Neuroprotection after status epilepticus by targeting protein interactions with postsynaptic density protein 95.

N-methyl-D-aspartate receptors (NMDARs) mediate essential neuronal excitation, but overactivation of NMDARs results in excitotoxic cell death in a variety of pathologic conditions, including status epilepticus (SE). Although NMDAR antagonists attenuate SE-induced brain injury, undesirable side effects have limited their clinical efficacy. Tat-NR2B9c was designed to disrupt protein interactions involving postsynaptic density protein 95 in the NMDAR signaling complex while not interfering with function of the NMDAR ion channel. We examined the ability of Tat-NR2B9c to provide neuroprotection in the hippocampus of rats after 60 minutes of SE induced by the repeated injection of low doses of pilocarpine (10 mg/kg). Tat-NR2B9c was administered 3hours after the termination of SE, and neuronal densities were assessed 14 days later by stereologic analysis of NeuN-positive cells. After SE, pyramidal cell densities were reduced by 70% in CA1, 34% in CA3, 58% in CA4, and 88% in the piriform cortex. In Tat-NR2B9c-treated rats, neuronal densities in CA1, a subregion of CA3, and CA4 were decreased by only 38%, 4%, and 26%, respectively. Tat-NR2B9c did not reduce cell loss in the posterior piriform cortex. The results indicate that targeted disruption of the NMDAR signaling complex represents a potential therapeutic approach for limiting neuronal cell loss after SE.

Distinct properties of murine alpha 5 gamma-aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopy.

Gamma-aminobutyric acid type A receptors (GABA(A)Rs) that contain the alpha 5 subunit are expressed predominantly in the hippocampus, where they regulate learning and memory processes. Unlike conventional postsynaptic receptors, GABA(A)Rs containing the alpha 5 subunit (alpha 5 GABA(A)Rs) are localized primarily to extrasynaptic regions of neurons, where they generate a tonic inhibitory conductance. The unique characteristics of alpha 5 GABA(A)Rs have been examined with pharmacological, immunostaining, and electrophysiological techniques; however, little is known about their biochemical properties. The aim of this study was to modify existing purification and enrichment techniques to isolate alpha 5 GABA(A)Rs preferentially from the mouse hippocampus and to identify the alpha 5 subunit by using tandem mass spectroscopy (MS/MS). The results showed that the detergent solubility of the alpha 5 subunits was distinct from that of alpha1 and alpha2 subunits, and the relative distribution of the alpha 5 subunits in Triton X-100-soluble fractions was correlated with that of the extracellular protein radixin but not with that of the postsynaptic protein gephyrin. Mass spectrometry identified the alpha 5 subunit and showed that this subunit associates with multiple alpha, beta, and gamma subunits, but most frequently the beta 3 subunit. Thus, the alpha 5 subunits coassemble with similar subunits as their synaptic counterparts yet have a distinct detergent solubility profile. Mass spectroscopy now offers a method for detecting and characterizing factors that confer the unique detergent solubility and possibly cellular location of alpha 5 GABA(A)Rs in hippocampal neurons.

Ischemia and status epilepitcus result in enhanced phosphorylation of calcium and calmodulin-stimulated protein kinase II on threonine 253.

Ca2+-stimulated protein kinase II (CaMKII) is critically involved in the regulation of synaptic function and is implicated in the neuropathology associated with ischemia and status epilepticus (SE). The activity and localization of CaMKII is regulated by multi-site phosphorylation. In the present study we investigated the effects of global ischemia followed by reperfusion and of SE on the phosphorylation of CaMKII on T253 in rat forebrains and compared this to the phosphorylation of T286. Both ischemia and SE resulted in marked increases in the phosphorylation of T253, and this was particularly marked in the postsynaptic density (PSD). Phosphorylation of T286 decreased rapidly towards basal levels following ischemia whereas phosphorylation of T253 remained elevated for between 1 and 6 h before decreasing to control values. Following SE, phosphorylation of T253 remained elevated for between 1 and 3 h before decreasing to control levels. In contrast, phosphorylation of T286 remained elevated for at least 24 h following the termination of SE. Total CaMKII associated with PSDs transiently increased 10 min following ischemia, but only several hours following SE. The results demonstrate that phoshorylation of CaMKII on T253 is enhanced following both ischemia/reperfusion and SE and indicate that the phosphorylation of T253 and T286 are differentially regulated.

Increase in tyrosine phosphorylation of the NMDA receptor following the induction of status epilepticus.

The administration of lithium followed by pilocarpine induces status epilepticus (SE) that produces neurodegeneration and the subsequent development of spontaneous recurrent seizures. We have reported that tyrosine phosphorylation of the NMDA receptor is elevated over controls for several hours following 60 min of SE. In the current study, we assessed the temporal relationship between tyrosine phosphorylation of the NMDA receptor and the onset of SE. SE was induced using the Li/pilocarine model and phosphorylation of the NMDA receptor subunits NR2A and NR2B determined. Tyrosine phosphorylation of the NMDAR remained unchanged prior to the onset of SE and increased gradually thereafter. The onset of SE was accompanied by activation of Src-family tyrosine kinases and Pyk2 in the post-synaptic density, consistent with a role for these enzymes in SE-induced tyrosine phosphorylation. The results indicate that tyrosine phosphorylation of the NMDAR closely parallels the activation of Src-family kinases and follows, rather than precedes, the onset of SE.

Lateral hypothalamic signaling mechanisms underlying feeding stimulation: differential contributions of Src family tyrosine kinases to feeding triggered either by NMDA injection or by food deprivation.

In rats, feeding can be triggered experimentally using many approaches. Included among these are (1) food deprivation and (2) acute microinjection of the neurotransmitter l-glutamate (Glu) or its receptor agonist NMDA into the lateral hypothalamic area (LHA). Under both paradigms, the NMDA receptor (NMDA-R) within the LHA appears critically involved in transferring signals encoded by Glu to stimulate feeding. However, the intracellular mechanisms underlying this signal transfer are unknown. Because protein-tyrosine kinases (PTKs) participate in NMDA-R signaling mechanisms, we determined PTK involvement in LHA mechanisms underlying both types of feeding stimulation through food intake and biochemical measurements. LHA injections of PTK inhibitors significantly suppressed feeding elicited by LHA NMDA injection (up to 69%) but only mildly suppressed deprivation feeding (24%), suggesting that PTKs may be less critical for signals underlying this feeding behavior. Conversely, food deprivation but not NMDA injection produced marked increases in apparent activity for Src PTKs and in the expression of Pyk2, an Src-activating PTK. When considered together, the behavioral and biochemical results demonstrate that, although it is easier to suppress NMDA-elicited feeding by PTK inhibitors, food deprivation readily drives PTK activity in vivo. The latter result may reflect greater PTK recruitment by neurotransmitter receptors, distinct from the NMDA-R, that are activated during deprivation-elicited but not NMDA-elicited feeding. These results also demonstrate how the use of only one feeding stimulation paradigm may fail to reveal the true contributions of signaling molecules to pathways underlying feeding behavior in vivo.

Trapping-induced changes in expression of the N-methyl-D-aspartate receptor in the hippocampus of snowshoe hares.

Live-trapping of animals in natural populations is one of the main ways to determine population processes. We examined the effects of live-trapping on the expression of N-methyl-aspartate (NMDA) receptor subunits in the hippocampus of snowshoe hares. Snowshoe hares were obtained either with or without the stress of live-trapping. The CA1, CA3 and dentate gyrus were dissected and analyzed for the presence of NMDA receptor subunits. Trapping resulted in a significant reduction of NMDA receptor 1 (NR1) in each of the regions examined but did not affect the levels of either NMDA receptor 2A or B (NR2A or NR2B). Co-immunoprecipitation analysis showed that the association between NR1 and NR2A was decreased in the trapped animals. These results suggest that stress associated with the trapping experience may adversely affect the structure and/or function of the NMDA receptor.

Developmental changes in the association of NMDA receptors with lipid rafts.

Lipid rafts (LR) are lipid microdomains present in the cell surface membrane that are organizational platforms involved in protein trafficking and formation of cell signaling complexes. In the adult brain, NMDA receptors (NMDAR) and receptor-associated proteins such as membrane-associated guanylate kinases (PSD-95 and SAP102), are distributed between the postsynaptic density (PSD) and lipid rafts. However, the time course of the association of NMDAR with LR during neural development is not known. We therefore investigated the effect of development on the association of NMDAR with LR prepared from rat brains ranging in postnatal age from 1-35 days and compared this with their expression in PSDs. LR and PSD fractions were prepared by extraction of P2 membranes with Tx-100 followed by sucrose density gradient centrifugation. The yield of LR, as reflected by levels of protein, Thy-1, and flotillin-1 increased during postnatal development. NR2A was associated predominantly with the lipid raft fraction at all ages examined whereas NR2B underwent a gradual shift from PSDs to lipid rafts during the first 3 weeks after birth. These changes in the distribution of NR2A and NR2B were paralleled by changes in the distribution of PSD-95 and SAP102 respectively. Tyrosine-phosphorylated proteins, including NR2A and NR2B, were preferentially associated with lipid rafts in older, as compared to younger, animals. These results show that the association of NMDAR with LR is regulated developmentally.

Isolation and characterization of LCHN: a novel factor induced by transient global ischemia in the adult rat hippocampus.

Using mRNA differential display to identify cerebral ischemia-responsive mRNAs, we isolated and cloned a cDNA derived from a novel gene, that has been designated LCHN. Antisense mRNA in situ hybridization and immunoblotting confirmed LCHN expression to be induced in the rat hippocampus following transient forebrain ischemia. The deduced amino acid sequence of the novel LCHN cDNA contains an open reading frame of 455 amino acids, encoding a protein with a predicted molecular mass of approximately 51 kDa. Although LCHN is highly conserved between rat, mouse, and human, the deduced amino acid sequence of LCHN does not possess significant homology to other known genes. LCHN immunoreactivity is detected within the somatodendritic compartment of neurons, is also present on dendritic growth cones, but is not detected on astrocytes. The induction of LCHN in the hippocampus following ischemic injury may have functional consequences, as the ectopic over-expression of LCHN generated neurons with longer and more branched axons and dendrites. Taken together, these data suggest that LCHN could play a role in neuritogenesis, as well as in neuronal recovery and/or restructuring in the hippocampus following transient cerebral ischemia.

Phosphorylation of CaMKII at Thr253 occurs in vivo and enhances binding to isolated postsynaptic densities.

Autophosphorylation of Ca(2+)-calmodulin stimulated protein kinase II (CaMKII) at two sites (Thr286 and Thr305/306) is known to regulate the subcellular location and activity of this enzyme in vivo. CaMKII is also known to be autophosphorylated at Thr253 in vitro but the functional effect of phosphorylation at this site and whether it occurs in vivo, is not known. Using antibodies that specifically recognize CaMKII phosphorylated at Thr253 together with FLAG-tagged wild type and phospho- and dephospho-mimic mutants of alpha-CaMKII, we have shown that Thr253 phosphorylation has no effect on either the Ca(2+)-calmodulin dependent or autonomous kinase activity of recombinant alpha-CaMKII in vitro. However, the Thr253Asp phosphomimic mutation increased alpha-CaMKII binding to subcellular fractions enriched in post-synaptic densities (PSDs). The increase in binding was similar in extent, and additive, to that produced by phosphorylation of Thr286. Thr253 phosphorylation was dynamically regulated in intact hippocampal slices. KCl induced depolarisation increased Thr253 phosphorylation and the phospho-Thr253-CaMKII was specifically recovered in the subcellular fraction enriched in PSDs. These results identify Thr253 as an additional site at which CaMKII is phosphorylated in vivo and suggest that this dynamic phosphorylation may regulate CaMKII function by altering its distribution within the cell.

Differential effects of hypoxia-ischemia on phosphorylation of the N-methyl-D-aspartate receptor in one- and three-week-old rats.

The effects of transient cerebral hypoxia-ischemia (HI) on phosphorylation of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor were investigated in 7 (P7)- and 21 (P21)-day-old rats. Unilateral HI was induced by ligation of the right common carotid artery and exposure to 8% O(2)/92% N(2) for 120 (P7) or 90 (P21) min. Phosphorylation by protein kinase A (PKA; S897) and PKC (S896 and S890) was depressed in the ipsilateral hemisphere relative to both naïve controls and the contralateral hemisphere immediately following HI at both ages. At P7, but not P21, reperfusion resulted in an initial recovery to control phosphorylation levels at all 3 sites followed by a secondary decline. At both ages, pS896 was less than control values after 24 h of recovery, whereas pS890 had returned to control levels by this time. pS897 recovered to control levels by 24 h in P21 animals but not in P7 animals. Differential effects of HI on phosphorylation of the NMDA receptor at P7 and P21 may contribute to age-related changes in sensitivity to HI.

Association of heat shock proteins and neuronal membrane components with lipid rafts from the rat brain.

Lipid rafts are specialized plasma membrane microdomains enriched in cholesterol and sphingolipids that serve as major assembly and sorting platforms for signal transduction complexes. Constitutively expressed heat shock proteins Hsp90, Hsc70, Hsp60, and Hsp40 and a range of neurotransmitter receptors are present in lipid rafts isolated from rat forebrain and cerebellum. Depletion of cholesterol dissociates these proteins from lipid rafts. After hyperthermic stress, flotillin-1, a lipid raft marker protein, does not show major change in levels. Stress-inducible Hsp70 is detected in lipid rafts at 1 hr posthyperthermia, with the peak levels attained at 24 hr, suggesting that Hsp70 may play roles in maintaining the stability of lipid raft-associated signal transduction complexes following neural stress.

Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain.

Ischemia results in increased phosphorylation of NMDA receptors. To investigate the possible role of lipid rafts in this increase, lipid rafts and post-synaptic densities (PSDs) were isolated by the extraction of rat brain synaptosomes with Triton X-100 followed by sucrose density gradient centrifugation. Lipid rafts accounted for the majority of PSD-95, whereas SAP102 was predominantly located in PSDs. Between 50 and 60% of NMDA receptors were associated with lipid rafts. Greater than 85-90% of Src and Fyn were present in lipid rafts, whereas Pyk2 was mainly associated with PSDs. Lipid rafts and PSDs were isolated from animals subjected to 15 min of global ischemia followed by 6 h of recovery. Ischemia did not affect the yield, density, flotillin-1 or cholesterol content of lipid rafts. Following ischemia, the phosphorylation of NR1 by protein kinase C and tyrosine phosphorylation of NR2A and NR2B was increased in both lipid rafts and PSDs, with a greater increase in tyrosine phosphorylation occurring in the raft fraction. Following ischemia, NR1, NR2A and NR2B levels were elevated in PSDs and reduced in lipid rafts. The findings are consistent with a model involving close interaction between lipid rafts and PSDs and a role for lipid rafts in ischemia-induced signaling pathways.

Inhibition of protein kinase C reduces ischemia-induced tyrosine phosphorylation of the N-methyl-d-aspartate receptor.

The role of protein kinase C (PKC) in tyrosine phosphorylation of the N-methyl-d-aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four-vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src-family kinases (SFKs) and Pyk2 were increased in post-synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post-ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia-induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia-induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia-induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src-family kinases.

Differential effects of hypoxia-ischemia on subunit expression and tyrosine phosphorylation of the NMDA receptor in 7- and 21-day-old rats.

The effect of cerebral hypoxia-ischemia (HI) on levels and tyrosine phosphorylation of the NMDA receptor was examined in 7- (P7) and 21 (P21)-day-old rats. Unilateral HI was administered by ligation of the right common carotid artery and exposure to an atmosphere of 8% O2/92% N2 for 2 (P7) or 1.5 (P21) h. This duration of HI produces significant infarction in nearly all of the survivors with damage being largely restricted to the cortex, striatum, and hippocampus of the hemisphere ipsilateral to the carotid artery ligation. NR2A levels in the right hemisphere of P7 pups were markedly reduced after 24 h of recovery, while NR1 and NR2B remained unchanged. In contrast, NR2B, but not NR2A, was reduced after HI at P21. At both ages, HI resulted in a transient increase in tyrosine phosphorylation of a number of forebrain proteins that peaked between 1 and 6 h of recovery. At both P7 and P21, tyrosine phosphorylation of NR2B was enhanced 1 h after HI and had returned to basal levels by 24 h. HI induced an increase in tyrosine phosphorylation of NR2A in 21 day, but not in 7-day-old animals. The differential effects of HI on the NMDA receptor at different post-natal ages may contribute to changing sensitivity to hypoxia-ischemia.

Treatment of ischemic brain damage by perturbing NMDA receptor- PSD-95 protein interactions.

N-methyl-D-aspartate receptors (NMDARs) mediate ischemic brain damage but also mediate essential neuronal excitation. To treat stroke without blocking NMDARs, we transduced neurons with peptides that disrupted the interaction of NMDARs with the postsynaptic density protein PSD-95. This procedure dissociated NMDARs from downstream neurotoxic signaling without blocking synaptic activity or calcium influx. The peptides, when applied either before or 1 hour after an insult, protected cultured neurons from excitotoxicity, reduced focal ischemic brain damage in rats, and improved their neurological function. This approach circumvents the negative consequences associated with blocking NMDARs and may constitute a practical stroke therapy.