Contrasting patterns of Bim induction and neuroprotection in Bim-deficient mice between hippocampus and neocortex after status epilepticus.

Prolonged seizures (status epilepticus) are associated with brain region-specific regulation of apoptosis-associated signaling pathways. Bcl-2 homology domain 3-only (BH3) members of the Bcl-2 gene family are of interest as possible initiators of mitochondrial dysfunction and release of apoptogenic molecules after seizures. Previously, we showed that expression of the BH3-only protein, Bcl-2 interacting mediator of cell death (Bim), increased in the rat hippocampus but not in the neocortex after focal-onset status epilepticus. In this study, we examined Bim expression in mice and compared seizure damage between wild-type and Bim-deficient animals. Status epilepticus induced by intra-amygdala kainic acid (KA) caused extensive neuronal death within the ipsilateral hippocampal CA3 region. Hippocampal activation of factors associated with transcriptional and posttranslational activation of Bim, such as CHOP and c-Jun NH(2)-terminal kinases, was significant within 1 h. Upregulation of bim mRNA was evident after 2 h and Bim protein increased between 4 and 24 h. Hippocampal CA3 neurodegeneration was reduced in Bim-deficient mice compared with wild-type animals after seizures in vivo, and short interfering RNA molecules targeting bim reduced cell death after KA treatment of hippocampal organotypic cultures. In contrast, neocortical Bim expression declined after status epilepticus, and neocortex damage in Bim-deficient mice was comparable with that in wild-type animals. These results show region-specific differential contributions of Bim to seizure-induced neuronal death.

Microarray profile of seizure damage-refractory hippocampal CA3 in a mouse model of epileptic preconditioning.

A neuroprotected state can be acquired by preconditioning brain with a stimulus that is subthreshold for damage (tolerance). Acquisition of tolerance involves coordinate, bi-directional changes to gene expression levels and the re-programmed phenotype is determined by the preconditioning stimulus. While best studied in ischemic brain there is evidence brief seizures can confer tolerance against prolonged seizures (status epilepticus). Presently, we developed a model of epileptic preconditioning in mice and used microarrays to gain insight into the transcriptional phenotype within the target hippocampus at the time tolerance had been acquired. Epileptic tolerance was induced by an episode of non-damaging seizures in adult C57Bl/6 mice using a systemic injection of kainic acid. Neuron and DNA damage-positive cell counts 24 h after status epilepticus induced by intraamygdala microinjection of kainic acid revealed preconditioning given 24 h prior reduced CA3 neuronal death by approximately 45% compared with non-tolerant seizure mice. Microarray analysis of over 39,000 transcripts (Affymetrix 430 2.0 chip) from microdissected CA3 subfields was undertaken at the point at which tolerance was acquired. Results revealed a unique profile of small numbers of equivalently up- and down-regulated genes with biological functions that included transport and localization, ubiquitin metabolism, apoptosis and cell cycle control. Select microarray findings were validated post hoc by real-time polymerase chain reaction and Western blotting. The present study defines a paradigm for inducing epileptic preconditioning in mice and first insight into the global transcriptome of the seizure-damage refractory brain.

Ca2+ -permeable acid-sensing ion channels and ischemic brain injury.

Acidosis is a common feature of brain in acute neurological injury, particularly in ischemia where low pH has been assumed to play an important role in the pathological process. However, the cellular and molecular mechanisms underlying acidosis-induced injury remain unclear. Recent studies have demonstrated that activation of Ca(2+)-permeable acid-sensing ion channels (ASIC1a) is largely responsible for acidosis-mediated, glutamate receptor-independent, neuronal injury. In cultured mouse cortical neurons, lowering extracellular pH to the level commonly seen in ischemic brain activates amiloride-sensitive ASIC currents. In the majority of these neurons, ASICs are permeable to Ca(2+), and an activation of these channels induces increases in the concentration of intracellular Ca(2+) ([Ca(2+)](i)). Activation of ASICs with resultant [Ca(2+)](i) loading induces time-dependent neuronal injury occurring in the presence of the blockers for voltage-gated Ca(2+) channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing [Ca(2+)](o). In focal ischemia, intracerebroventricular administration of ASIC1a blockers, or knockout of the ASIC1a gene protects brain from injury and does so more potently than glutamate antagonism. Furthermore, pharmacological blockade of ASICs has up to a 5 h therapeutic time window, far beyond that of glutamate antagonists. Thus, targeting the Ca(2+)-permeable acid-sensing ion channels may prove to be a novel neuroprotective strategy for stroke patients.

Activation of the caspase 8 pathway mediates seizure-induced cell death in cultured hippocampal neurons.

In response to harmful stresses, cells induce programmed cell death (PCD) or apoptosis. Seizures can induce neural damage and activate biochemical pathways associated with PCD. Since seizures trigger intracellular calcium overload, it has been presumed that the intrinsic cell death pathway mediated by mitochondrial dysfunction would modulate cell death following seizures. However, previous work suggests that the extrinsic cell death pathway may initiate the damage program. Here we investigate intrinsic versus extrinsic cell death pathway activation using caspase cleavage as a marker for activation of these pathways in a rat in vitro model of seizures. Hippocampal cells, chronically treated with kynurenic acid, had kynurenic acid withdrawn to induce seizure-like activity for 40 min. Subjecting rat hippocampal cultures to seizures increased cell death and apoptosis-like DNA fragmentation using TUNEL staining. Seizure-induced cell death was blocked by both MK801 (10 microM) and CNQX (40 microM), which suggests multiple glutamate receptors regulate seizure-induced cell death. Cleavage of the initiator caspases, caspase 8 and 12 were increased 4h following seizure, and cleavage of the quintessential executioner caspase, caspase 3 was increased 4h following seizure. In contrast, caspase 9 cleavage only increased 24h following seizure. Using an affinity labeling approach to trap activated caspases in situ, we show that caspase 8 is the apical caspase activated following seizures. Finally, we show that the caspase 8 inhibitor Ac-IETD-CHO was more effective at blocking seizure-induced cell death than the caspase 9 inhibitor Ac-LEHD-CHO. Taken together, our data suggests the extrinsic cell death pathway-associated caspase 8 is activated following seizures in vitro.

Conjugated linoleic acid impairs endothelial function.

OBJECTIVE: To determine the effect of dietary supplementation with conjugated linoleic acid (CLA) on body mass index (BMI), body fat distribution, endothelial function, and markers of cardiovascular risk. METHODS AND RESULTS: Forty healthy volunteers with BMI >27 kg/m2 were randomized to receive a CLA isomeric mixture or olive oil in a 12-week double-blind study. Subcutaneous body fat and abdominal/hepatic fat content were assessed using skin-fold thicknesses and computed tomography scanning, respectively. Endothelial function was assessed by brachial artery flow-mediated dilatation (FMD). Plasma isoprostanes were measured as an index of oxidative stress. CLA supplementation did not result in a significant change in BMI index or total body fat. There was a significant decrease in limb (-7.8 mm, P<0.001), but not torso skin-fold thicknesses or abdominal or liver fat content. Brachial artery FMD declined (-1.3%, P=0.013), and plasma F2-isoprostanes increased (+91 pg/mL, P=0.042). CONCLUSIONS: A CLA isomeric mixture had at most modest effects on adiposity and worsened endothelial function. On the basis of these results, the use of the isomeric mixture of CLA as an aid to weight loss cannot be recommended.

Fibrinogen, homocyst(e)ine, and C-reactive protein concentrations relative to sex and socioeconomic status in British young people.

This study assesses the prevalence of recently identified coronary heart disease (CHD) risk factors in young people of differing socioeconomic status (SES). From November 2001 through March 2002, 100 boys and 108 girls, of age 12.9 +/- 0.3 years, selected from differing SES were assessed for CHD risk factors. Measurements included fibrinogen (Fg), homocyst(e)ine (Hcy), and C-reactive protein (CRP). Fibrinogen was significantly greater among boys from a higher SES compared with those from a low SES (P < or = 0.05). Differences according to sex (P < or = 0.05) were identified for Fg and CRP. The data indicate the prevalence of recently identified CHD risk factors in this cohort of British schoolchildren. For the purpose of this article, the phrase "young people" embraces both children and adolescents.

Seizure-like activity leads to the release of BAD from 14-3-3 protein and cell death in hippocampal neurons in vitro.

Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.

Adeno-associated virus-mediated delivery of BCL-w gene improves outcome after transient focal cerebral ischemia.

A recombinant adeno-associated virus (rAAV) vector was used to overexpress the anti-apoptotic Bcl-2-family protein, BCL-w, in rat brain. Three weeks after injecting the vector into cerebral cortex and striatum on one side, temporary focal ischemia was induced by occlusion of the ipsilateral middle cerebral artery for 90 min, followed by reperfusion for 24 h. BCL-w expression was increased in cerebral cortex and striatum--and in neurons, astroglia and endothelial cells--in the brains of rats that received the rAAV-BCL-w vector, compared to rats given phosphate-buffered saline or a control vector containing the gene for green fluorescent protein. Recipients of the rAAV-BCL-w vector also showed a 30% reduction in infarct size and a 33-40% improvement in neurological function, compared to the control groups. These results provide evidence for a role of BCL-w in regulating histological and functional outcome after focal cerebral ischemia.

cDNA microarray analysis of changes in gene expression induced by neuronal hypoxia in vitro.

We used cDNA microarray gene expression profiling to characterize the transcriptional response to exposure of cultured mouse cerebral cortical neurons to hypoxia for 24 hr. Of 11,200 genes examined, 1,405 (12.5%) were induced or repressed at least 1.5-fold, whereas 26 known genes were induced and 20 known genes were repressed at least 2.5-fold. The most strongly induced genes included genes coding for endoplasmic reticulum proteins (Ero1L/Giig11, Sac1p, Ddit3/Gadd153), proteins involved in ubiquitination (Arih2, P4hb), proteins induced by hypoxia in non-neuronal systems (Gpi1, Aldo1, Anxa2, Hig1), and proteins that might promote cell death (Gas5, Egr1, Ndr1, Vdac2). These findings reinforce the importance of endoplasmic reticulum-based mechanisms and of protein-ubiquitination pathways in the neuronal response to hypoxia.

Formation of the Apaf-1/cytochrome c complex precedes activation of caspase-9 during seizure-induced neuronal death.

In this study we examine the in vivo formation of the Apaf-1/cytochrome c complex and activation of caspase-9 following limbic seizures in the rat. Seizures were elicited by unilateral intraamygdala microinjection of kainic acid to induce death of CA3 neurons within the hippocampus of the rat. Apaf-1 was found to interact with cytochrome c within the injured hippocampus 0-24 h following seizures by co-immunoprecipitation analysis and immunohistochemistry demonstrated Apaf-1/cytochrome c co-localization. Cleavage of caspase-9 was detected approximately 4 h following seizure cessation within ipsilateral hippocampus and was accompanied by increased cleavage of the substrate Leu-Glu-His-Asp-p-nitroanilide (LEHDpNA) and subsequent strong caspase-9 immunoreactivity within neurons exhibiting DNA fragmentation. Finally, intracerebral infusion of z-LEHD-fluoromethyl ketone increased numbers of surviving CA3 neurons. These data suggest seizures induce formation of the Apaf-1/cytochrome c complex prior to caspase-9 activation and caspase-9 may be a potential therapeutic target in the treatment of brain injury associated with seizures.

Fas (CD95) may mediate delayed cell death in hippocampal CA1 sector after global cerebral ischemia.

Cell death-regulatory genes like caspases and bcl-2 family genes are involved in delayed cell death in the CA1 sector of hippocampus after global cerebral ischemia, but little is known about the mechanisms that trigger their expression. The authors found that expression of Fas and Fas-ligand messenger ribonucleic acid and protein was induced in vulnerable CA1 neurons at 24 and 72 hours after global ischemia. Fas-associating protein with a novel death domain (FADD) also was upregulated and immunoprecipitated and co-localized with Fas. Caspase-10 was activated and interacted with FADD protein to an increasing extent as the duration of ischemia increased. Moreover, caspase-10 co-localized with both FADD and caspase-3. These findings suggest that Fas-mediated death signaling may play an important role in signaling hippocampal neuronal death in CA1 after global cerebral ischemia.

Altered expression of the neuropeptide-processing enzyme carboxypeptidase E in the rat brain after global ischemia.

Carboxypeptidase E, an exoprotease involved in the processing of bioactive peptides released by a regulated secretory pathway, was identified in a subtractive complementary DNA library derived from an ischemic rat brain by differential screening. In situ hybridization and immunocytochemical analysis showed the presence of carboxypeptidase E messenger RNA and protein in the cerebral cortex, thalamus, striatum, and hippocampus of a healthy rat brain. After 15 minutes of transient global ischemia followed by 8 hours of reperfusion, increased levels of carboxypeptidase E messenger RNA and protein were observed in the hippocampal CA1 and CA3 regions and in the cortex, as detected by Northern and Western blot analyses and in situ hybridization. After extended reperfusion (24 to 72 hours), both carboxypeptidase E messenger RNA and protein levels were decreased. The ischemia-induced changes in carboxypeptidase E expression suggest that this enzyme may play a role in modulating the brain's response to ischemia.

Global ischemia induces expression of acid-sensing ion channel 2a in rat brain.

Acid-sensing ion channels (ASICs) are ligand-gated cation channels that respond to acidic stimuli. They are expressed throughout the mammalian nervous system. In the peripheral nervous system, ASICs act as nociceptors, responding to the tissue acidosis that accompanies ischemic and inflammatory conditions. The function of ASICs in the central nervous system is not known. In this article, the authors present evidence that transient global ischemia induces ASIC 2a protein expression in neurons that survive ischemia. Western blot analysis with an anti-ASIC 2a antibody revealed up-regulation of an 80 kD protein in ischemic rat brain. Immunohistochemical analysis showed that ASIC 2a protein expression increased in neurons of the hippocampus and cortex. Klenow fragment-mediated labeling of DNA strand breaks determined that ASIC 2a induction did not occur in cells with detectable DNA damage. The current results suggest a possible role for ASICs in mediating a cellular response to ischemia.

Cleavage of bid may amplify caspase-8-induced neuronal death following focally evoked limbic seizures.

The mechanism by which seizures induce neuronal death is not completely understood. Caspase-8 is a key initiator of apoptosis via extrinsic, death receptor-mediated pathways; we therefore investigated its role in mediating seizure-induced neuronal death evoked by unilateral kainic acid injection into the amygdala of the rat, terminated after 40 min by diazepam. We demonstrate that cleaved (p18) caspase-8 was detectable immediately following seizure termination coincident with an increase in cleavage of the substrate Ile-Glu-Thr-Asp (IETD)-p-nitroanilide and the appearance of cleaved (p15) Bid. Expression of Fas and FADD, components of death receptor signaling, was increased following seizures. In vivo intracerebroventricular z-IETD-fluoromethyl ketone administration significantly reduced seizure-induced activities of caspases 8, 9, and 3 as well as reducing Bid and caspase-9 cleavage, cytochrome c release, DNA fragmentation, and neuronal death. These data suggest that intervention in caspase-8 and/or death receptor signaling may confer protection on the brain from the injurious effects of seizures.

Microarray analysis of hippocampal gene expression in global cerebral ischemia.

The brain's response to ischemia, which helps determine clinical outcome after stroke, is regulated partly by competing genetic programs that respectively promote cell survival and delayed cell death. Many genes involved in this response have been identified individually or systematically, providing insights into the molecular basis of ischemic injury and potential targets for therapy. The development of microarray systems for gene expression profiling permits screening of large numbers of genes for possible involvement in biological or pathological processes. Therefore, we used an oligodeoxynucleotide-based microarray consisting of 374 human genes, most implicated previously in apoptosis or related events, to detect alterations in gene expression in the hippocampus of rats subjected to 15 minutes of global cerebral ischemia followed by up to 72 hours of reperfusion. We found 1.7-fold or greater increases in the expression of 57 genes and 1.7-fold or greater decreases in the expression of 34 genes at 4, 24, or 72 hours after ischemia. The number of induced genes increased from 4 to 72 hours, whereas the number of repressed genes decreased. The induced genes included genes involved in protein synthesis, genes mutated in hereditary human diseases, proapoptotic genes, antiapoptotic genes, injury-response genes, receptors, ion channels, and enzymes. We detected transcriptional induction of several genes implicated previously in cerebral ischemia, including ALG2, APP, CASP3, CLU, ERCC3, GADD34, GADD153, IGFBP2, TIAR, VEGF, and VIM, as well as other genes not so implicated. We also found coinduction of several groups of related genes that might represent functional modules within the ischemic neuronal transcriptome, including VEGF and its receptor, NRP1; the IGF1 receptor and the IGF1-binding protein IGFBP2; Rb, the Rb-binding protein E2F1, and the E2F-related transcription factor, TFDP1; the CACNB3 and CACNB4 beta-subunits of the voltage-gated calcium channel; and caspase-3 and its substrates, ACINUS, FEM1, and GSN. To test the hypothesis that genes identified through this approach might have roles in the pathophysiology of cerebral ischemia, we measured expression of the products of two induced genes not heretofore implicated in cerebral ischemia-GRB2, an adapter protein involved in growth-factor signaling pathways, and SMN1, which participates in RNA processing and is deleted in most cases of spinal muscular atrophy. Western analysis showed enhanced expression of both proteins in hippocampus at 24 to 72 hours after ischemia, and SMN1 was localized by immunohistochemistry to hippocampal neurons. These results suggest that microarray analysis of gene expression may be useful for elucidating novel molecular mediators of cell death and survival in the ischemic brain.

Bax kappa, a novel Bax splice variant from ischemic rat brain lacking an ART domain, promotes neuronal cell death.

Bax is a pro-apoptotic Bcl-2 family protein that regulates programmed cell death through homodimerization and through heterodimerization with Bcl-2. Bax alpha is encoded by six exons and undergoes alternative splicing. Bax kappa, a splice variant of Bax with conserved BH1, BH2 and BH3 binding domains and a C-terminal transmembrane domain (TM), but with an extra 446-bp insert between exons 1 and 2 leading to loss of an N-terminal ART domain, was identified from an ischemic rat brain cDNA library. Expression of Bax kappa mRNA and protein was up-regulated in hippocampus after cerebral ischemic injury. The increased Bax kappa mRNA was distributed mainly in selectively vulnerable hippocampal CA1 neurons that are destined to die after global ischemia. Overexpression of Bax kappa protein in HN33 mouse hippocampal neuronal cells induced cell death, which was partially abrogated by co-overexpression of Bcl-2. Moreover, co-overexpression of Bax kappa and Bax alpha increased HN33 cell death. The results suggest that the Bax kappa may have a role in ischemic neuronal death.

Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures.

Nuclear changes, including internucleosomal DNA fragmentation, are characteristic features of neuronal apoptosis resulting from transient cerebral ischemia and related brain insults for which the molecular mechanism has not been elucidated. Recent studies suggest that a caspase-3-mediated mechanism may be involved in the process of nuclear degradation in ischemic neurons. In this study, we cloned from rat brain a homolog cDNA encoding caspase-activated deoxyribonuclease (CAD)/DNA fragmentation factor 40 (DFF40), a 40 kDa nuclear enzyme that is activated by caspase-3 and promotes apoptotic DNA degradation. Subsequently, we investigated the role of CAD/DFF40 in the induction of internucleosomal DNA fragmentation in the hippocampus in a rat model of transient global ischemia and in primary neuronal cultures under ischemia-like conditions. At 8-72 hr after ischemia, CAD/DFF40 mRNA and protein were induced in the degenerating hippocampal CA1 neurons. CAD/DFF40 formed a heterodimeric complex in the nucleus with its natural inhibitor CAD (ICAD) and was activated after ischemia in a delayed manner (>24 hr) by caspase-3, which translocated into the nucleus and cleaved ICAD. Furthermore, an induced CAD/DFF40 activity was detected in nuclear extracts in both in vivo and in vitro models, and the DNA degradation activity of CAD/DFF40 was inhibited by purified ICAD protein. These results strongly suggest that CAD/DFF40 is the endogenous endonuclease that mediates caspase-3-dependent internucleosomal DNA degradation and related nuclear alterations in ischemic neurons.

Increased Bcl-w expression following focally evoked limbic seizures in the rat.

Control of seizure-induced neuronal death may involve members of the Bcl-2 family of cell death regulating proteins. Bcl-w is a newly described anti-apoptotic member of this family that may confer neuroprotective effects. We therefore investigated Bcl-w expression in rat brain following focally evoked limbic seizures. Seizures were induced by unilateral microinjection of kainic acid into the amygdala of the rat and terminated after 40 min by diazepam. Constitutive Bcl-w expression was detected by Western blotting and immunohistochemistry. Bcl-w expression was increased 4-72 h following seizures within the injured hippocampus. Immunohistochemistry determined Bcl-w was predominantly expressed in neurons and seizures increased Bcl-w immunoreactivity within piriform cortex and surviving regions of the injured hippocampus. These data suggest Bcl-w may be involved in the modulation of seizure-induced brain injury.

Cyclic AMP response element binding protein (CREB) and CREB binding protein (CBP) in global cerebral ischemia.

Cyclic AMP (cAMP) response element binding protein (CREB) is a transcription factor that has been implicated in neuronal responses to ischemia. We examined the effect of global cerebral ischemia in the rat on the expression of CREB, its transcriptionally active phosphorylated form (pCREB), and the nuclear adaptor protein, CREB binding protein (CBP). Global ischemia induced the expression of pCREB and CBP in vulnerable neurons of the hippocampal CA1 sector. In primary cultures of murine cortical neurons subjected to hypoxia, CBP was selectively expressed in cells with morphologically intact cell nuclei, and not in cells with condensed or fragmented nuclei indicative of irreversibly damaged neurons. These results support a role for transcriptional activation by CREB and CBP in neuronal cell-survival programs following cerebral ischemia.