Rat B2 Sequences Are Induced in the Hippocampal CA1 Region After Transient Global Cerebral Ischemia

Abstract

Global brain ischemia causes cell death in the CA1region of the hippocampus 3-5 days after reperfusion. The biological pathway leading to such delayed neuronal damage has not been established. By using differential display analysis, we examined expression levels of poly(A) RNAs isolated from hippocampal extracts prepared from rats exposed to global ischemia and found an up-regulated transcript, clone 17a. Northern blot analysis of clone 17a showed an approximately 35-fold increase in the ischemic brain at 24 h after four-vessel occlusion. Rapid amplification of cDNA ends of clone 17a revealed a family of genes (160 -540 base pairs) that had the characteristics of rodent B 2 sequences. In situ hybridization demonstrated that the elevated expression of this gene was localized predominantly in the CA1 pyramidal neurons. The level of expression in the CA1 region decreased dramatically between 24 and 72 h after ischemia. The elevated expression of clone 17a was not observed in four-vessel occlusion rats treated with the compound LY231617, an antioxidant known to exert neuroprotection in rats subjected to global ischemia. Since delayed neuronal death has the characteristics of apoptosis, we speculate that clone 17a may be involved in apoptosis. We examined the expression level of clone 17a in in vitro models of apoptosis using cerebellar granule neurons that were subjected to potassium removal, glutamate toxicity, or 6-hydroxydopamine treatment and found that clone 17a transcripts were induced in cerebellar granule neurons by glutamate or 6-hydroxydopamine stimulation but not potassium withdrawal.A short period of global cerebral ischemia in rodents causes neurons in the striatum, hippocampus, and lateral thalamus to die (1). Intriguingly, pyramidal neurons in the CA1 region of the hippocampus undergo delayed neuronal death 3-5 days after the insult (1, 2). A similar phenomenon occurs in human cerebral ischemia (3). This time lag provides a window of opportunity for therapeutic interventions after ischemic injury. However, the molecular mechanisms that trigger and lead to delayed neuronal death have not been well established, although many hypotheses have been proposed such as excitotoxicity of glutamate, disturbed calcium homeostasis, altered lipid metabolism, free radicals, and mitochondrial involvement (for reviews, see Refs. 4 and 5). There is a growing body of evidence suggesting that apoptotic events occur in both global and focal brain ischemia (6 -8). It is possible that many of these hypotheses may in fact represent different aspects of a common mechanism. One of the approaches that can be used to further our understanding of the molecular mechanism of delayed neuronal death is to establish the gene expression profile of the process. Differential expression of many genes has been observed in ischemic brains, including some immediate early genes, heat shock proteins, and factors controlling apoptosis such as Bcl2, Bcl-x, Bax, and caspases (for reviews, see Refs. 9 and 10).Differential display (DD) 1 is a techn...

Correlation between Delayed Neuronal Cell Death and Selective Decrease in Phosphatidylinositol 4-Kinase Expression in the CA1 Subfield of the Hippocampus after Transient Forebrain Ischemia

“…On the basis of these criteria, we have succeeded in isolating two genes through DD analysis. One is identical to the B2 sequences recently reported as an up-regulated gene (Liu et al, 1999). Unfortunately, the B2 sequences protein has not been detected yet; however, it is speculated to be involved in gene expression and RNA processing (Krayev et al, 1982).…”

“…1C). However, we have identified that the rat B2 sequence, which has been already reported, was transiently up-regulated, peaking at 1 day after brain ischemia in hippocampus but not cerebral cortex (data not shown) (Liu et al, 1999).…”

“…For example, in gerbil, a serine protease inhibitor gene (SPI-3) and zinc transporter gene (ZnT-1) were isolated from ischemic hippocampus with this method (Tsuda et al, 1996(Tsuda et al, , 1997. In addition, adrenomedullin and the rat B2 sequence were discovered from rat cortex and hippocampus in a middle cerebral artery occlusion model and fourvessel occlusion model, respectively (Liu et al, 1999;Wang et al, 1995). These particular genes were upregulated by each ischemic model and suggested to be involved in neuronal cell death.…”

Cloning and expression of short interspersed elements B1 and B2 in ischemic brain

“…In vitro , the SINE analogs are induced by several stressful stimuli, including heat shock (Fornace & Mitchell, 1986; Liu et al ., 1995), DNA damage (Rudin & Thompson, 2001), viral infection (Singh et al ., 1985; Carey et al ., 1986; Panning & Smiley, 1995; Russanova et al ., 1995) and translational inhibition (Liu et al ., 1995). In vivo , the SINE B1 and B2 genes are induced after hyperthermic shock and ethanol administration in liver, spleen, kidney and testis (Li et al ., 1999). With the exception of the brain‐specific SINE BC1 expression (Tiedge et al ., 1991), there is only a single report describing the inducibility of the SINE B2 in the central nervous system (Liu et al ., 1999).…”

“…In vivo , the SINE B1 and B2 genes are induced after hyperthermic shock and ethanol administration in liver, spleen, kidney and testis (Li et al ., 1999). With the exception of the brain‐specific SINE BC1 expression (Tiedge et al ., 1991), there is only a single report describing the inducibility of the SINE B2 in the central nervous system (Liu et al ., 1999). With regard to the B2 expression after global ischemia, our results are comparable with the previous study of Liu et al .…”