Transgenic overproduction of omega-3 polyunsaturated fatty acids provides neuroprotection and enhances endogenous neurogenesis after stroke.

Strokes are devastating as there are no current therapies to prevent the long term neurological deficits that they cause. Soon after ischemic stroke, there is proliferation and differentiation of neural stem/progenitor cells as an important mechanism for neuronal restoration. However, endogenous neurogenesis by itself is insufficient for effective brain repair after stroke as most newborn neurons do not survive. One fascinating strategy for stroke treatment would thus be maintaining the survival and/or promoting the differentiation of endogenous neural stem/progenitor cells. Using transgenic (Tg) mice over-expressing the C. elegans fat-1 gene encoding an enzyme that converts endogenous omega-6 to omega-3 polyunsaturated fatty acids (n-3 PUFAs), we showed that fat-1 Tg mice with chronically elevated brain levels of n-3 PUFAs exhibited less brain damage and significantly improved long-term neurological performance compared to wild type littermates. Importantly, post-stroke neurogenesis occurred more robustly in fat-1 Tg mice after focal ischemia. This was manifested by enhanced neural stem cell proliferation/differentiation and increased migration of neuroblasts to the ischemic sites where neuroblasts matured into resident neurons. Moreover, these neurogenic effects were accompanied by significantly increased oligodendrogenesis. Our results suggest that n-3 PUFA supplementation is a potential neurogenic and oligodendrogenic treatment to naturally improve post-stroke brain repair and long-term functional recovery.

HSP27: mechanisms of cellular protection against neuronal injury.

The heat shock protein (HSP) family has long been associated with a generalized cellular stress response, particularly in terms of recognizing and chaperoning misfolded proteins. While HSPs in general appear to be protective, HSP27 has recently emerged as a particularly potent neuroprotectant in a number of diverse neurological disorders, ranging from ALS to stroke. Although its robust protective effect on a number of insults has been recognized, the mechanisms and regulation of HSP27's protective actions are still undergoing intense investigation. On the basis of recent studies, HSP27 appears to have a dynamic and diverse range of function in cellular survival. This review provides a forum to compare and contrast recent literature exploring the protective mechanism and regulation of HSP27, focusing on neurological disorders in particular, as they represent a range from protein aggregate-associated diseases to acute stress.

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.

Characterization of the rat DNA fragmentation factor 35/Inhibitor of caspase-activated DNase (Short form). The endogenous inhibitor of caspase-dependent DNA fragmentation in neuronal apoptosis.

Nuclear changes, including internucleosomal DNA fragmentation, are classical manifestations of apoptosis for which the biochemical mechanisms have not been fully elucidated, particularly in neuronal cells. We have cloned the rat DNA fragmentation factor 35/inhibitor of caspase-activated DNase (short form) (DFF35/ICAD(S)) and found it to be the predominant form of ICAD present in rodent brain cells as well as in many other types of cells. DFF35/ICAD(S) forms a functional complex with DFF40/caspase-activated DNase (CAD) in the nucleus, and when its caspase-resistant mutant is over-expressed, it inhibits the nuclease activity, internucleosomal DNA fragmentation, and nuclear fragmentation but not the shrinkage and condensation of the nucleus, in neuron-differentiated PC12 cells in response to apoptosis inducers. DFF40/CAD is found to be localized mainly in the nucleus, and during neuronal apoptosis, there is no evidence of further nuclear translocation of this molecule. It is further suggested that inactivation of DFF40/CAD-bound DFF35 and subsequent activation of DFF40/CAD during apoptosis of neuronal cells may not occur in the cytosol but rather in the nucleus through a novel mechanism that requires nuclear translocation of caspases. These results establish that DFF35/ICAD(S) is the endogenous inhibitor of DFF40/CAD and caspase-dependent apoptotic DNA fragmentation in neurons.

10 kD mitochondrial matrix heat shock protein mRNA is induced following global brain ischemia in the rat.

Heat shock proteins (HSP's) are a family of highly conserved proteins whose expression is increased by stress. The expression of many HSP's is induced in neurons by ischemia; however, the response of the 10 kDa mitochondrial matrix HSP (HSP10) is less well characterized. To address this issue, asphyxial cardiac arrest was induced in 28 male Sprague-Dawley rats. Northern blot analysis revealed that hsp10 mRNA was increased 2.7-fold in asphyxiated rats compared to sham-operated controls. In situ hybridization demonstrated increased mRNA in the cortex, septal nuclei, hippocampus, thalamic nuclei, purkinje cell layer of the cerebellum, and isolated brainstem nuclei of asphyxiated rats. The increase of mRNA was most robust 8 h after the injury but remained increased for 72 h. These results show that hsp10 mRNA is increased following asphyxial cardiac arrest in rats and suggest that hsp10 could be another determinate of neuronal survival after ischemia.

Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia.

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.

Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia.

The inducible isoform of the enzyme cyclooxygenase-2 (COX2) is an immediate early gene induced by synaptic activity in the brain. COX2 activity is an important mediator of inflammation, but it is not known whether COX2 activity is pathogenic in brain. To study the role of COX2 activity in ischemic injury in brain, expression of COX2 mRNA and protein and the effect of treatment with a COX2 inhibitor on neuronal survival in a rat model of global ischemia were determined. Expression of both COX2 mRNA and protein was increased after ischemia in CA1 hippocampal neurons before their death. There was increased survival of CA1 neurons in rats treated with the COX2-selective inhibitor SC58125 [1-[(4-methylsulfonyl) phenyl]-3-trifluoro-methyl-5-[(4-fluoro)phenyl] pyrazole] before or after global ischemia compared with vehicle controls. Furthermore, hippocampal prostaglandin E2 concentrations 24 h after global ischemia were decreased in drug-treated animals compared with vehicle-treated controls. These results suggest that COX2 activity contributes to CA1 neuronal death after global ischemia.

Transient global ischemia triggers expression of the DNA damage-inducible gene GADD45 in the rat brain.

Using in situ hybridization, Northern blot analysis, Western blot analysis, and immunocytochemistry, mRNA and protein expression of the novel DNA damage-inducible gene GADD45 was examined in the rat brain at 0.5, 2, 4, 8, 16, 24, 48, and 72 hours after 15 minutes of transient global ischemia. Transient ischemia produced by the four-vessel occlusion method resulted in DNA double-strand breaks and delayed neuronal cell death in vulnerable neurons of the hippocampal CA1 sector, the hilus, dorsal caudate-putamen, and thalamus, as shown by in situ DNA nick end-labeling and histologic staining. GADD45 mRNA was transiently increased in less-vulnerable regions such as the parietal cortex (up to 8 hours after ischemia) and dentate granule cells (up to 24 hours after ischemia) but was persistently increased in vulnerable neurons such as CA1 pyramidal neurons (up to 48 hours). GADD45 immunoreactivity was increased in both vulnerable and less-vulnerable regions at earlier reperfusion periods (4 to 16 hours), but thereafter immunoreactivity was decreased below control levels in most vulnerable regions before delayed cell death and DNA double-strand breaks. At 72 hours after transient ischemia, a moderate increase in GADD45 immunoreactivity was still detectable in some CA3 neurons and in a few surviving neurons in the CA1 region. Double staining performed at 16 to 72 hours after ischemia revealed that GADD45 immunoreactivity was persistently increased in neurons that did not develop DNA damage. Because GADD45 protein may participate in the DNA excision repair process and because it has been shown that this protein is also overexpressed in neurons that survive focal ischemia and kainate-induced epileptic seizures, the results reported here support the hypothesis that GADD45 could have a protective role in neuronal injury.

Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia.

Delayed neuronal death after transient cerebral ischemia may be mediated, in part, by the induction of apoptosis-regulatory gene products. Caspase-3 is a newly characterized mammalian cysteine protease that promotes cell death during brain development, in neuronal cultures, and in other cell types under many different conditions. To determine whether caspase-3 serves to regulate neuronal death after cerebral ischemia, we have (1) cloned a cDNA encoding the rat brain caspase-3; (2) examined caspase-3 mRNA and protein expression in the brain using in situ hybridization, Northern and Western blot analyses, and double-labeled immunohistochemistry; (3) determined caspase-3-like activity in brain cell extracts; and (4) studied the effect of caspase-3 inhibition on cell survival and DNA fragmentation in the hippocampus in a rat model of transient global ischemia. At 8-72 hr after ischemia, caspase-3 mRNA and protein were induced in the hippocampus and caudate-putamen (CPu), accompanied by increased caspase-3-like protease activity. In the hippocampus, caspase-3 mRNA and protein were predominantly increased in degenerating CA1 pyramidal neurons. Proteolytic activation of the caspase-3 precursor was detected in hippocampus and CPu but not in cortex at 4-72 hr after ischemia. Double-label experiments detected DNA fragmentation in the majority of CA1 neurons and selective CPu neurons that overexpressed caspase-3. Furthermore, ventricular infusion of Z-DEVD-FMK, a caspase-3 inhibitor, decreased caspase-3 activity in the hippocampus and significantly reduced cell death and DNA fragmentation in the CA1 sector up to 7 d after ischemia. These data strongly suggest that caspase-3 activity contributes to delayed neuronal death after transient ischemia.

72-kDa heat shock protein and mRNA expression after controlled cortical impact injury with hypoxemia in rats.

As part of the stress response, the 72 kDa heat shock protein (hsp72) is induced in neurons after ischemic and traumatic brain injury (TBI). To examine the stress response after TBI with secondary insult, we examined the regional and cellular expression of hsp72 mRNA and protein after controlled cortical impact (CCI) injury with secondary hypoxemia and mild hypotension in rats. Rats were killed at 6, 8, 24, 72, or 168 h after trauma. Naive and sham-operated rats were used as controls. Brains were removed, and in situ hybridization (n = 2/group), immunocytochemistry (n = 4/group), and Western blot analysis (n = 3 to 5/group) for hsp72 was performed. Hsp72 mRNA was expressed in neurons in the ipsilateral cortex, CA3 region of the hippocampus, hilus, and dentate gyrus at 6 h. Hsp72 mRNA was expressed primarily in the ipsilateral cortex, at 24 h, and by 72 h hsp72 mRNA expression returned to near basal levels. Hsp72 protein was seen in ipsilateral cortical neurons, hilar neurons, and neurons in the medial aspect of the CA3 region of the hippocampus (CA3-c) at 24 h. At 72 h, hsp72 immunoreactivity was reduced versus 24 h in these same regions, but it was increased versus baseline. Western blot analysis confirmed an increase in hsp72 protein in the ipsilateral cortex. The regional pattern of hsp72 mRNA induction in neurons was similar to the pattern of protein expression after CCI, with the exceptions that hsp72 mRNA, but not protein, was expressed in the dentate gyrus and the lateral aspect of the CA3 region of the hippocampus (CA3-a). The stress response, as detected by hsp72 expression, is induced in some neurons in some regions that are selectively vulnerable to delayed neuronal death in this model of TBI. The failure to translate some proteins including hsp72 may be associated with delayed neuronal death in certain hippocampal regions after TBI.

Apoptosis-suppressor gene bcl-2 expression after traumatic brain injury in rats.

Neuronal death after experimental traumatic brain injury (TBI) has features of both apoptosis and necrosis. Neurons in the peritrauma cortex, hippocampus, and dentate gyrus are particularly vulnerable. The apoptosis-suppressor gene bcl-2 is induced in brain after ischemia and epilepsy-induced injury and may serve to regulate neuronal death. We studied expression of bcl-2 mRNA and protein after experimental TBI in rats. To determine whether bcl-2 protein expression occurred in cells with evidence of apoptosis, triple-labeling studies were performed using (1) antibody against bcl-2, (2) bis-benzimide dye to examine gross nuclear morphology, and (3) terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) to assess for DNA fragmentation. At 6 and 24 hr, bcl-2 mRNA was induced in ipsilateral peritrauma cortex, hippocampus, and dentate gyrus. By 72 hr the increase in bcl-2 mRNA was detected only in cortex. bcl-2 protein was induced at 8, 24, 72, and 168 hr in ipsilateral cortex and hippocampus. Cells expressing bcl-2 protein included neurons in the peritrauma cortex, hippocampus, hilus, and dentate gyrus. The gross nuclear morphology of neurons expressing bcl-2 appeared normal. Furthermore, biochemical evidence of DNA fragmentation, in a pattern characteristic of either apoptosis or necrosis, was seldom seen in neurons expressing bcl-2 protein (bcl-2 colocalized with TUNEL in 0-2% of TUNEL-positive cells observed). These data suggest that bcl-2 may play an important role in the regulation of neuronal death after TBI, and they support a role for bcl-2 as an inducible neuroprotective gene.

Kainate induces the expression of the DNA damage-inducible gene, GADD45, in the rat brain.

The expression of the novel growth arrest and DNA damage-inducible gene GADD45 was examined in kainate-induced epileptic brain damage in the rat using in situ hybridization, northern blot analysis, western blot analysis and immunocytochemistry. Systemic administration of kainate resulted in DNA damage and neuronal degeneration in vulnerable neurons of limbic regions, including the amygdala and hippocampal pyramidal layers, as shown by in situ DNA nick end-labelling and histological staining. GADD45 messenger RNA was transiently increased in non-vulnerable neurons (2-8 h after kainate injection) but was persistently elevated in vulnerable neurons (up to 24 h after injection) after kainate injection. GADD45 protein was elevated in both vulnerable and non-vulnerable neurons at 4 h, but levels decreased in vulnerable neurons thereafter, suggesting that translational blockage of GADD45 protein occurred in these cells. GADD45 protein was overexpressed in non-vulnerable neurons up to 72 h after kainate injection. Because GADD45 may participate in the DNA excision repair process and because it has been shown to be overexpressed in neurons that survive focal cerebral ischaemia, these results support the hypothesis that GADD45 may have a protective role in the injured brain.

Apoptosis repressor genes Bcl-2 and Bcl-x-long are expressed in the rat brain following global ischemia.

The proto-oncogenes bcl-2 and bcl-x-long have been shown to suppress apoptotic cell death in a variety of in vitro systems and cell lines, including neurons. An alternatively spliced from of bcl-x, bcl-x-short, is a promoter of apoptotic death. Whether these genes are induced after ischemia or play any role in determining the fate of ischemic neurons is unknown. To begin to address this issue, we studied the expression of bcl-2, and bcl-x mRNA and protein after global ischemia in the rat. Ischemia was induced in isoflurane-anesthetized rats by the four-vessel occlusion method. mRNA expression was studied by Northern blot analysis at 24 h after ischemia and by in situ hybridization at 2, 4, 8, 24, and 72 h after 15 min of global ischemia. Protein expression was studied using both immunocytochemistry at 4, 8, 16, 24, and 72 h after ischemia and Western blot analysis from tissue harvested at 16, 24, and 72 h after ischemia. Western blots showed that bcl-x-long is the predominant form of bcl-x protein expressed in both normal and ischemic brain. Both bcl-2 and bcl-x-long mRNA were expressed in CA1, CA3, and the molecular layer of the dentate after ischemia. However, bcl-2 and bcl-x protein were expressed only in CA3 and dentate. Thus, while bcl-2 and bcl-x-long mRNA were expressed in both surviving and dying neurons, their proteins were expressed in neurons destined to survive. These results support potential roles for these two apoptosis suppressor proteins in promoting survival after cerebral ischemia.

Expression of the apoptosis-effector gene, Bax, is up-regulated in vulnerable hippocampal CA1 neurons following global ischemia.

The observation that delayed death of CA1 neurons after global ischemia is inhibited by protein synthesis inhibitors suggests that the delayed death of these neurons is an active process that requires new gene expression. Delayed death in CA1 has some of the characteristics of apoptotic death; however, candidate proapoptotic proteins have not been identified in the CA1 after ischemia. We studied the expression of Bax protein and mRNA, a member of the bcl-2 family that is an effector of apoptotic cell death, after global ischemia in the four-vessel global ischemia model in the rat and compared these results with the expression of the antiapoptotic gene bcl-2. Bax mRNA and protein are both expressed in CA1 before delayed death, whereas bcl-2 protein is not expressed. Bcl-2 protein expression, but not that of Bax, is increased in CA3, a region that is ischemic but less susceptible to ischemic injury. In the dentate gyrus, both Bax and bcl-2 proteins are expressed. The selective expression of Bax in Ca1 supports the hypothesis that Bax could contribute to delayed neuronal death in these vulnerable neurons by an independent mechanism or by forming heterodimers with gene family members other than bcl-2.

Focal ischemia induces expression of the DNA damage-inducible gene GADD45 in the rat brain.

GADD45 is a DNA damage-inducible gene that accelerates DNA excision repair and can be induced by a variety of DNA-damaging stimuli in mammalian cells. We investigated the expression of GADD45 mRNA and protein using in situ hybridization and immunocytochemistry in rat brains after 2 h of temporal focal ischemia. The expression of GADD45 mRNA was induced in neurons throughout ischemic cortex 4 h after the onset of ischemia but was restricted to ischemic penumbra regions at 24 h after ischemia. The expression of GADD45 protein was increased only in sublethally injured neurons in the penumbra regions at both 4 h and 24 h following ischemia. These results suggest that GADD45 could have a protective role in ischemic neurons.

Expression of the proto-oncogene bcl-2 is increased in the rat brain following kainate-induced seizures.

The proto-oncogene bcl-2 is an important suppressor of apoptotic cell death in development and of both apoptotic and necrotic cell death in mature neurons. We studied expression of bcl-2 and the related gene, bax, which may promote cell death, after seizures induced by systemic kainate injection in rats. Expression of bcl-2 mRNA was studied by in situ hybridization. Bax and bcl-2 protein expression was studied by immunocytochemistry. Histologic analysis of cresyl violet-stained paraffin sections was performed at 72 h. bcl-2 protein was expressed in CA1 neurons, a region that is injured, yet survives after seizures. Bcl-2 mRNA was expressed in CA3, a region where there is extensive neuronal death at 72 h, but the bcl-2 protein was not translated. However, bax protein expression in CA3 was increased at 24 h. These results support a possible role for bcl-2 in promoting survival of CA3 after seizures.