Sharon E. Benashski

The Mouse t-Complex-encoded Protein Tctex-1 Is a Light Chain of Brain Cytoplasmic Dynein

Mammalian brain cytoplasmic dynein contains three light chains of Mr = 8,000, 14,000, and 22,000 (King, S. M., Barbarese, E., Dillman, J. F., III, Patel-King, R. S., Carson, J. H., and Pfister, K. K. (1996) J. Biol. Chem. 271, 19358-19366). Peptide sequence data (16/16 residues correct) implicate the Mr = 14,000 polypeptide as Tctex-1, a protein encoded within the mouse t-complex. Tctex-1 cosediments with microtubules and is eluted with ATP or salt but not with GTP as expected for a dynein subunit. The ATP-eluted protein precisely cosediments with known cytoplasmic dynein proteins in sucrose density gradients. Tctex-1 also is immunoprecipitated from brain and other tissue homogenates by a monoclonal antibody raised against the 74-kDa cytoplasmic dynein intermediate chain. Quantitative densitometry indicates that Tctex-1 is a stoichiometric component of the dynein complex. As Tctex-1 is a candidate for involvement in the transmission ratio distortion (meiotic drive) of mouse t-haplotypes, these results suggest that cytoplasmic dynein dysfunction may play an important role in non-mendelian chromosome segregation.

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.

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.

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.

Solution structure of a dynein motor domain associated light chain.

Dyneins are molecular motors that translocate towards the minus ends of microtubules. In Chlamydomonas flagellar outer arm dynein, light chain 1 (LC1) associates with the nucleotide binding region within the γ heavy chain motor domain and consists of a central leucine-rich repeat section that folds as a cylindrical right handed spiral formed from six β-β-α motifs. This central cylinder is flanked by terminal helical subdomains. The C-terminal helical domain juts out from the cylinder and is adjacent to a hydrophobic surface within the repeat region that is proposed to interact with the dynein heavy chain. The position of the C-terminal domain on LC1 and the unexpected structural similarity between LC1 and U2A′ from the human spliceosome suggest that this domain interacts with the dynein motor domain.

The Tctex1/Tctex2 Class of Dynein Light Chains DIMERIZATION, DIFFERENTIAL EXPRESSION, AND INTERACTION WITH THE LC8 PROTEIN FAMILY

The Tctex1/Tctex2 family of dynein light chains associates with the intermediate chains at the base of the soluble dynein particle. These components are essential for dynein assembly and participate in specific motor-cargo interactions. To further address the role of these light chains in dynein activity, the structural and biochemical properties of several members of this polypeptide class were examined. Gel filtration chromatography and native gel electrophoresis indicate that recombinantChlamydomonas flagellar Tctex1 exists as a dimer in solution. Furthermore, yeast two-hybrid analysis suggests that this association also occurs in vivo. In contrast, both murine and Chlamydomonas Tctex2 are monomeric. To investigate protein-protein interactions involving these light chains, outer arm dynein from Chlamydomonas flagella was cross-linked using dimethylpimelimidate. Immunoblot analysis of the resulting products revealed the interaction of LC2 (Tctex2) with LC6, which is closely related to the highly conserved LC8 protein found in many enzyme systems, including dynein. Northern dot blot analysis demonstrated that Tctex1/Tctex2 family light chains are differentially expressed both in a tissue-specific and developmentally regulated manner in humans. These data provide further support for the existence of functionally distinct populations of cytoplasmic dynein with differing light chain content.

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.

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.

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.

Post-Stroke Hypothermia Provides Neuroprotection through Inhibition of AMP-Activated Protein Kinase

Hypothermia is robustly protective in pre-clinical models of both global and focal ischemia, as well as in patients after cardiac arrest. Although the mechanism for hypothermic neuroprotection remains unknown, reducing metabolic drive may play a role. Capitalizing on the beneficial effects of hypothermia while avoiding detrimental effects such as infection will be the key to moving this therapy forward as a treatment for stroke. AMPK is a master energy sensor that monitors levels of key energy metabolites. AMPK is activated via phosphorylation (pAMPK) when cellular energy levels are low, such as that seen during ischemia. AMPK activation appears to be detrimental in experimental stroke, likely via exacerbating ischemia-induced metabolic failure. We tested the hypothesis that hypothermia reduces AMPK activation. First, it was found that hypothermia reduced infarct after middle cerebral artery occlusion. Second, induced hypothermia reduced brain pAMPK in both sham control and stroke mice. Third, hypothermic neuroprotection was ameliorated after administration of compound C, an AMPK inhibitor. Finally, deletion of one of the catalytic isoforms of AMPK completely reversed the effect of hypothermia on stroke outcome after both acute and chronic survival. These effects were mediated by a reduction in AMPK activation rather than a reduction in LKB1, an upstream AMPK kinase. In summary, these studies provide evidence that hypothermia exerts its protective effect in part by inhibiting AMPK activation in experimental focal stroke. This suggests that AMPK represents a potentially important biological target for stroke treatment.