GluR2 receptor targeting and trafficking presynaptic neurons at CA1 synapses. During the ischemic

H . Tanaka et al. Brain Research 886 2000 190 –207 195 which regulate the initiation and elongation of mature this mechanism comes from the finding of pools of transcripts, and 2 translational repressor and enhancer AMPARs within spines [118], the high concentration of factors. In addition, translational efficiency is influenced by NSF in the hippocampus [54,116], its high localization transcript-specific structural motifs, which serve as recog- within hippocampal PSDs [155], and its accumulation in nition sequences for the RNA binding proteins. These PSDs following transient cerebral ischemia [55]. include: 1 sequences residing in the 59 and 39 UTR; 2 alternate or additional 59-UTR AUG codons or their cognate short open reading frames; 3 consensus se-

4. Regulation of GluR2 expression in

quences for RNA binding proteins; and or 4 the exact neurodegenerative disorders nucleotide context of the initiator AUG [58]. The GluR2 transcript appears to be under negative 4.1. Ischemia translational control. The presence of the 59-UTR region in the GluR2 transcript suppresses translation of GluR2 in 4.1.1. Global ischemia-induced suppression of GluR2 Xenopus oocytes and in cell-free reticulocyte lysates [92]. mRNA and subunit expression The longest GluR2 transcripts identified in vivo, beginning Global ischemia during cardiac arrest affects 150,000 481 bases downstream from the AUG, exhibit greatly Americans each year and in many cases results in delayed reduced translational efficiency by about 40- to 50-fold onset of neurological deficits [112]. In addition, open heart relative to the GluR2 transcript containing only a seven- surgery can cause brain ischemia [14,119,140]. Transient, base leader when assayed in reticulocyte lysates [67,92]. severe global or forebrain ischemia, observed in patients The region with the greatest suppressor activity |20-fold during cardiorespiratory arrest, cardiac surgery or ex- overlaps all identified transcription initiation sites and perimentally in animals, induces selective and delayed includes two of the five upstream AUG codons and a neuronal death [14,119,140]. Pyramidal neurons in the 40-nucleotide imperfect GU repeat. Although the mecha- CA1 region of the hippocampus are particularly vulner- nism for translational suppression by the GluR2 59-UTR able. Histological evidence of degeneration, exhibiting the has yet to be identified, the upstream AUGs on their own hallmarks of apoptosis, is not observed until two to three exert only minor suppression of translation [91]. days after induction of ischemia in rats [25,64,115,121]. The delayed cell death after ischemia requires an early rise 21 21 in Ca in CA1 and initial translocation of Zn from

3. GluR2 receptor targeting and trafficking presynaptic neurons at CA1 synapses. During the ischemic

21 episode cells depolarize, exhibit a rise in intracellular Ca Trafficking and targeting of specific mRNAs and poly- and become inexcitable. Following reperfusion, cells ap- ribosomes to dendritic spines are thought to play an pear morphologically normal, exhibit normal intracellular 21 important role in the modification of synaptic strength Ca and regain the ability to generate action potentials for during synaptogenesis and in synaptic plasticity for 24 to 72 h after the ischemic insult. Ultimately, intracellu- 21 21 review, see [7]. New protein synthesis is essential to lar Ca and or Zn rises in vulnerable neurons and cell long-lasting modifications of synaptic strength [4] and the death ensues. maintenance phase of LTP [80]. Moreover, targeting of This delayed neurodegeneration, which may have clini- mRNAs to dendrites and dendritic spines can be regulated cal relevance, is thought to be triggered by a transient rise by synaptic transmission and plasticity [47,90,134,143]. in glutamate release during the ischemic episode [142], 21 Individual spines along a single dendrite may express followed by late and excessive Ca influx through AMPARs which differ in subunit composition and func- glutamate receptor channels [29,135]. Although NMDARs 21 21 tional properties including conductance, kinetics and Ca are highly Ca -permeable, there is now general consensus permeability [70,123]. Recent studies support an NSFN- that antagonists of AMPARs, like the quinoxalinedione methyl-maleimide sensitive fusion protein-dependent NBQX, appear to be much more effective than NMDA mechanism for insertion of GluR2-containing AMPARs antagonists in preventing CA1 cell death following severe and reveal a rapid recycling of functional AMPARs at CA1 global ischemia, even when given as late as 24 h after synapses under physiological conditions Fig. 3. Immuno- ischemia [18,94,133]. These observations indicate that precipitation experiments demonstrate that GluR2 exists AMPARs are critical mediators of ischemia-induced neuro- together with NSF in a complex in hippocampus nal death and that neuronal damage is not irreversible for [103,137]. Yeast two-hybrid studies reveal that GluR2 and 24 h after the insult. GluR4c bind directly to NSF via short segments within Brief forebrain or global ischemia triggers a suppression their cytoplasmic tails [137]. Loading of postsynaptic of GluR2 mRNA expression in pyramidal neurons of the hippocampal neurons with peptides corresponding to the hippocampal CA1 [44,49,110,111,113] Fig. 4. The sup- NSF binding site on GluR2 or with a monoclonal antibody pression of GluR2 mRNA is detectable in vulnerable against NSF causes rapid rundown ,15 min in the neurons as early as 12 h following the ischemic insult and amplitude of AMPAR EPSCs [95,137]. Further support for decreases to 30 of control levels within 24 h. GluR2 196 H Fig. 3. Model of NSF modulation of AMPA receptor function. A Regulation of receptor insertion. Interaction of NSF with C terminus of the GluR2 or GluR4 subunit in subsynaptic vesicles 1 may regulate the association or ‘docking’ 2 of the vesicles with the postsynaptic membrane fusion machinery and promote the insertion of AMPA receptors into the synaptic plasma membrane 3. B Regulation of receptor function or clustering. Interaction of NSF with the C terminus of the GluR2 or GluR4 subunit in the synaptic plasma membrane 1 regulates the conformation 2 of the GlurR subunits and modulates channel function and or the association of the receptors with synaptic proteins involved in synaptic targeting. Reprinted from [137]. mRNA downregulation directly precedes neural damage, lated into changes in subunit abundance. Studies involving immunofluorescent labeling of brain sections and Western and does not occur in the CA3 or dentate gyrus regions blot analysis of microdissected hippocampal subfields which are resistant to ischemia-induced damage. GluR3 indicate that global ischemia triggers a pronounced and mRNA levels also decrease, but to a lesser degree to cell-specific reduction in GluR2 subunit abundance in CA1 about 50 of control values; GluR1 and NR1 levels pyramidal neurons [101]. At 72 h after ischemia, virtually remain constant. The GluR1 GluR2 ratio is markedly all CA1 pyramidal neurons exhibited greatly reduced increased in CA1 at 24 h, but not in CA3 or dentate gyrus. GluR2 immunolabeling throughout their somata and de- Because the presence of the edited GluR2 subunit greatly 21 ndritic processes Fig. 5. GluR2 immunolabeling was reduces the Ca permeability of AMPA channels, these 21 unchanged in pyramidal cells of the CA3 and granule cells findings predict expression of functional of Ca -perme- of the dentate gyrus, regions resistant to ischemia-induced able AMPARs in CA1 neurons after global ischemia, but at damage. In contrast, immunolabeling of the AMPAR times preceding neuronal death. Moreover, timing of the GluR1 was unchanged in CA1, CA3 and DG. Western switch is consistent with a causal role for AMPAR-me- 21 analysis indicated that GluR2 subunit abundance was diated Ca influx in ischemia-induced damage. Since markedly reduced in CA1 at 60 and 72 h after the ischemic editing of GluR2 mRNA at the Q R site is unaltered after insult, times prior to the onset of neuronal death; GluR1 global ischemia [106,125,159], the change in AMPAR abundance was unchanged in all subfields at all times functional properties would appear to be a direct conse- examined. These findings, together with our previous quence of altered gene expression. 21 observation of enhanced AMPA-elicited Ca influx in Because AMPAR subunit expression is known to be post-ischemic CA1 neurons [44] demonstrate expression of under translational control, an important question was 21 whether suppression of GluR2 mRNA expression is trans- functional GluR2-lacking, Ca -permeable AMPARs in H . Tanaka et al. Brain Research 886 2000 190 –207 197 Fig. 5. Global ischemia induces downregulation of GluR2, but not GluR1, immunolabeling in CA1 pyramidal neurons. GluR1 and GluR2 immunoreactivity in brain sections from A, C a control sham-operated and B, D an experimental animal 72 h after ischemia. B GluR2 immunoreactivity decreased in CA1 pyramidal cells after ischemia, although D GluR1 immunoreactivity was unchanged. so: stratum oriens; sp: stratum pyramidale; sr: stratum radiatum. Similar results were observed in experimental and control animals. 21 acetyl spermine [147], channel blockers selective for Ca - permeable AMPARs [11,48]. These findings are consistent with a mechanism whereby post-ischemic CA1 neurons 21 generate slow EPSCs mediated by newly expressed Ca - permeable AMPARs. Many post-ischemic CA1 pyramidal Fig. 4. Expression of GluR2 mRNA is reduced specifically in CA1 after neurons are irreversibly depolarized by prolonged low ischemia. Photomicrographs of autoradiograms of GluR1, GluR2, a NR1 frequency stimulation of the Schaffer collateral commisur- mRNA in situ hybridization in coronal sections of gerbil brain at the level al input [149]. Moreover, post-ischemic neurons fail to of the dorsal hippocampus from control animals A, C, E and from exhibit long-term potentiation following tetanic stimulation experimental animals 72 h after ischemia B, D, F are shown. GluR2 [64]. Hippocampal neurons in culture subjected to subleth- mRNA was dramatically reduced in the pyramidal cell layer of the vulnerable CA1 but not in the pyramdial cell layer of CA3 or in the al oxygen–glucose deprivation exhibit increased AMPA- 21 granule cell layer of the dentate gyrus, areas that do not undergo or kainate-induced Ca accumulation, increased sensitivi- neurodegeneration. GluR1 and NR1 mRNAs were somewhat reduced in ty of AMPARs to Joro spider toxin, and increased vul- CA1 72 h after ischemia. Reprinted from [44]. nerability to AMPAR-mediated excitotoxity, suggesting 21 increased formation of GluR2-lacking, Ca -permeable AMPARs [159]. vulnerable neurons. Thus, the present study provides an To determine whether downregulation of GluR2 expres- 21 important link in the causal chain between global ischemia sion translates into enhanced Ca influx through AM- and delayed death of CA1 pyramidal neurons. PARs into CA1 neurons, we used a combination of electrophysiological intracellular recording and optical 21 4.1.2. Ischemia-induced changes in glutamate receptor imaging with the Ca indicator dye fura-2 [44]. In function hippocampal slices from gerbils 48 h after ischemia, 21 Global ischemia induces a number of functional changes AMPA elicits only a small rise in [Ca ] , which is not i 21 in the hippocampal CA1 indicative of increased expression significantly different from Ca rises in control neurons. 21 of Ca -permeable AMPARs. AMPAR-mediated excitat- At 72 h after the ischemic insult individual CA1 neurons ory postsynaptic currents EPSCs at the CA1 Schaffer that retain the ability to fire action potentials exhibit a 21 collateral synapse are prolonged after ischemia and exhibit greatly enhanced AMPA-elicited rise in [Ca ] . Basal i 21 increased sensitivity to Joro spider toxin and 1-naphthyl [Ca ] does not differ in control and post-ischemic i 198 H pyramidal cells Fig. 6. These findings indicate that AMPAR antagonist, 6-cyano-7-nitro-quinoxiline-2,3-dione 21 AMPAR functional responses are altered following global disodium, or of the Ca permeable AMPAR channel 21 ischemia and provide direct evidence for Ca -influx blocker, 1-naphthyl acetyl spermine, afforded protection directly through AMPARs in vulnerable CA1 neurons at against antisense-induced cell death. This finding indicates 21 times after brief global ischemia and preceding obvious that antisense-induced cell death is mediated by Ca cell loss. permeable AMPARs. GluR2 antisense and brief sublethal To examine whether acute downregulation of the GluR2 global ischemia acted synergistically to cause degeneration subunit, even in the absence of a neurological insult, can of pyramidal neurons, consistent with a common mecha- cause neuronal cell death, we performed GluR2 ‘knoc- nism of cell death. These findings demonstrate that dow- kdown’ experiments in rats and gerbils with antisense nregulation of GluR2 is sufficient to induce delayed death oligonucleotides targeted to GluR2 mRNA [100]. of specific neuronal populations. 21 Knockdown of receptor subunits has proven valuable in Recent evidence implicates Zn entry as a critical investigation of receptor function in vivo and in vitro. component of neuronal death after transient global is- 21 GluR2 antisense induced delayed death of pyramidal chemia in rats [66]. After a brief ischemic insult, Zn is neurons in CA1 and CA3. Antisense-induced neurodegene- translocated from presynaptic terminals and accumulates in ration was preceded by a reduction in GluR2 mRNA, as degenerating neurons in CA1 [66]. This accumulation indicated by in situ hybridization analysis, and in GluR2 precedes the onset of neurodegeneration and is prevented 21 protein, as indicated by Western blot analysis. GluR2 by intraventricular administration of the Zn chelator antisense suppressed GluR2 mRNA in the dentate gyrus, Ca-EDTA just prior to induction of ischemia. Ca-EDTA but did not cause cell death Fig. 7. Administration of the administration 1 h after reperfusion is not neuroprotective, 21 Fig. 6. AMPA-elicited inward current and rise in intracellular free Ca concentration in a CA1 pyramidal neuron after ischemia. A Inward current elicited by AMPA 30 mM with 10 mM cyclothiazide CTZ in a CA1 pyramidal neuron in a hippocampal slice from an animal 72 h after ischemia. 21 1 AMPA and CTZ were bath applied for 30 s red bar in saline containing NMDA, Ca and Na channel blockers. Then the AMPA was washed out with 21 1 saline containing the NMDA, Ca and Na channel blockers. After |5 min, CNQX 20 mM was added to the other blockers to cause more rapid recovery. In the control neuron illustrated in B, the AMPA-elicited inward current in the presence of 30 mM CTZ was of somewhat lower amplitude but similar in time course. B Optical imaging Optical imaging excitation wavelength, 350 nm of individual CA1 pyramidal neurons injected with fura-2 in hippocampal slices from a control animal upper row and an experimental animal 72 h after ischemia lower row, same cell as in A. a Image taken before bath application of agonist time indicated in current trace above; b image taken at peak inward current after application of AMPA 30 mM with 21 10 mM CTZ; c image taken after recovery to near baseline current. Color represents Ca concentration determined from the ratio of fluorescence 21 obtained at two excitation wavelengths 350 nm and 380 nm; calibration at right. AMPA elicited little change in intracellular free Ca in the control 21 neuron. In contrast, AMPA elicited a rise in Ca in the soma of the postischemic neuron and a smaller increase in its proximal dendrites. Red circles in the 21 350 nm excitation images indicate representative sites of Ca measurement in cell somata and dendrites. Reprinted from [44]. H . Tanaka et al. Brain Research 886 2000 190 –207 199 Fig. 7. GluR2 antisense and brief, sublethal ischemia induce cell death in the hippocampus of gerbils. Upper Toluidine blue labelling of coronal brain sections at the level of the dorsal hippocampus from gerbils at 7 days after 2 min ischemia associated with saline or GluR2 antisense AS2 injection.Top row, low magnification view of right hippocampus. Middle and bottom rows, higher magnification views of CA1 and CA3 respectively. A–C 2 min ischemia immediately after four injections of saline produced no neuronal death in any hippocampal region. D–F A single GluR2 antisense injection immediately followed by 2 min ischemia produced partial cell loss in the pyramidal cell layers of CA1 and CA3. G–H Four injections of antisense at 12 h intervals immediately followed by 2 min ischemia produced virtually complete loss of CA1 pyramidal cells and partial loss of CA3 pyramidal cells. Scale bars: 100 mm for A, D, G; 50 mm for B, C, E, F, H, I. Lower AMPA antagonists reduce antisense-induced neurodegeneration. Cell density cell number mm expressed as a percentage of the corresponding value for CA1 in untreated animals. Naspm and CNQX were injected into the right cerebral ventricle concomitantly with either the second or fourth injection of GluR2 antisense AS2 i.e., at 12 or 36 h after the first injection or 24 h after the last injection at 60 h after the first injection, see lower panel for timing. Animals were sacrificed at 7 days after the last antisense injection. Cell counts are normalized to the value for untreated rats defined as 100; n55 for each treatment and time point. Naspm and CNQX largely prevented antisense-induced delayed neurodegeneration in CA1 P,0.05, n55 for each antagonist compared to saline injection at the time of the second antisense injection, which had no protective effect. Protection was not complete at 12 or 26 h about 10 residual loss, P,0.05 for Naspm, P,0.01 for CNQX. Naspm or CNQX injected at 60 h after the first AS injection afforded no significant neuroprotection. The NMDA receptor antagonist AP5 administered at any of the three time points afforded no significant neuroprotection against GluR2 antisense-induced neurodegeneration. P,0.05 for antisense plus antagonist vs. antisense plus saline injected animals labeled saline. Reprinted from [100]. 200 H 21 indicating that Zn entry during or immediately after ischemia is toxic. Moreover, ischemia induces mRNA expression of the transporter gene ZNT-1 in CA1 neurons that are destined to die, presumably in response to the 21 21 Zn accumulation, since Zn induces expression of the gene in cultured neurons [150]. Although one can interpret the induction as a homeostatic response, it does not prevent cell death. Since GluR2-lacking AMPARs are permeable to 21 21 Zn [132], delayed entry of Zn may contribute to degeneration. If so, Ca-EDTA treatment delayed by several days after the ischemic insult would also be neuroprotec- tive. 21 Finally, evidence in support of a role for Ca -perme- able AMPARs in ischemia-induced damage comes from studies involving dissociated primary cultures of cortical 21 neurons [24]. This study reveals that activation of Ca - permeable AMPARs, in addition to activation of NMDA channels, leads to the generation of oxygen radicals, a well-established cause of cell damage. Together, these studies provide substantial evidence that suppression of GluR2 expression in CA1 after transient global ischemia leads to assembly and insertion of GluR2- lacking AMPARs at CA1 synapses. The change in subunit composition is consonant with observed changes in AMPAR functional properties and increased influx of toxic 21 21 Ca and Zn [9,109]. 4.2. Status epilepticus Kainic acid-induced status epilepticus leads to delayed, selective death of pyramidal neurons of the hippocampus. Status epilepticus in adult rats triggers a pronounced Fig. 8. Status epilepticus induces downregulation of GluR2, but not suppression of GluR2 mRNA in vulnerable CA1 and CA3 GluR1 mRNA in hippocampal CA1 and CA3. A GluR2 mRNA pyramidal neurons prior to the onset of neuronal death, as expression in rat hippocampus after status epilepticus. Upper Film assessed by in situ hybridization [38,113] Fig. 8. To autoradiograms of GluR2 mRNA expression in the hippocampus of status epilepticus and control rats. Downregulation of GluR2 mRNA expression examine whether the observed changes in GluR2 mRNA is first detected at 12 h after KA injection within CA1 and CA3, but are translated into changes in subunit expression, we remains stable in DG. Lower Time-course of GluR2 mRNA expression performed immunolabeling and Western blot analysis [45] after onset of status epilepticus. Mean densities in hippocampal CA1 and Fig. 9. Double immunolabeling revealed individual CA1 CA3 decreased and increased in DG. B GluR1 mRNA expression in rat and CA3 pyramidal neurons expressing abundant GluR1 hippocampus after status epilepticus. Upper Film autoradiograms of GluR1 mRNA expression at 6, 12, 16, 20, 24 h after KA-induced status and greatly reduced GluR2. GluR2 immunolabeling was epilepticus and in saline controls. GluR1 mRNA expression remains enhanced in granule cells of the dentate gyrus, a region stable at all time points assayed, although there is a modest decrease in resistant to seizure-induced damage. Quantitative Western CA1 and CA3 at 24 h. Lower Mean densities of GluR1 mRNA remain blot analysis revealed a reduction in GluR2 subunit unchanged. Values are expressed as a percentage of saline injected abundance by about 15 in CA1 and by about 30 in controls. Error bars represent the standard error of the mean. P,0.05, Student’s unpaired t test. Reprinted from [45]. CA3 at 20 h, with no change in dentate gyrus. Thus, GluR2 subunit abundance was regulated in a cell-specific manner. GluR1 subunit abundance was unchanged in all subfields at all times examined. These findings indicate induces status epilepticus, followed by delayed neurode- that AMPAR subunit composition is remodeled in response generation of pyramidal neurons in the hippocampal CA3. 21 to neuronal injury and support a role for Ca -permeable Status epilepticus triggered suppression of GluR2 mRNA, 21 AMPAR as critical mediators in the neuronal death associ- consistent with expression of GluR2-lacking, Ca -perme- ated with status epilepticus. able AMPARs in neurons destined to die. In pup rats, We also investigated molecular mechanisms underlying kainic acid induces status epilepticus, but not hippocampal the resistance of developing neurons to seizure-induced neurodegeneration. To investigate molecular mechanisms damage by in situ hybridization. In adult rats, kainic acid underlying the enhanced resistance of developing neurons H . Tanaka et al. Brain Research 886 2000 190 –207 201 Fig. 9. Status epilepticus downregulates GluR2 subunit expression in CA3. Upper Co-localization of GluR1 and GluR2 immunoreactivity in CA3 pyramidal neurons. Saline injected control sections stained for GluR1 A and GluR2 C. GluR2 immunoreactivity decreased in CA3 pyramidal cells 22h after kainate administration B, although GluR1 immunoreactivity was unchanged D. Co-localization of GluR1 and GluR2 occurred primarily in stratum pyramidale E,F. Lower Status epilepticus decreases GluR2 but not GluR1 protein levels in CA1 and CA3. Quantitative Western blot analysis of GluR1 and GluR2 protein expression in microdissected hippocampal subfields see Insert in A after status epilepticus. A GluR2 protein levels decreased in CA3a–b at 16 h after KA injections, and further declined at 20 and 24 h. Similar but smaller changes were seen in CA1, but were not significant until 24 h. No changes in the level of GluR2 protein were observed in DG-CA3c. The plotted ratios are the means of the ratios for individual animals. Error bars represent SEMs. P,0.001; P,0.05; Student’s upaired t test. Reprinted from [45]. to seizure-induced damage, AMPA, NMDA and GABA induced changes in AMPAR protein expression. Immuno- A receptor mRNA expression were examined by in situ cytochemistry with subunit-specific antibodies revealed a hybridization in postnatal days 5 P5 and 14 P14 pup marked reduction in GluR2 protein in CA3 pyramidal rats following status epilepticus. In young pups, status neurons after status epilepticus. In pup rats, GluR2 protein epilepticus did not alter expression of GluR1 or GluR2 in was unchanged in CA3 and dentate gyrus. These findings, vulnerable CA3, but increased expression of GluR1 and together with those of Friedman et al. [38,39] suggest that GluR2 in dentate gyrus. In adult rats, status epilepticus status epilepticus-induced alterations in GluR2 mRNA and 202 H subunit expression vary with development: GluR2 dow- in synaptic plasticity during brain development, and failure 21 nregulation occur only at those ages at which seizure- to decrease Ca -permeability of AMPARs at later stages induced damage is observed. These findings provide may lead to aberrant development. 21 evidence for Ca -permeable AMPARs in the causal chain Why do neurons which express little or no GluR2 e.g., of events between severe limbic seizures and delayed hippocampal interneurons and hippocampal pyramidal neuronal death. neurons of the GluR22 2 knockout mouse [59] survive? Viability of the GluR2 knock-out mice in contrast to that of the GluR2 editing deficient mice could occur for any of

5. Genetic approaches to the role of GluR2 in a number of reasons. These include 1 compensatory