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The BCL-2 family is centrally involved in the mechanism of cell death after cerebral ischemia. It is well known that the proteins of the BCL-2 family are key regulators of apoptosis through controlling mitochondrial outer membrane permeabilization. Recent findings suggest that many BCL-2 family members are also directly involved in controlling transmission of Ca 2+ from the endoplasmic reticulum (ER) to mitochondria through a specialization called the mitochondria-associated ER membrane (MAM). Increasing evidence supports the involvement of microRNAs (miRNAs), some of them targeting BCL-2 family proteins, in the regulation of cerebral ischemia. In this mini-review, after highlighting current knowledge about the multiple functions of BCL-2 family proteins and summarizing their relationship to outcome from cerebral ischemia, we focus on the regulation of BCL-2 family proteins by miRNAs, especially miR-29 which targets multiple BCL-2 family proteins. [ABSTRACT FROM AUTHOR]

It is commonly believed that sustained elevations in the mitochondrial matrix Ca2+ concentration are a major feature of the intracellular cascade of lethal events during cerebral ischemia. The physical association between the endoplasmic reticulum (ER) and mitochondria, known as the mitochondria-associated ER membrane (MAM), enables highly efficient transmission of Ca2+ from the ER tomitochondria under both physiological and pathological conditions.Molecular chaperones are well known for their protective effects during cerebral ischemia. It has been demonstrated recently that many molecular chaperones coexist with MAM and regulate the MAM and thus Ca2+ concentration inside mitochondria. Here, we review recent research on cerebral ischemia and MAM, with a focus on molecular chaperones and ER-mitochondrial calcium transfer. [ABSTRACT FROM AUTHOR]

Mitochondria are central to both apoptotic and necrotic cell death, as well as to normal physiological function. Astrocytes are crucial for neuronal metabolic, antioxidant, and trophic support, as well as normal synaptic function. In the setting of stress, such as during cerebral ischemia, astrocyte dysfunction may compromise the ability of neurons to survive. Despite their central importance, the response of astrocyte mitochondria to stress has not been extensively studied. Limited data already suggest clear differences in the response of neuronal and astrocytic mitochondria to oxygen–glucose deprivation (GD). Prominent mitochondrial alterations during stress that can contribute to cell death include changes in production of reactive oxygen species (ROS) and release of death regulatory and signaling molecules from the intermembrane space. In response to stress mitochondrial respiratory function and membrane potential also change, and these changes appear to depend on cell type. Bcl-2 family proteins are the best studied regulators of cell death, especially apoptosis, and mitochondria are a major site of action for these proteins. Although much data supports the role of Bcl-2 family proteins in the regulation of some of these mitochondrial alterations, this remains an area of active investigation. This mini-review summarizes current knowledge regarding mitochondrial control of cell survival and death in astrocytes and the effects of anti-apoptotic Bcl-2 proteins on astrocyte mitochondrial function. [Copyright &y& Elsevier]

Regulation of pH in the central nervous system is critical to normal brain function and response to pathophysiological conditions. Here we identify two novel variants of the sodium-driven chloride bicarbonate exchanger (NCBE) from brain. The developmental expression pattern seen by in situ hybridization for the 90-bp containing insert (insert A) reveals strong expression in spinal cord and brain beginning in embryonic development. High-level expression is seen in cerebellar Purkinje cells and principal cells in hippocampus. The variant missing a 39-bp insert at the 3′ end (insert B) encodes a protein in which the deduced carboxyterminal three amino acids are replaced with a unique 21 amino acid stretch terminating in a PDZ motif. rb2NCBE, the PDZ motif-encoding variant, is more highly expressed in astrocytes than is rb1NCBE. Both variants are expressed at similar levels in neurons. Expression varies with age and cell type. The FLAG epitope was fused in-frame at the amino terminus and each variant was expressed using a retroviral vector to study subcellular localization. Both variants were associated with the plasma membrane, but rb2NCBE colocalized with actin filaments to a greater extent, suggesting the PDZ form may interact with the cytoskeleton, whereas rb1NCBE was more often seen in intracellular vesicles. The PDZ motif-containing variant was much more active in pH regulation, with the expected ionic dependence on Na+, HCO3− and Cl− when expressed in 3T3 cells. These results are a first step towards understanding the regulation of expression and activity of this transporter in the brain. [ABSTRACT FROM AUTHOR]

Protection of hair cells by HSP70 released by supporting cells is reported by May et al. in this issue of the JCI. Their findings suggest a new way to reduce ototoxicity from therapeutic medications and raise larger questions about the role and integration of heat shock proteins in non–cell-autonomous responses to stress. Increasing evidence suggests an important role for extracellular heat shock proteins in both the nervous system and the immune system. The work also suggests that defective chaperones could cause ear disease and supports the potential use of chaperone therapeutics. [ABSTRACT FROM AUTHOR]

Protection of hair cells by HSP70 released by supporting cells is reported by May et al. in this issue of the JCI. Their findings suggest a new way to reduce ototoxicity from therapeutic medications and raise larger questions about the role and integration of heat shock proteins in non-cell-autonomous responses to stress. Increasing evidence suggests an important role for extracellular heat shock proteins in both the nervous system and the immune system. The work also suggests that defective chaperones could cause ear disease and supports the potential use of chaperone therapeutics. [ABSTRACT FROM AUTHOR]

Reports on the protection of neuronal cells in murine cortical cultures by glutamate antagonists from injury under acidic conditions typical of hypoxia-ischemia. Reduction of extracellular acidity; Reduction of neuronal death by N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-mthyl-4-isoxazolepropionate-kainate antagonism.

Background: Isoflurane induces cell death in neurons undergoing synaptogenesis via increased production of pro-brain-derived neurotrophic factor (proBDNF) and activation of postsynaptic p75 neurotrophin receptor (p75). Astrocytes express p75, but their role in neuronal p75-mediated cell death remains unclear. The authors investigated whether astrocytes have the capacity to buffer increases in proBDNF and protect against isoflurane/p75 neurotoxicity.Methods: Cell death was assessed in day in vitro (DIV) 7 mouse primary neuronal cultures alone or in co-culture with age-matched or DIV 21 astrocytes with propidium iodide 24 h after 1 h exposure to 2% isoflurane or recombinant proBDNF. Astrocyte-targeted knockdown of p75 in co-culture was achieved with small-interfering RNA and astrocyte-specific transfection reagent and verified with immunofluorescence microscopy. proBDNF levels were assessed by enzyme-linked immunosorbent assay. Each experiment used six to eight replicate cultures/condition and was repeated at least three times.Results: Exposure to isoflurane significantly (P < 0.05) increased neuronal cell death in primary neuronal cultures (1.5 ± 0.7 fold, mean ± SD) but not in co-culture with DIV 7 (1.0 ± 0.5 fold) or DIV 21 astrocytes (1.2 ± 1.2 fold). Exogenous proBDNF dose dependently induced neuronal cell death in both primary neuronal and co-cultures, an effect enhanced by astrocyte p75 inhibition. Astrocyte-targeted p75 knockdown in co-cultures increased media proBDNF (1.2 ± 0.1 fold) and augmented isoflurane-induced neuronal cell death (3.8 ± 3.1 fold).Conclusions: The presence of astrocytes provides protection to growing neurons by buffering increased levels of proBDNF induced by isoflurane. These findings may hold clinical significance for the neonatal and injured brain where increased levels of proBDNF impair neurogenesis. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA), miR-181a, is enriched in the brain, and inhibition of miR-181a reduced astrocyte death in vitro and infarct volume after stroke in vivo. This study investigated the role of miR-181a in neuronal injury in vitro and hippocampal neuronal loss in vivo after forebrain ischemia. miR-181a levels were altered by transfection with mimic or antagomir. N2a cells subjected to serum deprivation and oxidative stress showed less cell death when miR-181a was reduced and increased death when miR-181a increased; protection was associated with increased Bcl-2 protein. In contrast, transfected primary neurons did not show altered levels of cell death when miR-181a levels changed. Naive male rats and rats stereotactically infused with miR-181a antagomir or control were subjected to forebrain ischemia and cornus ammonis (CA)1 neuronal survival and protein levels were assessed. Forebrain ischemia increased miR-181a expression and decreased Bcl-2 protein in the hippocampal CA1 region. miR-181a antagomir reduced miR-181a levels, reduced CA1 neuronal loss, increased Bcl-2 protein, and significantly prevented the decrease of glutamate transporter 1. Thus, miR-181a antagomir reduced evidence of astrocyte dysfunction and increased CA1 neuronal survival. miR-181a inhibition is thus a potential target in the setting of forebrain or global cerebral ischemia as well as focal ischemia. [ABSTRACT FROM AUTHOR]

Abstract: The Na+-driven exchanger (NCBE) plays an important role in the regulation of intracellular pH (pHi). We previously identified two variants of NCBE from rat brain of which the variant with a carboxyterminal PSD-95/Dlg/ZO-1 (PDZ) motif (rb2NCBE) colocalized with the actin cytoskeleton. Increased rb2NCBE activity by PKA inhibition and reduction by forskolin and cAMP agonist suggest PKA regulation of NCBE. Disruption of actin filaments also decreased rb2NCBE activity. EBP50 and FLAG-rb2NCBE were reciprocally co-immunoprecipitated from rb2NCBE transfected cells. It is concluded that NCBE activity is inhibited by PKA and depends on the integrity of the actin cytoskeleton within a multiprotein complex at the plasma membrane. [Copyright &y& Elsevier]

In cytoplasm, nuclear factor-κB (NF-κB) is associated with the inhibitory protein, IκBα. On activation by H2O2, IκBα is phosphorylated and degraded, exposing the nuclear localization signals on the NF-κB heterodimer. Cyclooxygenase-2 (COX-2), which mediates prostaglandin synthesis during inflammation, is induced by oxidative stress mediated by NF-κB. We investigated whether the NF-κB signaling pathway affected cell death and COX-2 expression after hypoxia-induced oxidative stress in wild-type (WT) and copper/zinc-superoxide dismutase transgenic (SOD1 Tg) astrocytes. In WT astrocytes, phospho-IκBα was highly expressed after oxygen–glucose deprivation (OGD) and 2 h of reperfusion, concomitant with the decrease in IκBα. The NF-κB p50 level increased similarly in WT and SOD1 Tg astrocytes (1.2-/1.4-fold) after OGD. Electrophoretic mobility shift assay showed higher DNA-binding activity of NF-κB p50 in WT than in SOD1 Tg astrocytes 6 h after 4 h of OGD. The COX-2 level was induced by 2.7- and 1.3-fold after OGD in WT and SOD1 Tg astrocytes, and an antioxidant protected both groups against OGD injury. Superoxide dismutase transgenic cells were 23% more protective against OGD injury than WTs when assessed by lactate dehydrogenase release. However, transfection of NF-κB small interfering RNAs in SOD1 Tg astrocytes aggravated cell death and increased COX-2 expression. These results suggest that the NF-κB signaling pathway induced COX-2 expression and promoted cell death in WTs after OGD injury; however, NF-κB activation protected cells and decreased COX-2 expression in SOD1 Tg astrocytes. This biphasic role of NF-κB might be coordinately regulated by reactive oxygen species levels in astrocytes, thereby functioning as a regulator of cell death/survival.Journal of Cerebral Blood Flow & Metabolism (2006) 26, 1076–1088. doi:10.1038/sj.jcbfm.9600261; published online 14 December 2005 [ABSTRACT FROM AUTHOR]

Abstract: Our prior work demonstrated that geldanamycin (GA) reduced injury due to oxygen-glucose deprivation (OGD) in primary astrocyte cultures. Using medium with an ionic composition similar to that observed during in vivo global ischemia, the selectivity and temporal profile of CA1 neuronal damage seen in vivo was mimicked with OGD in mouse hippocampal organotypic slice cultures. The present study tested the ability of GA to reduce delayed neuronal death in such cultures. Treating organotypic cultures with 100nM GA for 24h prior to OGD induced Hsp70 and significantly reduced CA1 neuronal damage. Staining with ubiquitin to identify protein aggregates revealed reduced redistribution of ubiquitin, consistent with reduced protein aggregation likely due at least in part to induction of Hsp70 by GA. [Copyright &y& Elsevier]

Abstract: Overexpression of copper/zinc superoxide dismutase (SOD1) in transgenic mice protects from transient focal cerebral ischemia in adult animals, but increases oxidative injury in perinatal mice. The effect of SOD1 overexpression on astrocytes subjected to ischemia-like insults has not yet been determined. Overexpression of human SOD1 in astrocytes resulted in a 3-fold increase in SOD1 activity without coupled up-regulation of catalase or glutathione peroxidase activities. Cells subjected to oxygen-glucose deprivation (OGD) or glucose deprivation to mimic ischemic injury were protected by SOD1 overexpression. OGD injury was reduced 47.6±9.3%, assessed by release of lactate dehydrogenase. OGD also caused a significant increase in catalase activity which was moderated by SOD1 overexpression. The level of glutathione in astrocytes overexpressing SOD1 was maintained at higher levels following 5 h OGD compared to control cultures under the same conditions. Reduction of glutathione prior to OGD significantly increased cell death of SOD1-overexpressing astrocytes as well as controls, but SOD1 still provided significant protection, suggesting that both GSH-dependent scavenging and GSH-independent scavenging are relevant to SOD1 protection in astrocytes. [Copyright &y& Elsevier]

Many studies have demonstrated the protective effects of Bcl-xL against both apoptotic and necrotic cell death, but the mode of action of Bcl-xL remains unclear. This work analyzed effects of Bcl-xL overexpression on cellular levels of reactive oxygen species (ROS), intracellular calcium ([Ca2+]i), and mitochondrial membrane potential (ΔΨm) in cultured mouse primary astrocytes after exposure to glucose deprivation (GD) or hydrogen peroxide (H2O2). Upon exposure to GD or H2O2, uninfected and Lac-Z-expressing astrocytes showed an immediate, rapid increase in ROS accumulation that was slowed and or reduced by Bcl-xL. Changes in ΔΨm in response to the two insults differed. H2O2 induced a decrease in ΔΨm that was initially greater in Bcl-xL cells, but then held stable. ΔΨm in control and Lac-Z-expressing cells initially declined more slowly, but after about 20 min showed rapid deterioration. Five hours of GD caused mitochondrial membrane hyperpolarization followed by a decrease in ΔΨm, which was not observed with Bcl-xL overexpression. Bcl-xL failed to inhibit the calcium dysregulation seen in control cells exposed to 400 μM H2O2, but still improved cell survival. There was no increase in [Ca2+]i with 5 h of GD. These data thus dissociate the effect of Bcl-xL on calcium homeostasis from effects on ROS, ΔΨm, and for H2O2 exposure, cell survival. [Copyright &y& Elsevier]

• Pre-treatment with anti-miR-338 reduced stroke severity and improved neurobehavior. • Silencing miR-338 in vivo and in vitro augmented COX4I1 and improved cell survival. • MiR-338 inhibition preserved ATP and cytochrome c oxidase in astrocytes and neurons. Brain-enriched microRNA-338 (miR-338) is known to play a central role in brain mitochondrial function, however the role of miR-338 in stroke injury remains unknown. This study investigated the role of miR-338 in injury from transient focal cerebral ischemia in mice, and in cell survival and mitochondrial function after in vitro ischemia in astrocyte and neuronal cultures. Pre-treatment of mice with intracerebroventricular injection of miR-338 antagomir 24 h prior to 1 h of middle cerebral artery occlusion (MCAO) significantly reduced infarct size and improved neurological score at both 24 h and 7d after injury. Levels of the miR-338 target cytochrome-c oxidase subunit 4I1 (COX4I1), which plays an essential role in maintaining brain mitochondrial ATP production, were increased in miR-338 antagomir-treated mice. Mouse primary astrocyte cell cultures subjected to glucose deprivation exhibited increased cell survival when pre-treated with miR-338 inhibitor, and greater cell death with miR-338 mimic. Decreased miR-338 levels were associated with increased ATP production, augmented cytochrome c oxidative (CcO) activity and preservation of COX4I1. In vitro protection with miR-338 inhibitor was blocked by concurrent knockdown of COX4I1 with small interfering RNA. Parallel studies in mouse neuronal N2a cultures resulted in preserved ATP content and CcO activity with miR-338 inhibition, indicating a shared miR-338-dependent response to ischemic stress between brain cell types. These results suggest that miR-338 inhibition and/or COX4I1-targeted therapies may be novel clinical strategies to protect against stroke injury via preservation of mitochondrial function in multiple cell types. [ABSTRACT FROM AUTHOR]

Whether the effect of miR-181a is sexually dimorphic in stroke is unknown. Prior work showed protection of male mice with miR-181a inhibition. Estrogen receptor-α (ERα) is an identified target of miR181 in endometrium. Therefore we investigated the separate and joint effects of miR-181a inhibition and 17β-estradiol (E2) replacement after ovariectomy. Adult female mice were ovariectomized and implanted with an E2- or vehicle-containing capsule for 14d prior to 1 h middle cerebral artery occlusion (MCAO). Each group received either miR-181a antagomir or mismatch control by intracerebroventricular injection 24 h before MCAO. After MCAO neurologic deficit and infarct volume were assessed. Primary male and female astrocyte cultures were subjected to glucose deprivation with miR-181a inhibitor or transfection control, and E2 or vehicle control, with/without ESRα knockdown with small interfering RNA. Cell death was assessed by propidium iodide staining, and lactate dehydrogenase assay. A miR-181a/ERα target site blocker (TSB), with/without miR-181a mimic, was used to confirm targeting of ERα by miR-181a in astrocytes. Individually, miR-181a inhibition or E2 decreased infarct volume and improved neurologic score in female mice, and protected male and female astrocyte cultures. Combined miR-181a inhibition plus E2 afforded greater protection of female mice and female astrocyte cultures, but not in male astrocyte cultures. MiR-181a inhibition only increased ERα levels in vivo and in female cultures, while ERα knockdown with siRNA increased cell death in both sexes. Treatment with ERα TSB was strongly protective in both sexes. In conclusion, the results of the present study suggest miR-181a inhibition enhances E2-mediated stroke protection in females in part by augmenting ERα production, a mechanism detected in female mice and female astrocytes. Sex differences were observed with combined miR-181a inhibition/E2 treatment, and miR-181a targeting of ERα. [ABSTRACT FROM AUTHOR]

Neurogenesis is essential to brain development and plays a central role in the response to brain injury. Stroke and head trauma stimulate proliferation of endogenous neural stem cells (NSCs); however, the survival of young neurons is sharply reduced by postinjury inflammation. Cellular mitochondria are critical to successful neurogenesis and are a major target of inflammatory injury. Mitochondrial protection was shown to improve survival of young neurons. This study tested whether reducing cellular microRNA-210 (miR-210) would enhance mitochondrial function and improve survival of young murine neurons under inflammatory conditions. Several studies have demonstrated the potential of miR-210 inhibition to enhance and protect mitochondrial function through upregulation of mitochondrial proteins. Here, miR-210 inhibition significantly increased neuronal survival and protected the activity of mitochondrial enzymes cytochrome c oxidase and aconitase in differentiating NSC cultures exposed to inflammatory mediators. Unexpectedly, we found that reducing miR-210 significantly attenuated NSC proliferation upon induction of differentiation. Further investigation revealed that increased mitochondrial function suppressed the shift to primarily glycolytic metabolism and reduced mitochondrial length characteristic of dividing cells. Activation of AMP-regulated protein kinase--retinoblastoma signaling is important in NSC proliferation and the reduction of this activation observed by miR-210 inhibition is one mechanism contributing to the reduced proliferation. Postinjury neurogenesis occurs as a burst of proliferation that peaks in days, followed by migration and differentiation over weeks. Our studies suggest that mitochondrial protective miR-210 inhibition should be delayed until after the initial burst of proliferation, but could be beneficial during the prolonged differentiation stage. [ABSTRACT FROM AUTHOR]

Neurons in the cornu ammonis 1 (CA1) region of the hippocampus are vulnerable to cerebral ischemia, while dentate gyrus (DG) neurons are more resistant. This effect is mediated by local astrocytes, and may reflect differences in subregional hippocampal expression of miR-29a. We investigated the role of miR-29a on survival of hippocampal astrocytes cultured selectively from CA1 and DG in response to glucose deprivation (GD). CA1 astrocytes exhibited more cell death and a greater decrease in miR-29a than DG astrocytes. A reciprocal change was observed in the mitochondrial voltage dependent cation channel-1 (VDAC1), a regulator of mitochondria and target of miR-29a. In CA1 astrocytes, increasing miR-29a decreased VDAC1 and improved cell survival, while knockdown of VDAC1 improved survival. Finally, the protective effect of miR-29a was eliminated by inhibition of miR-29a/VDAC1 binding. These findings suggest that the selective vulnerability of the CA1 to injury may be due in part to a limited miR-29a response in CA1 astrocytes, allowing a greater increase in VDAC1-mediated cellular dysfunction in CA1 astrocytes. [ABSTRACT FROM AUTHOR]

miR-181 has deleterious effects on stroke outcome, and reducing miR-181a levels prior to middle cerebral artery occlusion (MCAO) was shown previously to be protective. Here we tested the effect of post-ischemic treatment with miR-181a antagomir by intracerebroventricular and intravenous routes of administration on infarct size, neurological outcome, inflammatory response and long term behavioral outcome. Post-treatment with miR-181a antagomir significantly reduced infarction size, improved neurological deficits and reduced NF-κB activation, numbers of infiltrating leukocytes and levels of Iba1. Targets affected by miR-181a antagomir administered after stroke onset include BCL2 and X-linked inhibitor of apoptosis protein (XIAP). Post-treatment with miR-181a antagomir significantly improved behavioral outcome assessed by rotarod at one month. These findings indicate that post-treatment with miR-181a antagomir has neuroprotective effects against ischemic neuronal damage and neurological impairment in mice, and the protection is long lasting including recovery of motor function and coordination over one month. The ability to protect the brain with post-treatment with miR-181a antagomir with long lasting effect makes this a promising therapeutic target and may be an innovative and effective new approach for stroke therapy. [ABSTRACT FROM AUTHOR]

Significance: Cerebral ischemia is a major cause of death and disability throughout the world, yet therapeutic options remain limited. The interplay between the cellular redox state and the immune response plays a critical role in determining the extent of neural cell injury after ischemia and reperfusion. Excessive amounts of reactive oxygen species (ROS) generated by mitochondria and other sources act both as triggers and effectors of inflammation. This review will focus on the interplay between these two mechanisms. Recent Advances: MicroRNAs (miRNAs) are important post-transcriptional regulators that interact with multiple target messenger RNAs coordinately regulating target genes, including those involved in controlling mitochondrial function, redox state, and inflammatory pathways. This review will focus on the regulation of mitochondria, ROS, and inflammation by miRNAs in the chain of deleterious intra- and intercellular events that lead to brain cell death after cerebral ischemia. Critical Issues: Although pretreatment using miRNAs was effective in cerebral ischemia in rodents, testing treatment after the onset of ischemia is an essential next step in the development of acute stroke treatment. In addition, miRNA formulation and delivery into the CNS remain a challenge in the clinical translation of miRNA therapy. Future Directions: Future research should focus on post-treatment and potential clinical use of miRNAs. Antioxid. Redox Signal. 22, 187-202. [ABSTRACT FROM AUTHOR]

Highlights: [•] Astrocytes and miRNAs are key players in cerebral ischemia. [•] Some miRNAs are astrocyte-enriched. [•] Targeting astrocyte-enriched miRNAs protects neurons from ischemia. [Copyright &y& Elsevier]

Overexpression of the inducible heat shock protein 70, Hsp72, has broadly cytoprotective effects and improves outcome following stroke. A full understanding of how Hsp72 protects cells against injury is elusive, though several distinct mechanisms are implicated. One mechanism is its anti-inflammatory effects. We study the effects of Hsp72 overexpression on activation of the transcription factor NF-κB in microglia combining experimentation and mathematical modeling, using TNFα to stimulate a microglial cell line stably overexpressing Hsp72. We find that Hsp72 overexpression reduces the amount of NF-κB DNA binding activity, activity of the upstream kinase IKK, and amount of IκBα inhibitor phosphorylated following TNFα application. Simulations evaluating several proposed mechanisms suggest that inhibition of IKK activation is an essential component of its regulatory activities. Unexpectedly we find that Hsp72 overexpression reduces the initial amount of the RelA/p65 NF-κB subunit in cells, contributing to the attenuated response. Neither mechanism in isolation, however, is sufficient to attenuate the response, providing evidence that Hsp72 relies upon multiple mechanisms to attenuate NF-κB activation. An additional observation from our study is that the induced expression of IκBα is altered significantly in Hsp72 expressing cells. While the mechanism responsible for this observation is not known, it points to yet another means by which Hsp72 may alter the NF-κB response. This study illustrates the multi-faceted nature of Hsp72 regulation of NF-κB activation in microglia and offers further clues to a novel mechanism by which Hsp72 may protect cells against injury. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies suggest a link between mitochondria and proinflammatory cytokine generation. We previously demonstrated that overexpression of mitochondrial chaperone glucose-regulated protein75 (Grp75/mortalin) protects mitochondria. In this study we investigated the modulation of the lipopolisaccharide (LPS)-induced inflammatory response of microglial BV-2 cells by Grp75. We demonstrate that LPS-induced activation promotes significant metabolic changes suppressing mitochondrial function and increasing glycolysis. Overexpression of Grp75 attenuates the LPS-induced oxidative and metabolic responses, and suppresses proinflammatory activation, which depends on both NF-κB activation and lactate. Thus overexpression of Grp75 provides a novel strategy to modulate proinflammatory cytokine production of relevance to inflammation-associated pathologies. [Copyright &y& Elsevier]

Abstract: Astrocyte activation is a hallmark of the response to brain ischemia consisting of changes in gene expression and morphology. Heat shock protein 72 (Hsp72) protects from cerebral ischemia, and although several protective mechanisms have been investigated, effects on astrocyte activation have not been studied. To identify potential mechanisms of protection, microarray analysis was used to assess gene expression in the ischemic hemispheres of wild-type (WT) and Hsp72-overexpressing (Hsp72Tg) mice 24h after middle cerebral artery occlusion or sham surgery. After stroke both genotypes exhibited changes in genes related to apoptosis, inflammation, and stress, with more downregulated genes in Hsp72Tg and more inflammation-related genes increased in WT mice. Genes indicative of astrocyte activation were also upregulated in both genotypes. To measure the extent and time course of astrocyte activation after stroke, detailed histological and morphological analyses were performed in the cortical penumbra. We observed a marked and persistent increase in glial fibrillary acidic protein (GFAP) and a transient increase in vimentin. No change in overall astrocyte number was observed based on glutamine synthetase immunoreactivity. Hsp72Tg and WT mice were compared for density of astrocytes expressing activation markers and astrocytic morphology. In animals with comparable infarct size, overexpression of Hsp72 reduced the density of GFAP- and vimentin-expressing cells, and decreased astrocyte morphological complexity 72h following stroke. However, by 30days astrocyte activation was similar between genotypes. These data indicate that early modulation of astrocyte activation provides an additional novel mechanism associated with Hsp72 overexpression in the setting of ischemia. [Copyright &y& Elsevier]

Reactive astrogliosis is characterized by a profound change in astrocyte phenotype in response to all CNS injuries and diseases. To better understand the reactive astrocyte state, we used Affymetrix GeneChip arrays to profile gene expression in populations of reactive astrocytes isolated at various time points after induction using two mouse injury models, ischemic stroke and neuroinflammation. We find reactive gliosis consists of a rapid, but quickly attenuated, induction of gene expression after insult and identify induced Lcn2 and Serpina3n as strong markers of reactive astrocytes. Strikingly, reactive astrocyte phenotype strongly depended on the type of inducing injury. Although there is a core set of genes that is upregulated in reactive astrocytes from both injury models, at least 50% of the altered gene expression is specific to a given injury type. Reactive astrocytes in ischemia exhibited a molecular phenotype that suggests that they may be beneficial or protective, whereas reactive astrocytes induced by LPS exhibited a phenotype that suggests that they may be detrimental. These findings demonstrate that, despite well established commonalities, astrocyte reactive gliosis is a highly heterogeneous state in which astrocyte activities are altered to respond to the specific injury. This raises the question ofhowmanysubtypes of reactive astrocytes exist. Our findings provide transcriptome databases for two subtypes of reactive astrocytes that will be highly useful in generatingnewand testable hypotheses of their function, as well as for providingnew markers to detect different types of reactive astrocytes in human neurological diseases. [ABSTRACT FROM AUTHOR]

Summary: Recovery from stroke engages mechanisms of neural plasticity. Here we examine a role for MHC class I (MHCI) H2-Kb and H2-Db, as well as PirB receptor. These molecules restrict synaptic plasticity and motor learning in the healthy brain. Stroke elevates neuronal expression not only of H2-Kb and H2-Db, but also of PirB and downstream signaling. KbDb knockout (KO) or PirB KO mice have smaller infarcts and enhanced motor recovery. KO hippocampal organotypic slices, which lack an intact peripheral immune response, have less cell death after in vitro ischemia. In PirB KO mice, corticospinal projections from the motor cortex are enhanced, and the reactive astrocytic response is dampened after MCAO. Thus, molecules that function in the immune system act not only to limit synaptic plasticity in healthy neurons, but also to exacerbate brain injury after ischemia. These results suggest therapies for stroke by targeting MHCI and PirB. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondria are central to the execution of apoptosis, and the Bcl-2 protein family of pro- and anti-apoptotic proteins interacts with mitochondria to regulate apoptosis. Using bioinformatics we predicted that miR-181, a microRNA expressed in brain, could target the 3′UTRs of Bcl-2 family members Bcl-2-L11/Bim, Mcl-1, and Bcl-2. Using the luciferase reporter assay we confirmed these targets. We used mimic and inhibitor to alter miR-181a levels in primary astrocyte cultures and found miR-181a reduction was associated with increased Bcl-2 and Mcl-1 protein levels. Decreased miR-181a levels reduced glucose deprivation induced apoptosis, mitochondrial dysfunction, and loss of mitochondrial membrane potential in astrocytes. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNA) are short (~22nt) single stranded RNAs that downregulate gene expression. Although recent studies indicate extensive miRNA changes in response to ischemic brain injury, there is currently little information on the roles of specific miRNAs in this setting. Heat shock proteins (HSP) of the HSP70 family have been extensively studied for their multiple roles in cellular protection, but there is little information on their regulation by miRNAs. We used bioinformatics to identify miR-181 as a possible regulator of several HSP70 family members. We validated GRP78/BIP as a target by dual luciferase assay. In response to stroke in the mouse we find that miR-181 increases in the core, where cells die, but decreases in the penumbra, where cells survive. Increased levels of miR-181a are associated with decreased GRP78 protein levels, but increased levels of mRNA, implicating translational arrest. We manipulated levels of miR-181a using plasmid overexpression of pri-miR-181ab or mimic to increase, and antagomir or inhibitor to reduce levels. Increased miR-181a exacerbated injury both in vitro and in the mouse stroke model. Conversely, reduced levels were associated with reduced injury and increased GRP78 protein levels. Studies in C6 cells show that if GRP78 levels are maintained miR-181a no longer exerts a toxic effect. These data demonstrate that miR-181 levels change in response to stroke and inversely correlate with levels of GRP78. Importantly, reducing or blocking miR-181a protects the brain from stroke. [Copyright &y& Elsevier]

Reactive gliosis is a hallmark of brain pathology and the injury response, yet the extent to which astrocytes proliferate, and whether this is central to astrogliosis is still controversial. We determined the fraction of mature astrocytes that proliferate in a mouse stroke model using unbiased stereology as a function of distance from the infarct edge. Cumulatively 11.1±1.2% of Aldh1l1+ astrocytes within 400 ¼m in the cortical penumbra incorporate BrdU in the first week following stroke, while the overall number of astrocytes does not change. The number of astrocytes proliferating fell sharply with distance with more than half of all proliferating astrocytes found within 100 ¼m of the edge of the infarct. Despite extensive cell proliferation primarily of microglia and neutrophils/monocytes in the week following stroke, few mature astrocytes re-enter cell cycle, and these are concentrated close to the infarct boundary. [ABSTRACT FROM AUTHOR]

Objective: Reperfusion after stroke leads to infiltration of inflammatory cells into the ischemic brain. Nicotinamide adenine dinucleotide phosphate oxidase (NOX2) is a major enzyme system that generates superoxide in immune cells. We studied the effect of NOX2 derived from the immune cells in the brain and in blood cells in experimental stroke.Methods: To establish whether NOX2 plays a role in brain ischemia, strokes were created in mice, then mice were treated with the NOX2 inhibitor apocynin or vehicle and compared to mice deficient in NOX2's gp91 subunit and their wild-type littermates. To determine whether NOX2 in circulating cells versus brain resident cells contribute to ischemic injury, bone marrow chimeras were generated by transplanting bone marrow from wild-type or NOX2-deficient mice into NOX2 or wild-type hosts, respectively.Results: Apocynin and NOX2 deletion both significantly reduced infarct size, blood-brain barrier disruption, and hemorrhagic transformation of the infarcts, compared to untreated wild-type controls. This was associated with decreased matrix metalloproteinase 9 expression and reduced loss of tight junction proteins. NOX2-deficient mice receiving wild-type marrow had better outcomes compared to the wild-type mice receiving wild-type marrow. Interestingly, wild-type mice receiving NOX2-deficient marrow had even smaller infarct sizes and less hemorrhage than NOX2-deficient mice receiving wild-type marrow.Interpretation: This indicates that NOX2, whether present in circulating cells or brain resident cells, contributes to ischemic brain injury and hemorrhage. However, NOX2 from the circulating cells contributed more to the exacerbation of stroke than that from brain resident cells. These data suggest the importance of targeting the peripheral immune system for treatment of stroke. [ABSTRACT FROM AUTHOR]

Background: Problems with learning and memory are common after surgery in the elderly and are associated with high morbidity. Heat shock protein 72 (Hsp72) confers neuroprotection against acute neurologic injury. We hypothesized that overexpression of Hsp72 would prevent the development of postoperative memory loss.Methods: C57BL/6 wild-type and Hsp72 overexpressing transgenic mice were randomly allocated to the following: control, isoflurane anesthesia alone, or tibial fracture during isoflurane anesthesia. Animals were trained 24 h before surgery using a fear conditioning protocol and assessed in their training environment and in a novel context on posttreatment days 1, 3, and 7. Microglial activation was assessed by immunostaining.Results: Adult male C57BL/6 wild-type mice exhibited reduced memory evidenced by a decreased percentage freezing time on days 1 and 3 after anesthesia alone (58.8 ± 5, 46.5 ± 5 mean ± SEM) and after surgery (53.4 ± 6, 44.1 ± 7), compared with controls (78.8 ± 5, 63.4 ± 6; P < 0.05 and P < 0.001, respectively). Hsp72 mice showed no difference by treatment on any day. Similarly, nonhippocampal-dependent memory was significantly impaired on days 1 and 3 after surgery and day 3 after anesthesia. The genotype effect was significant on days 1 and 7. CD68-immunopositive activated microglia in the hippocampus varied modestly with subregion and time; on day 7, there was a significant treatment effect with no genotype effect, with more activated microglia after surgery in all regions.Conclusion: Hsp72 overexpression is associated with prevention of postoperative hippocampal-dependent and -independent memory deficit induced by anesthesia and/or surgery. Memory deficit is not correlated with numbers of activated hippocampal microglia. [ABSTRACT FROM AUTHOR]

Abstract: Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria at contact sites between the organelles can induce mitochondrial dysfunction and programmed cell death after stress. The ER-localized chaperone glucose-regulated protein 78kDa (GRP78/BiP) protects neurons against excitotoxicity and apoptosis. Here we show that overexpressing GRP78 protects astrocytes against ischemic injury, reduces net flux of Ca2+ from ER to mitochondria, increases Ca2+ uptake capacity in isolated mitochondria, reduces free radical production, and preserves respiratory activity and mitochondrial membrane potential after stress. We conclude that GRP78 influences ER-mitochondrial Ca2+ crosstalk to maintain mitochondrial function and protect astrocytes from ischemic injury. [Copyright &y& Elsevier]

The impairment of hippocampal neurogenesis has been linked to the pathogenesis of neurological disorders from chronic neurodegenerative disease to the progressive cognitive impairment of children who receive brain irradiation. Numerous studies provide evidence that inflammation downregulates neurogenesis, with multiple factors contributing to this impairment. Although mitochondria are one of the primary targets of inflammatory injury, the role of mitochondrial function in the modulation of neurogenesis remains relatively unstudied. In this study, we used neurosphere-derived cells to show that immature doublecortin (Dcx)-positive neurons are uniquely sensitive to mitochondrial inhibition, demonstrating rapid loss of mitochondrial potential and cell viability compared with glial cells and more mature neurons. Mitochondrial inhibition for 24 h produced no significant changes in astrocyte or oligodendrocyte viability, but reduced viability of mature neurons by 30%, and reduced survival of Dcx+ cells by 60%. We demonstrate that protection of mitochondrial function with mitochondrial metabolites or the mitochondrial chaperone mtHsp75/mortalin partially reverses the inflammation associated impairment of neurogenesis in vitro and in irradiated mice in vivo. Our findings highlight mitochondrial mechanisms involved in neurogenesis and indicate mitochondria as a potential target for protective strategies to prevent the impairment of neurogenesis by inflammation. [ABSTRACT FROM AUTHOR]

Mitochondria are known to be central to the cell's response to ischemia, because of their role in energy generation, in free radical generation, and in the regulation of apoptosis. Heat shock protein 75 (Hsp75/Grp75/mortalin/TRAP1) is a member of the HSP70 chaperone family, which is targeted to mitochondria. Overexpression of Hsp75 was achieved in rat brain by DNA transfection, and expression was observed in both astrocytes and neurons. Rats were subjected to 100 mins middle cerebral artery occlusion followed by assessment of infarct volume, neurological score, mitochondrial function, and levels of oxidative stress at 24 h reperfusion. Overexpression of Hsp75 reduced infarct area from 44.6%±21.1% to 25.7%±12.1% and improved neurological outcome significantly. This was associated with improved mitochondrial function as shown by protection of complex IV activity, marked reduction of free radical generation detected by hydroethidine fluorescence, reduction of lipid peroxidation detected by 4-hydroxy-2-nonenol immunoreactivity, and increased preservation of ATP levels. This suggests that targeting mitochondria for protection may be a useful strategy to reduce ischemic brain injury.Journal of Cerebral Blood Flow & Metabolism (2009) 29, 365–374; doi:10.1038/jcbfm.2008.125; published online 5 November 2008 [ABSTRACT FROM AUTHOR]

Abstract: We previously showed that hypothermia attenuates inflammation in focal cerebral ischemia (FCI) by suppressing activating kinases of nuclear factor-kappa B (NFκB). Here we characterize the inflammatory response in global cerebral ischemia (GCI), and the influence of mild hypothermia. Rodents were subjected to GCI by bilateral carotid artery occlusion. The inflammatory response was accompanied by microglial activation, but not neutrophil infiltration, or blood brain barrier disruption. Mild hypothermia reduced CA1 damage, decreased microglial activation and decreased nuclear NFκB translocation and activation. Similar anti-inflammatory effects of hypothermia were observed in a model of pure brain inflammation that does not cause brain cell death. Primary microglial cultures subjected to oxygen glucose deprivation (OGD) or stimulated with LPS under hypothermic conditions also experienced less activation and less NFκB translocation. However, NFκB regulatory proteins were not affected by hypothermia. The inflammatory response following GCI and hypothermia''s anti-inflammatory mechanism is different from that observed in FCI. [Copyright &y& Elsevier]

Although heat shock proteins have been studied for decades, new intracellular and extracellular functions in a variety of diseases continue to be discovered. Heat shock proteins function within networks of interacting proteins; they can alter cellular physiology rapidly in response to stress without requiring new protein synthesis. This review focuses on the heat shock protein 70 family and considers especially the functions of the inducible member, heat shock protein 72, in the setting of cerebral ischemia. In general, inhibiting apoptotic signaling at multiple points and up-regulating survival signaling, heat shock protein 70 has a net prosurvival effect. Heat shock protein 70 has both antiinflammatory and proinflammatory effects depending on the cell type, context, and intracellular or extracellular location. Intracellular effects are often antiinflammatory with inhibition of nuclear factor-kappaB signaling. Extracellular effects can lead to inflammatory cytokine production or induction of regulatory immune cells and reduced inflammation. [ABSTRACT FROM AUTHOR]

Mitochondrial heat shock protein 70 (mtHsp70/Hsp75/Grp75/mortalin/TRAP-1/PBP74) is an essential mitochondrial chaperone and a member of the heat shock protein 70 (HSP70) family. Although many studies have shown the protective properties of overexpression of the cytosolic inducible member of the HSP70 family, Hsp72, few studies have investigated the protective potential of Hsp75 against ischemic injury. Mitochondria are one of the primary targets of ischemic injury in astrocytes. In this study, we analyzed the effects of Hsp75 overexpression on cellular levels of reactive oxygen species (ROS), mitochondrial membrane potential, ATP levels, and viability during the ischemia-like conditions of oxygen–glucose deprivation (OGD) or glucose deprivation (GD) in primary astrocytic cultures. We show that Hsp75 overexpression decreases ROS production and preserves mitochondrial membrane potential during GD, and preserves ATP levels and cell viability during OGD. These findings indicate that Hsp75 can provide protection against ischemia-like in vitro injury and suggest that it should be further studied as a potential candidate for protection against ischemic injury.Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1009–1016; doi:10.1038/sj.jcbfm.9600600; published online 19 December 2007 [ABSTRACT FROM AUTHOR]

Much evidence suggests that astrocytes protect neurons against ischemic injury. Although astrocytes are more resistant to some insults than neurons, few studies offer insight into the real time changes of astrocytic protective functions with stress. Mitochondria are one of the primary targets of ischemic injury in astrocytes. We investigated the time course of changes in astrocytic ATP levels, plasma membrane potential, and glutamate uptake, a key protective function, induced by mitochondrial inhibition. Our results show that significant functional change precedes reduction in astrocytic viability with mitochondrial inhibition. Using the mitochondrial inhibitor fluorocitrate (FC, 0.25 mmol/L) that is preferentially taken by astrocytes we found that inhibition of astrocyte mitochondria increased vulnerability of co-cultured neurons to glutamate toxicity. In our studies, the rates of FC-induced astrocytic mitochondrial depolarization were accelerated in mixed astrocyte/neuron cultures. We hypothesized that the more rapid mitochondrial depolarization was promoted by an additional energetic demand imposed be the co-cultured neurons. To test this hypothesis, we exposed pure astrocytic cultures to 0.01–1 mmol/L aspartate as a metabolic load. Aspartate application accelerated the rates of FC-induced mitochondrial depolarization, and, at 1 mmol/L, induced astrocytic death, suggesting that strong energetic demands during ischemia can compromise astrocytic function and viability. [ABSTRACT FROM AUTHOR]

Abstract: Both nitric oxide and asymmetric dimethylarginine (ADMA) play a critical role in the regulation of cerebral blood flow, though their neuroprotective and cytotoxic effects are still under investigation. In this study, we found that nitrate/nitrite (NO x ) levels in plasma, ischemic brain tissue, and cerebrospinal fluid (CSF) increased significantly 24h after 2h transient middle cerebral artery occlusion (MCAO) in rats. ADMA levels were unchanged in plasma, but decreased significantly in CSF 24h following MCAO. The CSF ADMA/NO x ratio decreased markedly following ischemia. Rats protected by expression of the chaperonin GroEL or its folding deficient mutant D87K had lower plasma NO x levels at 24h reperfusion. ADMA, NO, and their ratio in CSF merit further study as biomarkers for ischemic brain injury. [Copyright &y& Elsevier]

Transient global ischemia, as with cardiac arrest, causes loss ofCA1hippocampal neurons 2- 4 d later, whereas nearby dentate gyrus (DG) neurons are relatively resistant. Whether differential astrocyte vulnerability in ischemic injury contributes to CA1 neuronal death is uncertain. Here, we find that CA1 astrocytes are more sensitive to ischemia than DG astrocytes. In rats subjected to transient forebrain ischemia, CA1 astrocytes lose glutamate transport activity and immunoreactivity for GFAP, S100β, and glutamate transporter GLT-1 within a few hours of reperfusion, but without astrocyte cell death. Oxidative stress may contribute to the observed selective CA1 changes, because CA1 astrocytes show early increases in mitochondrial free radicals and reduced mitochondrial membrane potential. Similar changes were not observed in DG astrocytes. Upregulation of GLT-1 expression in astrocytes with ceftriaxone protected CA1 neurons from forebrain ischemia. Wesuggest that greater oxidative stress and loss of GLT-1 function selectively in CA1 astrocytes is central to the well known delayed death of CA1 neurons. [ABSTRACT FROM AUTHOR]

Heat shock protein (Hsp)70 can suppress both necrosis and apoptosis induced by various injuries in vivo and in vitro. However, the relative importance of different functions and binding partners of Hsp70 in ischemic protection is unknown. To explore this question, we tested the ability of Hsp70-K71E, an adenosine triphosphate (ATP)ase-deficient point mutant, and Hsp70-381–640, a deletion mutant lacking the ATPase domain and encoding the carboxyl-terminal portion, to protect against ischemia-like injury in vivo and in vitro. Heat shock protein 70-wild type (-WT), -K71E, -381–640, and control vector plasmid LXSN were expressed in primary murine astrocyte cultures. Astrocytes overexpressing Hsp70-WT, -K71E, or -381–640 were all significantly protected from 4 h combined oxygen–glucose deprivation and 24 h reperfusion when assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or propidium iodide staining and cell counting (P<0.05). Brains of rats were transfected with plasmids encoding Hsp70-WT, -K71E, -381–640, or LXSN 24 h before 2 h middle cerebral artery occlusion followed by 24 h reperfusion. Animals that overexpressed either of the mutant proteins or Hsp70-WT had significantly better neurological scores and smaller infarcts than control animals. Protection by both mutants was associated with reduced protein aggregation, as assessed by ubiquitin immunohistochemistry and reduced nuclear translocation of apoptosis-inducing factor. The results show that the carboxyl-terminal portion of Hsp70 is sufficient for neuroprotection. This indicates that neither the ability to fold denatured proteins nor interactions with cochaperones or other proteins that bind the amino-terminal half of Hsp70 are essential to ischemic protection.Journal of Cerebral Blood Flow & Metabolism (2006) 26, 937–950. doi:10.1038/sj.jcbfm.9600246; published online 16 November 2005 [ABSTRACT FROM AUTHOR]

Abstract: Protein aggregation and misfolding are central mechanisms of both acute and chronic neurodegeneration. Overexpression of chaperone Hsp70 protects from stroke in animal and cell culture models. Although it is accepted that chaperones protect cells, the mechanism of protection by chaperones in ischemic injury is poorly understood. In particular, the relative importance of preventing protein aggregation compared to facilitating correct protein folding during ischemia and recovery is not known. To test the importance of protein folding and minimize interaction with co-chaperones we studied the bacterial chaperonin GroEL (HSPD1) and a folding-deficient mutant D87K. Both molecules protected cells from ischemia-like injury, and reduced infarct volume and improved neurological outcome after middle cerebral artery occlusion in rats. Protection was associated with reduced protein aggregation, assessed by ubiquitin immunohistochemistry. Marked neuroprotection by the folding-deficient chaperonin demonstrates that inhibition of aggregation is sufficient to protect the brain from ischemia. This suggests that strategies to maintain protein solubility and inhibit aggregation in the face of acute insults such as stroke may be a useful protective strategy. [Copyright &y& Elsevier]

Activation of ε protein kinase C (εPKC) has been shown to protect cardiac myocytes against ischemia and reperfusion injury. However, the role of PKC in ischemic brain injury is less well defined. Western blot analysis of murine neurons and astrocytes in primary culture demonstrated ε- and βIPKC expression in both cell types. Activation of εPKC increased in neuronal cultures in response to the ischemia-like insult of oxygen–glucose deprivation (OGD). Isozyme-specific peptide activators or inhibitors of PKC were applied at various times before, during and after the OGD period. Neuron–astrocyte mixed cultures pretreated with a selective εPKC activator peptide showed a significant reduction in neuronal injury after OGD and reperfusion, compared to cultures pretreated with control peptide. The εPKC activator peptide counteracted the increased damage induced by pretreatment with the εPKC-selective inhibitor peptide in relatively pure neuronal cultures subjected to OGD. Neither εPKC activator nor inhibitor peptides affected injury of neurons when applied after OGD onset. In contrast, the βIPKC-selective inhibitor peptide increased injury in astrocyte cultures exposed to OGD at all application times tested. Our data demonstrate protection of neurons by selective activation of εPKC but enhanced astrocyte cell death with selective inhibition of βIPKC. Thus PKC isozymes exhibit cell type-specific effects on ischemia-like injury. [Copyright &y& Elsevier]

Overexpression of the proto-oncogene bcl-2 has been shown to protect a variety of cell types from oxidative and non-oxidative injury, blocking apoptotic and necrotic types of cell death. Retroviral vectors were used to stably overexpress bcl-2 in primary murine astrocyte cultures with more than 95% efficiency. Compared to β-galactosidase-expressing and uninfected control cells, bcl-2 overexpressing astrocytes suffered < 40% injury after 24 h glucose deprivation, while controls were essentially completely injured. After exposure to 0.2 mm hydrogen peroxide, the bcl-2 overexpressing astrocytes suffered < 40% the injury seen in controls. In contrast, when the cultures were injured by combined oxygen–glucose deprivation, no difference in the extent or time course of injury was found between cells overexpressing bcl-2 and those expressing β-galactosidase. To investigate one possible mechanism of bcl-2 protection, we measured the levels of glutathione and three antioxidant enzymes. Astrocytes overexpressing bcl-2 had elevated glutathione levels (130–200%), increased superoxide dismutase (170%) and glutathione peroxidase (140%) activities compared with β-galactosidase-expressing controls. Bcl-2 overexpressing astrocytes suffered less lipid peroxidation after glucose deprivation, as assessed by cis-parinaric acid fluorescence, and demonstrated more rapid removal of hydrogen peroxide from the medium. When glutathione levels were decreased 80% by pretreatment with buthionine sulfoximine, the extent of protection from glucose deprivation of bcl-2 overexpressing cells was decreased by about half. Increased antioxidant defence contributes to protection from glucose deprivation in bcl-2 overexpressing astrocytes. [ABSTRACT FROM AUTHOR]