Inhibition of glycogen synthase kinase-3ß enhances cognitive recovery after stroke: the role of TAK1.

Memory deficits are common among stroke survivors. Identifying neuroprotective agents that can prevent memory impairment or improve memory recovery is a vital area of research. Glycogen synthase kinase-3ß (GSK-3ß) is involved in several essential intracellular signaling pathways. Unlike many other kinases, GSK-3ß is active only when dephosphorylated and activation promotes inflammation and apoptosis. In contrast, increased phosphorylation leads to reduced GSK-3ß (pGSK-3ß) activity. GSK-3ß inhibition has beneficial effects on memory in other disease models. GSK-3ß regulates both the 5'AMP-activated kinase (AMPK) and transforming growth factor-ß-activated kinase (TAK1) pathways. In this work, we examined the effect of GSK-3ß inhibition, both independently, in conjunction with a TAK inhibitor, and in AMPK-α2 deficient mice, after stroke to investigate mechanistic interactions between these pathways. GSK-3ß inhibition was neuroprotective and ameliorated stroke-induced cognitive impairments. This was independent of AMPK signaling as the protective effects of GSK-3ß inhibition were seen in AMPK deficient mice. However, GSK-3ß inhibition provided no additive protection in mice treated with a TAK inhibitor suggesting that TAK1 is an upstream regulator of GSK-3ß. Targeting GSK-3ß could be a novel therapeutic strategy for post-stroke cognitive deficits.

Sex differences in ischemic stroke sensitivity are influenced by gonadal hormones, not by sex chromosome complement.

Epidemiologic studies have shown sex differences in ischemic stroke. The four core genotype (FCG) mouse model, in which the testes determining gene, Sry, has been moved from Y chromosome to an autosome, was used to dissociate the effects of sex hormones from sex chromosome in ischemic stroke outcome. Middle cerebral artery occlusion (MCAO) in gonad intact FCG mice revealed that gonadal males (XXM and XYM) had significantly higher infarct volumes as compared with gonadal females (XXF and XYF). Serum testosterone levels were equivalent in adult XXM and XYM, as was serum estrogen in XXF and XYF mice. To remove the effects of gonadal hormones, gonadectomized FCG mice were subjected to MCAO. Gonadectomy significantly increased infarct volumes in females, while no change was seen in gonadectomized males, indicating that estrogen loss increases ischemic sensitivity. Estradiol supplementation in gonadectomized FCG mice rescued this phenotype. Interestingly, FCG male mice were less sensitive to effects of hormones. This may be due to enhanced expression of the transgene Sry in brains of FCG male mice. Sex differences in ischemic stroke sensitivity appear to be shaped by organizational and activational effects of sex hormones, rather than sex chromosomal complement.

NF-κB contributes to the detrimental effects of social isolation after experimental stroke.

Social isolation (SI) is increasingly recognized as a risk factor for stroke. Individuals with lack of social support systems have an increased incidence of stroke, poorer recovery, and greater functional decline after injury compared to individuals with social support. Attesting to the importance of social factors in stroke outcome is that these same effects can be reproducibly demonstrated in animals; social interaction improves behavioral deficits and reduces damage after experimental stroke, whereas SI enhances injury. The mechanism by which SI exacerbates injury is unclear. We investigated the role of nuclear factor-kappaB (NF-κB) signaling in male mice that were pair housed (PH) with an ovariectomized female prior to random assignment into continued PH or SI for 7 days prior to middle cerebral artery occlusion. The effects of SI on infarct volume and functional recovery were assessed at 72 h post-stroke. Nuclear NF-κB levels and activity were assessed by Western blot and transcriptional assays. SI significantly exacerbated infarct size in both male and female mice compared to PH mice. SI mice had delayed functional recovery compared to PH mice. An elevation of systemic IL-6 levels, increased nuclear NF-κB transcriptional activity, and enhanced nuclear translocation of NF-κB was seen in SI stroke animals. Interference with NF-κB signaling using either a pharmacological inhibitor or genetically engineered NF-κB p50 knockout mice abolished the detrimental effects of SI on both infarct size and functional recovery. This suggests that NF-κB mediates the detrimental effects of SI.

Age-related changes in AMP-activated protein kinase after stroke.

Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved energy sensor sensitive to changes in cellular AMP/ATP ratio which is activated by phosphorylation (pAMPK). pAMPK levels decrease in peripheral tissues with age, but whether this also occurs in the aged brain, and how this contributes to the ability of the aged brain to cope with ischemic stress is unknown. This study investigated the activation of AMPK and the response to AMPK inhibition after induced stroke in both young and aged male mice. Baseline levels of phosphorylated AMPK were higher in aged brains compared to young mice. Stroke-induced a robust activation of AMPK in young mice, yet this response was muted in the aged brain. Young mice had larger infarct volumes compared with aged animals; however, more severe behavioral deficits and higher mortality were seen in aged mice after stroke. Inhibition of AMPK with Compound C decreased infarct size in young animals, but had no effect in aged mice. Compound C administration led to a reduction in brain ATP levels and induced hypothermia, which led to enhanced neuroprotection in young but not aged mice. This work demonstrates that aging increases baseline brain pAMPK levels; aged mice have a muted stroke-induced pAMPK response; and that AMPK inhibition and hypothermia are less efficacious neuroprotective agents in the aged brain. This has important translational relevance for the development of neuroprotective agents in preclinical models and our understanding of the enhanced metabolic stress experienced by the aged brain.

miR-23a regulation of X-linked inhibitor of apoptosis (XIAP) contributes to sex differences in the response to cerebral ischemia.

It is increasingly recognized that the mechanisms underlying ischemic cell death are sexually dimorphic. Stroke-induced cell death in males is initiated by the mitochondrial release of apoptosis-inducing factor, resulting in caspase-independent cell death. In contrast, ischemic cell death in females is primarily triggered by mitochondrial cytochrome c release with subsequent caspase activation. Because X-linked inhibitor of apoptosis (XIAP) is the primary endogenous inhibitor of caspases, its regulation may play a unique role in the response to injury in females. XIAP mRNA levels were higher in females at baseline. Stroke induced a significant decrease in XIAP mRNA in females, whereas no changes were seen in the male brain. However, XIAP protein levels were decreased in both sexes after stroke. MicroRNAs (miRNAs) predominantly induce translational repression and are emerging as a major regulators of mRNA and subsequent protein expression after ischemia. The miRNA miR-23a was predicted to bind XIAP mRNA. miR-23a directly bound the 3' UTR of XIAP, and miR-23a inhibition led to an increase in XIAP mRNA in vitro, demonstrating that XIAP is a previously uncharacterized target for miR-23a. miR-23a levels differed in male and female ischemic brains, providing evidence for sex-specific miRNA expression in stroke. Embelin, a small-molecule inhibitor of XIAP, decreased the interaction between XIAP and caspase-3 and led to enhanced caspase activity. Embelin treatment significantly exacerbated stroke-induced injury in females but had no effect in males, demonstrating that XIAP is an important mediator of sex-specific responses after stroke.

Sex differences in the response to poly(ADP-ribose) polymerase-1 deletion and caspase inhibition after stroke.

BACKGROUND AND PURPOSE: Emerging data suggest that the molecular cell death pathways triggered by ischemic insults differ in the male and female brain. Cell death in males is initiated by poly(ADP-ribose) polymerase-1 (PARP-1) activation; however, manipulation of this pathway paradoxically increases ischemic damage in females. In contrast, females are exquisitely sensitive to caspase-mediated cell death. The effect of caspase inhibition in PARP-1 knockout mice was evaluated to determine if the detrimental effects of PARP deletion in females were secondary to increased caspase activation. METHODS: Focal stroke was induced by transient or permanent middle cerebral artery occlusion (MCAO) in wild-type (WT) and PARP-1(-/-) mice of both sexes. The pan-caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh), was administered 90 minutes after middle cerebral artery occlusion. Infarct size and neurological sores were assessed. Separate cohorts were used for protein analysis for PAR, Apoptosis inducing factor (AIF), caspase-9, and caspase-3. RESULTS: WT mice of both sexes had increased nuclear AIF after stroke compared to PARP-1(-/-) mice. PARP-1(-/-) females had higher mitochondrial cytochrome C and activated caspase-9 and -3 levels than WT female mice. PARP-1(-/-) females also had an increase in stroke-induced cytosolic cytochrome C release compared with WT females, which was not seen in males. Q-VD-OPh decreased caspase-9 in both males and females but only led to reduction of infarct in females. PARP-1(-/-) males had smaller infarcts, whereas PARP-1(-/-) females had larger strokes compared with WT. Q-VD-OPh significantly decreased infarct in both WT and PARP-1(-/-) females in both transient and permanent MCAO models, but had no effect in males. CONCLUSIONS: Deletion of PARP-1 reduces infarct in males but exacerbates injury in females. PARP-1(-/-) females have enhanced caspase activation. The detrimental effects of PARP loss in females can be reversed with caspase inhibition.

Effects of metformin in experimental stroke.

BACKGROUND AND PURPOSE: Adenosine 5'-monophosphate-activated protein kinase (AMPK) is an important sensor of energy balance. Stroke-induced AMPK activation is deleterious because both pharmacological inhibition and genetic deletion of AMPK are neuroprotective. Metformin is a known AMPK activator but reduces stroke incidence in clinical populations. We investigated the effect of acute and chronic metformin treatment on infarct volume and AMPK activation in experimental stroke. METHODS: Male mice were subjected to middle cerebral artery occlusion after acute (3 days) or chronic (3 weeks) administration of metformin. Infarct volumes, AMPK activation, lactate accumulation, and behavioral outcomes were assessed. The roles of neuronal nitric oxide synthase and AMPK were examined using mice with targeted deletion of AMPK or neuronal nitric oxide synthase. RESULTS: Acute metformin exacerbated stroke damage, enhanced AMPK activation, and led to metabolic dysfunction. This effect was lost in AMPK and neuronal nitric oxide synthase knockout mice. In contrast, chronic metformin given prestroke was neuroprotective, improved stroke-induced lactate generation, and ameliorated stroke-induced activation of AMPK. Similarly, the neuroprotective effect of chronic prestroke metformin was lost in neuronal nitric oxide synthase knockout mice. CONCLUSIONS: AMPK is an important potential target for stroke treatment and prevention. These studies show that the timing, duration, and amount of AMPK activation are key factors in determining the ultimate downstream effects of AMPK on the ischemic brain.

Adenosine monophosphate activated protein kinase inhibition is protective in both sexes after experimental stroke.

Sex differences in clinical and experimental stroke are now well recognized. Adenosine monophosphate activated protein kinase (AMPK) is an important energy sensor that is activated in times of energy demand. Increasing AMPK is deleterious in experimental cerebral ischemia, at least in males. Interestingly, studies in peripheral tissues have suggested that there are sex differences in the regulation of AMPK in muscle after exercise. PolyADP ribose polymerase (PARP), a key mediator of ischemic cell death, stimulates AMPK activation, yet activation of PARP appears to be selectively detrimental in male brain. As interference with sex specific cell death pathways can determine the efficacy of experimental neuroprotective agents, and AMPK inhibition is a novel neuroprotective target, we examined the effect of AMPK inhibition in male and female mice. In this study, AMPK alpha2 gene expression (mRNA) and pAMPK protein levels were examined and found to be comparable between both sexes after transient middle cerebral artery occlusion (MCAO). Treatment with the AMPK inhibitor Compound C at stroke onset significantly reduced infarct size and neurological deficits 24h after stroke in ovariectomized female mice. Finally, genetic deletion of AMPK alpha2 in ovariectomized females was neuroprotective as assessed by smaller infarct volumes and improved neurological deficits when compared to wild type littermates. This work demonstrates that AMPK activation is deleterious in experimental stroke, and this effect is independent of sex.

Expression of Na-K-Cl cotransporter and edema formation are age dependent after ischemic stroke.

Age is the most important independent risk factor for stroke; however aging animals are rarely used in stroke studies. Previous work demonstrated that young male mice had more edema formation after an induced stroke than aging animals. An important contributor to cerebral edema formation is the Na-K-Cl cotransporter (NKCC). We examined the expression of NKCC in young (10-12 weeks) and aging (15-16 months) C57BL6 male mice after middle cerebral artery occlusion (MCAO) and investigated the effect of pharmacological inhibition of NKCC with Bumetanide on cerebral edema formation. Both immunofluorescent staining and Western blotting analysis showed that NKCC expression was significantly higher in the ischemic penumbra of young compared to aging mice after stroke. Edema formation was significantly more robust in young mice and was reduced with Bumetanide. Bumetanide had no effect on cerebral edema in aging mice after MCAO. This suggests that NKCC expression and edema formation are age dependent after ischemic stroke.

Changes in experimental stroke outcome across the life span.

Acute ischemic stroke is a leading cause of mortality and disability in the elderly. Age is the most important nonmodifiable risk factor for stroke, yet many preclinical models continue to examine only young male animals. It remains unclear how experimental stroke outcomes change with aging and with biologic sex. If sex differences are present, it is not known whether these reflect an intrinsic differing sensitivity to stroke or are secondary to the loss of estrogen with aging. We subjected both young and aging mice of both sexes to middle cerebral artery occlusion (MCAO). Young female mice had smaller strokes compared with age-matched males, an effect that was reversed by ovariectomy. Stroke damage increased with aging in female mice, whereas male mice had decreased damage after MCAO. Blood-brain barrier (BBB) permeability changes are correlated with infarct size. However, aging mice had significantly less edema formation, an effect that was independent of sex and histologic damage. Differences in the cellular response to stroke occur across the life span in both male and female mice. These differences need to be considered when developing relevant therapies for stroke patients, the majority of whom are elderly.

Redox-based control of the gamma heavy chain ATPase from Chlamydomonas outer arm dynein.

The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these proteins belong to a distinct subgroup within the thioredoxin family. This observation suggested that some aspect of dynein activity might be modulated through redox poise. To test this, we have examined the effect of sulfhydryl oxidation on the ATPase activity of isolated dynein and axonemes from wildtype and mutant strains lacking various heavy chain combinations. The outer, but not inner, dynein arm ATPase was stimulated significantly following treatment with low concentrations of dithionitrobenzoic acid; this effect was readily reversible by dithiol, and to a lesser extent, monothiol reductants. Mutational and biochemical dissection of the outer arm revealed that ATPase activation in response to DTNB was an exclusive property of the gamma heavy chain, and that enzymatic enhancement was modulated by the presence of other dynein components. Furthermore, we demonstrate that the LC5 thioredoxin-like light chain binds to the N-terminal stem domain of the alpha heavy chain and that the beta heavy chain-associated LC3 protein also interacts with the gamma heavy chain. These data suggest the possibility of a dynein-associated redox cascade and further support the idea that the gamma heavy chain plays a key regulatory role within the outer arm.

A bipartite Ca2+-regulated nucleoside-diphosphate kinase system within the Chlamydomonas flagellum. The regulatory subunit p72.

Regulation of flagellar activity in Chlamydomonas involves both Ca(2+) and cAMP-mediated signaling pathways. However, Chlamydomonas and sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular protein (p72) from the Chlamydomonas flagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca(2+). There are close homologues of p72 in both mammalian and insect genomes. The p72 protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that p72 interacts with two or three additional axonemal polypeptides. The sensitivity of p72 to tryptic digestion differed considerably in the presence and the absence of Ca(2+), suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca(2+) regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.