaq-linked G protein-coupled receptors linked to phospholipase C beta1-induced hydrolysis of PIP(2). Furthermore, suppression of TRPM7 expression in hippocampal neurons strongly depressed the inward currents evoked by lowering extracellular divalents. Finally, we show that activation of TRPM7 channels by lowering divalents significantly contributes to cell death. Together, the results demonstrate that TRPM7 contributes to the mechanism by which hippocampal neurons "detect" reductions in extracellular divalents and provide a means by which TRPM7 contributes to neuronal death during transient brain ischemia.     

Interaction of the AMPA receptor subunit GluR2/3 with PDZ domains regulates hippocampal long-term depression

The interaction of PDZ domain-containing proteins with the C termini of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors has been suggested to be important in the regulation of receptor targeting to excitatory synapses. Recent studies have shown that the rapid internalization of AMPA receptors at synapses may mediate, at least in part, the expression of long-term depression (LTD). We have previously shown that phosphorylation of Ser-880 on the AMPA receptor GluR2 subunit differentially regulated the interaction of GluR2 with the PDZ domain-containing proteins GRIP1 and PICK1. Here, we show that induction of LTD in hippocampal slices increases phosphorylation of Ser-880 within the GluR2 C-terminal PDZ ligand, suggesting that the modulation of GluR2 interaction with GRIP1 and PICK1 may regulate AMPA receptor internalization during LTD. Moreover, postsynaptic intracellular perfusion of GluR2 C-terminal peptides that disrupt GluR2 interaction with PICK1 inhibit the expression of hippocampal LTD. These results suggest that the interaction of GluR2 with PICK1 may play a regulatory role in the expression of LTD in the hippocampus.     

Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD

Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of ~44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands (K_i~10.1 μM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.     

Chronic ethanol upregulates NMDA and AMPA, but not kainate receptor subunit proteins in rat primary cortical cultures

The present study examined the effects of chronic ethanol exposure on the expression of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA) and kainate receptor subunit proteins in rat cortical neuronal cultures grown in media containing 2 mM (high) or 0.1 mM (low) glutamine. Immunoblot analysis of NMDA (NR1, NR2A, NR2B, and NR2D), AMPA (GluR1 and GluR2/3), and kainate (GluR6/7) subunit polypeptides in 3-, 5-, 8-, 10-, and 12 day-old-cultures showed that NMDA receptor subunits NR1, NR2A, and NR2B and AMPA receptor subunits GluR2/3 progressively increased as a function of time, whereas levels of NMDA subunit NR2D were high at day 3 and progressively declined to barely detectable levels by day 12. Levels of AMPA subunit GluR1 and the kainate subunit GluR6/7 remained stable throughout the time course. Replacing the culture media with low glutamine media at culture day 5 did not alter the levels of subunit proteins measured at culture days 9 and 13. However, exposure of low glutamine cultures to 100 mM ethanol for 4 days (starting at culture day 9) significantly increased the levels of NMDA receptor subunits (NR1, NR2A, and NR2B) and AMPA receptor subunits (GluR1 and GluR2/3), but had no effect upon kainate receptor subunits (GluR6/7) or the synapse-associated proteins synapsin I and PSD-95. In contrast, chronic ethanol did not alter the levels of any of these subunit proteins in cells grown in high glutamine. These data demonstrate that under certain experimental conditions, prolonged exposure to ethanol upregulates NMDA and AMPA receptor subunit proteins, but has no effect upon kainate receptor subunit proteins. Because we have previously shown that acute ethanol can inhibit NMDA and AMPA, but not kainate, receptor function in these cultures, the increase in subunit expression likely reflects an adaptive response to the inhibitory effects of ethanol and suggests that both NMDA and AMPA receptors may play an important role in adaptation of the CNS to chronic ethanol.     

Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a

Acid-sensing ion channel (ASIC) 1a subunit is expressed in synapses of central neurons where it contributes to synaptic plasticity. However, whether these channels can conduct Ca(2+) and thereby raise the cytosolic Ca(2+) concentration, [Ca(2+)](c), and possibly alter neuronal physiology has been uncertain. We found that extracellular acidosis opened ASIC1a channels, which provided a pathway for Ca(2+) entry and elevated [Ca(2+)](c) in wild-type, but not ASIC1(-/-), hippocampal neurons. Acid application also raised [Ca(2+)](c) and evoked Ca(2+) currents in heterologous cells expressing ASIC1a. Although ASIC2a is also expressed in central neurons, neither ASIC2a homomultimeric channels nor ASIC1a/2a heteromultimers showed H(+)-activated [Ca(2+)](c) elevation or Ca(2+) currents. Because extracellular acidosis accompanying cerebral ischemia contributes to neuronal injury, we tested the effect of acidosis on cell death measured as lactate dehydrogenase release. Eliminating ASIC1a from neurons or treating ASIC1a-expressing cells with the ASIC blocker amiloride attenuated acidosis-induced cell injury. These results indicate that ASIC1a provides a non-voltage-gated pathway for Ca(2+) to enter neurons. Thus, it may provide a target for modulation of [Ca(2+)](c).     

Glutamate regulates intracellular calcium and gene expression in oligodendrocyte progenitors through the activation of DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

Oligodendrocytes and their progenitors (O-2A) express functional kainate- and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring glutamate receptors. The physiological consequences of activation of these receptors were studied in purified rat cortical O-2A progenitors and in the primary oligodendrocyte cell line CG-4. Changes in the mRNA levels of a set of immediate early genes were studied and were correlated to intracellular Ca2+ concentration, as measured by fura-2 Ca2+ imaging. Both in CG-4 and in cortical O-2A progenitors, basal mRNA levels of NGFI-A were much higher than c-fos, c-jun, or jun-b. Glutamate, kainate, and AMPA greatly increased NGFI-A mRNA and protein by activation of membrane receptors in a Ca(2+)-dependent fashion. Agonists at non-N-methyl-D-aspartate receptors promoted transmembrane Ca2+ influx through voltage-dependent channels as well as kainate and/or AMPA channels. The influx of Ca2+ ions occurring through glutamate-gated channels was sufficient by itself to increase the expression of NGFI-A mRNA. AMPA receptors were found to be directly involved in intracellular Ca2+ and NGFI-A mRNA regulation, because the effects of kainate were greatly enhanced by cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA but not kainate receptors. Our results indicate that glutamate acting at AMPA receptors regulates immediate early gene expression in cells of the oligodendrocyte lineage by increasing intracellular calcium. Consequently, modulation of these receptor channels may have immediate effects at the genomic level and regulate oligodendrocyte development at critical stages.