Calpain activation is involved in early caspase-independent neurodegeneration in the hippocampus following status epilepticus

Evidence for increased calpain activity has been described in the hippocampus of rodent models of temporal lobe epilepsy. However, it is not known whether calpains are involved in the cell death that accompanies seizures. In this work, we characterized calpain activation by examining the proteolysis of calpain substrates and in parallel we followed cell death in the hippocampus of epileptic rats. Male Wistar rats were injected with kainic acid (10 mg/kg) intraperitoneally and killed 24 h later, after development of grade 5 seizures. We observed a strong Fluoro-Jade labeling in the CA1 and CA3 areas of the hippocampus in the rats that received kainic acid, when compared with saline-treated rats. Immunohistochemistry and western blot analysis for the calpain-derived breakdown products of spectrin showed evidence of increased calpain activity in the same regions of the hippocampus where cell death is observed. No evidence was found for caspase activation, in the same conditions. Treatment with the calpain inhibitor MDL 28170 significantly prevented the neurodegeneration observed in CA1. Taken together, our data suggest that early calpain activation, but not caspase activation, is involved in neurotoxicity in the hippocampus after status epilepticus .     

Phosphoinositide 3-kinase accelerates calpain-dependent proteolysis of fodrin during hypoxic cell death

We have shown recently that phosphoinositide 3-kinase (PI 3-kinase) accelerates the hypoxia-induced necrotic cell death of H9c2, derived from rat cardiomyocytes, by enhancing metabolic acidosis. Here we show the downstream events of acidosis that cause hypoxic cell death. Hypoxia induces the proteolysis of fodrin, a substrate of calpain. Intracellular Ca(2+) chelation by BAPTA, and the addition of SJA6017, a specific peptide inhibitor of calpain, also reduces cell death and fodrin proteolysis, indicating that Ca(2+) influx and calpain activation might be involved in these events. The overexpression of wild type PI 3-kinase accelerates fodrin proteolysis, while dominant-negative PI 3-kinase reduces it. Both (N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)/H(+) exchanger, and KB-R7943, an inhibitor of the Na(+)/Ca(2+) exchanger, reduce hypoxic cell death and fodrin proteolysis. The depletion of intracellular Ca(2+ )stores by thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase, also reduces cell death and fodrin proteolysis, indicating that Ca(2+ )release from intracellular Ca(2+ )stores might be also involved. These results indicate that PI 3-kinase might accelerate hypoxic cell death by enhancing the calpain-dependent proteolysis of fodrin.     

Differential response to ischemia in adjacent hippocampalsectors: neuronal death in CA1 versus neurogenesis in dentate gyrus

Two hippocampal sectors show distinct responses to transient ischemia: the cornu Ammonis (CA)1 sector undergoes a delayed neuronal death followed by a lack of neuronal generation, while the dentate gyrus (DG) shows slight postischemic damage followed by an increased neurogenesis. Using the monkey experimental paradigm of transient whole brain global ischemia, the 'calpain-cathepsin hypothesis' was formulated in 1998. This hypothesis proposes that following ischemia calpain compromises the integrity of lysosomal membrane, causing a leakage of degrading hydrolytic enzymes--cathepsins--into the cytoplasm. Ischemia induces Ca(2+) mobilization, calpain activation, lysosomal membrane disruption, and cathepsin release, which all occur specifically in the CA1 sector and cause neuronal death. In the postischemic DG, a vascular niche has been implicated in adult neurogenesis, in that adventitial cells of the DG microvascular environment provoke postischemic up-reguation of neurogenesis with the aid of brain-derived neurotrophic factor and polysialylated form of the neural cell adhesion molecule. In parallel, Down's syndrome cell adhesion molecule has recently been shown to be expressed specifically in the neural progenitor cells of DG. In this review, we focus on the monkey experimental paradigm to reveal the remarkable contrasts between CA1 and DG in response to the ischemic insult.     

Translocation and Down-Regulation of Protein Kinase C-α, -β, and -γ Isoforms During Ischemia-Reperfusion in Rat Brain

We investigated the distribution of protein kinase C (PKC) isoforms in the subcellular fractions (P1, 1,000-g pellet; P2, 10,000-g pellet; P3, 100,000-g pellet; S, 100,000-g supernatant) of rat forebrain after ischemia or reperfusion by immunoblotting. PKC-delta and -epsilon isoforms were predominant in the P2 (synaptosome-rich) fraction, whereas PKC-alpha, -beta, -gamma, -epsilon, and -zeta isoforms were rich in the S (cytosolic) fraction. With time of ischemia (5-30 min), PKC-alpha, -beta, and -gamma translocated to the P2 and P3 fractions, whereas reperfusion for 60 min after 30 min of ischemia reduced PKC-beta activity greatly and PKC-alpha and -gamma activities to a lesser extent. There was no redistribution of PKC-delta, -epsilon, and -zeta after ischemia or reperfusion. A calpain inhibitor, acetylleucylleucylnorleucinal, inhibited the down-regulation of PKC-beta, through intravenous injection. The PKC translocation to the P2 fraction was accompanied by their dephosphorylation, transition of PKC-alpha from dimer to trimer, and the decrease in activity. These data show that PKC-alpha, -beta, and -gamma isoforms translocate chiefly to the synaptosome in ischemic brain in association with the dephosphorylation, multimeric change, and inactivation, followed by the proteolysis of PKC-beta by calpain after postischemic reperfusion.     

Protein kinase C beta in postischemic brain mitochondria

PKC is implicated in the regulation of mitochondrial metabolism. We examined the association of PKCβ with mitochondria and followed postischemic changes in its amount in mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant (CA2-4,DG) hippocampus in gerbil model of transient brain ischemia. Our observations suggest that transient ischemic episode induces a significant, rapid and long lasting increase of PKCβ in mitochondria in CA2-4,DG, which may bespeak neuroprotection. In organotypic hippocampal culture (OHC) model of neurodegeneration, PKCβ inhibition imposed over NMDA toxicity extended the death area beyond the CA1. These results suggest that PKCβ might have a protective effect against excitotoxic damage in rat OHC. The pull-down method and LC-MS/MS analysis revealed mitochondrial proteins that can bind directly with PKCβΙ. The proteins were parts of i) mitochondrial redox carriers forming the electron transport chain including ATP synthase and ii) MPTP: ANT and creatine kinase. PKCβ acting through mitochondrial proteins could play a role in protecting the cells from death by e.g. influencing ROS and ATP production after ischemia in CA2-4,DG region of the hippocampus.     

Iron Deposition after Transient Forebrain Ischemia in Rat Brain

In this study, we investigated the iron deposition in the cerebral cortex, hippocampus CA1 area and corpus striatum pars dorsolateralis in a rat model of cerebral ischemia. Forebrain ischemia was induced by four-vessel occlusion for 20 min. Using iron histochemistry, regional changes were examined from 1 to 8 weeks of postischemic recirculation. Neuronal death was demonstrated in pyramidal cells of the hippocampal CA1 area and in the dorsolateral part of the corpus striatum, which are known as areas most vulnerable to ischemia. Iron deposition in hippocampal CA1 area was coupled to delayed pyramidal cell death. Perl's reaction with DAB intensification revealed of the 1 week iron deposits in the CA1 area, which gradually increased and formed clusters by 8 weeks. In the corpus striatum, strong iron staining was observed in injured cellular layer pars dorsolateralis 1 week after recirculation. Granular iron was deposited in the cytoplasm of pyramidal cells in layers III and V of the frontal cortex after 2 weeks of recirculation. In contrast to the hippocampus and striatum, the cerebral cortex did not develop severe neuronal cell death and atrophy immediately after the ischemic insult, which suggest that the neuronal cell death in the cerebral cortex occurs extremely late.     

Involvement of mu- and m-calpains and protein kinase C isoforms in L8 myoblast differentiation

The objectives were to investigate the roles of different calpains and protein kinase C (PKC) isoforms in muscle differentiation. Concentrations of mu- and m-calpain increased significantly whereas PKCalpha and delta declined significantly during L8 myoblast differentiation. Both mu-calpain and m-calpain antisense oligonucleotides inhibited myotube formation and creatine kinase activity during L8 myoblast differentiation. These results implied that both mu- and m-calpain were involved in L8 myoblast differentiation. To investigate the involvement of calpain in regulation of PKC concentrations, mu-calpain antisense oligonucleotides were added to L8 myoblasts. PKCalpha remained unchanged and PKCdelta declined. By adding m-calpain antisense oligonucleotides instead, PKCalpha level remained unchanged and PKCdelta concentrations increased significantly during differentiation. These results suggest that PKCalpha, but not PKCdelta, is the substrate for mu-calpain and PKCalpha and delta are the substrates for the m-calpain. In addition, more phosphorylated myogenin was found in day 2 antisense oligonucleotides treated L8 cells. It is concluded that the decline of PKCalpha mediated by m- and mu-calpain is essential for L8 myoblast differentiation. The decline of PKC during myoblast differentiation may cause hypo-phosphorylation of myogenin, which in turn activates muscle-specific genes during myogenesis.     

Expression and changes of endogenous insulin-like growth factor-1 in neurons and glia in the gerbil hippocampus and dentate gyrus after ischemic insult

In the present study, we focused upon expression and changes of endogenous insulin-like growth factor-1 (IGF-1) in the hippocampus of the Mongolian gerbil after ischemic insult. In sham-operated animals, IGF-1 immunoreactivity was absent from the hippocampus. IGF-1-immunoreactive (IR) neurons were detected at 12 h and 1 day after ischemic insult. In the hippocampal CA1 area, the IGF-IR neurons were non-pyramidal cells (GABAergic neurons). In the hippocampal CA2/3 areas, the IGF-1-IR neurons were pyramidal and non-pyramidal cells, and in the dentate gyrus the IGF-1-IR neurons were hilar neurons. Four days after ischemia-reperfusion, IGF-1 immunoreactivity disappeared from neurons, and significantly increased in astrocytes and microglia. These results suggest that the induction of IGF-1 in the CA1 area during the early stage (12-24 h after ischemic insult) is associated with the relative vulnerabilities of pyramidal glutamatergic neurons and non-pyramidal GABAergic neurons. The later increase (4 days after ischemic insult) of IGF-1 expression and protein content was found to promote the activities of astrocytes and microglia. These increases of IGF-1 in astrocytes and in microglia are associated with mechanisms that compensate for the effects of delayed neuronal death.     

Early calpain-mediated proteolysis following AMPA receptor activation compromises neuronal survival in cultured hippocampal neurons

In this work, we investigated the involvement of calpains in the neurotoxicity induced by short-term exposure to kainate (KA) in non-desensitizing conditions of AMPA receptor activation (cyclothiazide present, CTZ), in cultured rat hippocampal neurons. The calpain inhibitor MDL28170 had a protective effect in cultures treated with KA plus CTZ (p < 0.01), preventing the decrease in MTT reduction caused by exposure to KA (p < 0.001). Caspase inhibition by ZVAD-fmk was not neuroprotective against the toxic effect of KA. At 1 h after treatment, we could already observe significantly increased calpain activity, which was prevented by MDL 28170 and NBQX. Western blot analysis of calpain substrates, GluR1, neuronal nitric oxide synthase (nNOS) and nonerythroid spectrin (fodrin), showed a time-dependent and MDL 28170-sensitive proteolysis of these proteins. This effect was due to calpains, but not caspases, since ZVAD-fmk was ineffective in preventing proteolytic events. Breakdown products of fodrin (BDPs) were detected as early as 15 min after exposure to KA. Overall, these results show early activation of calpains following activation of AMPA receptors as well as compromise of neuronal survival, likely due to proteolytic events that affect proteins involved in neuronal signaling.     

Comparative Effect of Transient Global Ischemia on Extracellular Levels of Glutamate, Glycine, and γ-Aminobutyric Acid in Vulnerable and Nonvulnerable Brain Regions in the Rat

We evaluated whether regional differences in the magnitude of glutamate, gamma-aminobutyric acid (GABA), and glycine release could explain why some regions are vulnerable to ischemia whereas others are spared. By means of the microdialysis technique, the temporal profile of ischemia-induced changes in extracellular levels of glutamate, GABA, and glycine was compared in regions that demonstrate differing susceptibilities to a 10- and 20-min ischemic insult (dorsal hippocampus, anterior thalamus, somatosensory cortex, and dorsolateral striatum). The degree of ischemia (as established by local cerebral blood flow reduction) and the magnitude of histopathological neuronal damage were also evaluated in these regions. The blood flow reduction was severe and uniform in all regions; however, the histopathological outcome illustrated a different pattern. Whereas the CA1 sector of the hippocampus was severely damaged, the thalamus and cortex were relatively spared from both 10 and 20 min of ischemia. Striatal neurons were resistant to a 10-min insult but severely damaged after 20 min of ischemia. Ischemia-induced increase in glutamate and GABA content were of a similar magnitude and temporal profile in all four brain regions. A uniform increase in extracellular glycine levels was also observed in all four brain structures. The postischemic response, however, was different. Glycine levels remained twofold higher than baseline in the hippocampus but fell to baseline in the cortex and thalamus after both 10- and 20-min insults. In the striatum, glycine levels returned to baseline after 10 min of ischemia but remained relatively high after a 20-min insult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of L-arginine transporters with fodrin: implications for hypoxic inhibition of arginine uptake

In this study, we investigated the possible interaction between the cationic amino acid transporter (CAT)-1 arginine transporter and ankyrin or fodrin. Because ankyrin and fodrin are substrates for calpain and because hypoxia increases calpain expression and activity in pulmonary artery endothelial cells (PAEC), we also studied the effect of hypoxia on ankyrin, fodrin, and CAT-1 contents in PAEC. Exposure to long-term hypoxia (24 h) inhibited L-arginine uptake by PAEC, and this inhibition was prevented by calpain inhibitor 1. The effects of hypoxia and calpain inhibitor 1 were not associated with changes in CAT-1 transporter content in PAEC plasma membranes. However, hypoxia stimulated the hydrolysis of ankyrin and fodrin in PAEC, and this could be prevented by calpain inhibitor 1. Incubation of solubilized plasma membrane proteins with anti-fodrin antibodies resulted in a 70% depletion of CAT-1 immunoreactivity and in a 60% decrease in L-arginine transport activity in reconstituted proteoliposomes (3,291 +/- 117 vs. 8,101 +/- 481 pmol. mg protein(-1). 3 min(-1) in control). Incubation with anti-ankyrin antibodies had no effect on CAT-1 content or L-arginine transport in reconstituted proteoliposomes. These results demonstrate that CAT-1 arginine transporters in PAEC are associated with fodrin, but not with ankyrin, and that long-term hypoxia decreases L-arginine transport by a calpain-mediated mechanism that may involve fodrin proteolysis.     

Changes in Extracellular Amino Acids and Spontaneous Neuronal Activity During Ischemia and Extended Reflow in the CA1 of the Rat Hippocampus

This study addresses the possible involvement of an agonist-induced postischemic hyperactivity in the delayed neuronal death of the CA1 hippocampus in the rat. In two sets of experiments, dialytrodes were implanted into the CA1 either acutely or chronically (24 h of recovery). During 20 min of cerebral ischemia (four-vessel occlusion model) and 8 h of reflow, we followed extracellular amino acids and multiple-unit activity. Multiple-unit activity ceased within 20 sec of ischemia and remained zero during the ischemic insult and for the following 1 h of reflow. During ischemia, extracellular aspartate, glutamate, taurine, and gamma-aminobutyric acid increased in both acute and chronic experiments (seven- to 26-fold). Multiple-unit activity recovered to preischemic levels following 4-6 h of reflow. In the group with dialytrodes implanted acutely, the continuous increase in multiple-unit activity reached 110% of basal at 8 h of reflow. In the group with dialytrodes implanted chronically, multiple-unit activity recovered faster and reached 140% of control at 8 h, paralleled by an increase in extracellular aspartate (5.5-fold) and glutamate (twofold). In conclusion, the postischemic increase of excitatory amino acids and the recovery of the neuronal activity may stress the CA1 pyramidal cells, which could be detrimental in combination with, e.g., postsynaptic impairments.     

Noradrenaline Metabolism in Neocortex and Hippocampus Following Transient Forebrain Ischemia in Rats: Relation to Development of Selective Neuronal Necrosis

Noradrenaline (NA) metabolism in the neocortex and hippocampus was examined in rats at 1, 24, and 48 h following 15 min of reversible forebrain ischemia. As assessed by the ratio of accumulated 3,4-dihydroxyphenylalanine (DOPA) to the tissue NA level after inhibition of DOPA decarboxylase, the NA turnover rates were markedly increased (120-148% above the control) at 1 h postischemia in both the neocortex and hippocampal formation (CA1 and CA3 plus dentate gyrus). The DOPA:NA ratio went back to control levels after longer postischemic survival times. The ratio between levels of the deaminated NA metabolite, 3,4-dihydroxyphenylethyleneglycol (DOPEG), and NA, which gives another measure of NA turnover rate, showed similar changes. In the neocortex and the CA3 plus dentate gyrus, the DOPEG:NA ratio was markedly increased (89-118%) 1 h after the ischemia, but this change had disappeared at 24 and 48 h. Thus, both the DOPA accumulation experiments and the NA and DOPEG measurements indicate that following transient forebrain ischemia, there is an increased NA turnover in the hippocampus and cortex only in the early recirculation period and not after longer postischemic survival times. The degree of neuronal necrosis in the CA1 region was examined light microscopically on celestine blue-acid fuchsin-stained sections at 24, 48, and 96 h following the ischemic insult. The neuronal damage in CA1 was sparse after 24 h of recovery, had increased markedly after 48 h, and was very pronounced at 96 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered protein kinase C activity in different subfields of hippocampus following cerebral ischemia

Protein kinase C (PKC) activity was determined in different (membrane, nuclear and soluble) subcellular fractions prepared separately from the CA1 and CA3 subfields ofMongolian gerbils hippocampus at various time intervals following a single 5-min occlusion of the common carotid arteries.Soluble andnuclear PKC activities of the CA1 sector were found to be elevated at 24 hours following the ischemic injury, while PKC activities did not increase in the CA3 region until the 3rd day after ischemia. The ratio ofsoluble/membrane-associated PKC activities followed a similar pattern, predominantly because the activation/elevation and then down regulation of the cytosolic enzyme pool changing correspondingly to the ongoig pathological processes. PKC activity returned to the normal level in each subfraction of the CA3 subfield by the 7th day. However, PKC activity remained elevated in the soluble fraction of the CA1 sector even after the delayed death of pyramidal neurons, presumably because of the reactive response of astrocytes. Conceivably, the transient activation and rapid down regulation of PKC in the CA1 sector may contribute to the initiation of postischemic neuronal death in the CA1 subfield.Abbreviations BSA bovine serum albumin - GFAP glial fibrillary acidic protein - PKC Ca²⁺/phospholipid-dependent protein kinase - PMSE phenylmethylsulfonyl fluoride     

Enhancement of Neurite Outgrowth Following Calpain Inhibition Is Mediated by Protein Kinase C

Abstract: We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH-SY-5Y human neuroblastoma cells. SH-SY-5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum-containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell-permeant cysteine protease inhibitors N-acetyl-leucyl-leucyl-norleucinal (“C1”) and calpeptin or by the phospholipid-mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti-calpain antisera. Calpain inhibition in intact cells was confirmed by immunoblot analysis showing inhibition of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule-associated protein 1. The above inhibitory peptides and antiserum did not induce neurites in medium containing serum but lacking hirudin, suggesting that increased surface protein adhesiveness is a prerequisite for enhancement of neurite outgrowth by calpain inhibition. Treatment of cells with the PKC inhibitor H7, staurosporine, or sphingosine induced neurite outgrowth independently of serum concentration. Because calpain is thought to regulate PKC activity, we examined this potential interrelationship during neurite outgrowth. Simultaneous treatment with calpain and PKC inhibitors did not produce additive or synergistic effects on neurite outgrowth. PKC activation by 2-O-tetradecanoylphorbol 13-acetate (TPA) prevented and reversed both neurite initiation by serum deprivation and its enhancement by calpain inhibitors. Treatment of cells with the calpain inhibitor C1 retarded PKC down-regulation following TPA treatment. Cell-free analyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of millimolar calcium-requiring calpain to cleave the SH-SY-5Y PKC regulatory subunit from the catalytic subunit, yielding a free catalytic subunit (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that of surface adhesiveness and calpain activity.     

凝闭双侧椎动脉预处理对大鼠全脑缺血损伤的防护作用

目的：观察凝闭双侧椎动脉与夹闭双侧颈总动脉之间的不同时间间隔对Pulsinelli四血管闭塞法全脑缺血模型的影响、以及在凝闭单侧椎动脉的基础上夹闭双侧颈总动脉后的脑缺血的特点。方法：84只Wistar大鼠．随机分为以下4组：对照组、双侧椎动脉凝闭组、全脑缺血组、单侧椎动脉凝闭＋双侧颈总动脉夹闭组。全脑缺血组中,根据凝闭双侧椎动脉与夹闭双侧颈总动脉之间的时间间隔不同,又分为24h间隔、48h间隔和72h间隔3个亚组。观察大鼠脑缺血过程中的反应包括瞳孔散大、对光反射等情况,脑缺血后恢复翻正反射所需要的时间、以及动物的一般状况,并应用硫堇染色法观察海马CA1区锥体神经元迟发性死亡的情况：结果：全脑缺血72h间隔亚组的大鼠,脑缺血过程中的反应、脑缺血后的一般状况和锥体神经元迟发性死亡程度均明显重于全脑缺血24h间隔亚组及48h间隔亚组,但24h间隔亚组与48h间隔亚组之间无显著差异一单侧椎动脉凝闭＋双侧颈总动脉夹闭组大鼠的凝闭侧瞳孔散大、对光反射消失、海马CA1区神经元大量死亡;而未凝闭侧未见上述相关变化。结论：凝闭双侧椎动脉本身也具有脑缺血预处理样作用,对其后48h内夹闭双侧颈总动脉所致的严重脑缺血具有一定程度的保护作用;大鼠椎动脉对脑干及海马的血液供应均存在明显的同侧优势效应,     

胶质细胞谷氨酸转运体-1反义寡核苷酸对脑缺血预处理神经保护效应的抑制作用

本研究应用胶质细胞谷氨酸转运体-1(glial glutamate transporter-1,GLT-1)的反义寡核苷酸(antisense oligo-deoxynucleotides,AS-ODNs)抑制Wistar大鼠GLT-1蛋白的表达,观察其对脑缺血预处理(cerebral ischemic preconditioning.CIP)增强脑缺血耐受作用的影响,探讨GLT-1在CIP诱导的脑缺血耐受中的作用.将凝闭双侧椎动脉的Wistar大鼠随机分为7组:(1)Sham组:只暴露双侧颈总动脉,不阻断血流;(2)CIP组:夹闭双侧颈总动脉3 min;(3)脑缺血打击组:夹闭双侧颈总动脉8 min;(4)CIP 脑缺血打击组:夹闭双侧颈总动脉3 min作为CIP,再灌注2 d后,夹闭双侧颈总动脉8min;(5)双蒸水组:于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射双蒸水,每次5 μL,其它同sham组;(6)AS-ODNs组:于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射GLT-1 AS-ODNs溶液,每次5 μL,其它同sham组,再根据AS-ODNs的剂量进一步分为9 nmol和18 nmol 2个亚组;(7)AS-ODNs CIP 脑缺血打击组:于CIP前12 h、后12 h及后36 h右侧脑室注射GLT-1 AS-ODNs溶液,每次5 μL,其它同CIP 脑缺血打击组,根据AS-ODNs的剂量进一步分为9 nmol和18 nmol 2个亚组.Western blot分析法观察GLT-1蛋白的表达,硫堇染色观察海马CA1区锥体神经元迟发性死亡(delayed neuronal death,DND)情况.Western blot分析显示,侧脑室注射GLT-1 AS-ODNs可剂量依赖性地抑制大鼠海马CA1区GLT-1蛋白表达.硫堇染色显示,sham组和CIP组海马CA1区未见明显的DND;脑缺血打击组海马CA1区有明显的DND:预先给予CIP可显著对抗脑缺血打击引起的DND,表明CIP可以诱导海马CA1区神经元产生缺血性耐受,对抗脑缺血打击引起的DND;而在GLT-1 AS-ODNs CIP 脑缺血打击组,侧脑室注射GLT-1 AS-ODNs后,大鼠海马CA1区出现了明显的DND,表明GLT-1 AS-ODNs通过抑制大鼠GLT-1蛋白表达从而减弱CIP对抗脑缺血打击的神经保护作用.以上结果进一步证实了GLT-1参与CIP诱导的脑缺血耐受.     

Neuronal fodrin proteolysis occurs independently of excitatory amino acid-induced neurotoxicity.

In cultured cerebellar granule cells, the total amount of fodrin alpha subunit increased 3-fold between 0 and 10 days in vitro and fodrin mRNA increased 5-fold. The exposure of cerebellar neurons to NMDA induced the accumulation of a 150 kd proteolytic fragment of fodrin. The NMDA-induced breakdown of fodrin was time-, concentration-, and Ca2(+)-dependent and was inhibited by APV, Mg2+, or the calpain I inhibitor N-acetyl-Leu-Leu-norleucinal. Kainate caused fodrin proteolysis through indirect activation of NMDA receptors. Quisqualate was ineffective. The NMDA-induced degradation of fodrin occurred under conditions that did not cause degeneration of cultured cerebellar neurons. These results show that Ca2+/calpain I-dependent proteolysis of fodrin is selectively associated with NMDA receptor activation; however, fodrin proteolysis per se does not play a causal role in NMDA-induced toxicity in cerebellar granule cells.     

Casein Kinase II Activity in the Postischemic Rat Brain Increases in Brain Regions Resistant to Ischemia and Decreases in Vulnerable Areas

Abstract: Casein kinase II (CKII) is a protein kinase acting in the intracellular cascade of reactions activated by growth factor receptors, and that has a profound influence on cell proliferation and survival. In this investigation, we studied the changes in the activity and levels of CKII in the rat brain exposed to 10. 15 and 20 min of transient forebrain ischemia followed by variable periods of reperfusion. The cytosolic CKII activity decreased during reperfusion by ∼ 30 and ∼ 50% in the selectively vulnerable areas, striatum and the CA1 region of the hippocampus, respectively. In the resistant CA3 region of hippocampus and neocortex, the activity increased by ∼ 20 and ∼ 60%, respectively. The postischemic changes in CKII activity were dependent on the duration of the ischemic insult. The levels of CKII did not change after ischemia, suggesting that the enzyme is modulated by covalent modification or is interacting with an endogenous inhibitor/activator. Treatment of the cytosolic fraction from cortex of rats exposed to ischemia and 1 h of reperfusion with agarose-bound phosphatase decreased the activity of CKII to control levels, suggesting that CKII activation after ischemia involves a phosphorylation of the enzyme. The correlation between postischemic CKII activity and neuronal survival implies that preservation or activation of CKII activity may be important for neuronal survival after cerebral ischemia.