Sex differences in the response to activation of the poly (ADP-ribose) polymerase pathway after experimental stroke

https://doi.org/10.1016/j.expneurol.2009.02.012Get rights and content

Abstract

It is increasingly recognized that histological and functional outcomes after stroke are shaped by biologic sex. Emerging data suggests that ischemic cell death pathways are sexually dimorphic (Hurn, P., Vannucci, S., Hagberg, H. (2005) Adult or perinatal brain injury: does sex matter?. Stroke 36, 193–195 ; Lang, J.T., McCullough, L.D. (2008) Pathways to ischemic neuronal cell death: are sex differences relevant?. J. Transl. Med. 6). Reducing neuronal nitric oxide (NO) or poly-ADP-ribose polymerase (PARP1) activation protects only the male brain (Hagberg, H., et al. PARP-1 gene disruption in mice preferentially protects males from perinatal brain injury. J. Neurochem. 90, 1068–1075 (2004)), and paradoxically enhances ischemic injury in females (McCullough, L.D., et al. Ischemic nitric oxide and poly (ADP-ribose) polymerase-1 in cerebral ischemia: male toxicity, female protection. J. Cereb. Blood Flow Metab. 25, 502–512 (2005)). In this study, we examined downstream mediators of NO/PARP activation to investigate possible mediators of ischemic sexual dimorphism. Nuclear translocation of Apoptosis Inducing Factor (AIF) was equivalent in wild type males and females after stroke and was unaffected by estrogen exposure. Deletion of PARP1 led to a dramatic reduction in stroke-induced poly (ADP-ribose) polymerase (PAR) formation and AIF translocation in both sexes, yet ischemic damage was reduced only in males. Subsequent examination of AIF-deficient Harlequin mice demonstrated that male Harlequin mice had less PAR formation, reduced AIF translocation and less ischemic damage than male wild type mice. In contrast, female Harlequin mice had no neuroprotective effect of gene deletion despite robust reductions in PAR formation and AIF translocation. Although equivalent activation of this cell death pathway occurs in both sexes after ischemia, detrimental effects are only present in males. AIF translocation and PAR formation do not mediate ischemic injury in the female brain, therefore agents designed to reduce PARP1 activation are unlikely to benefit females.

Introduction

Stroke affects 15 million people worldwide each year, and is the leading cause of long-term disability in the United States (Rosamond, et al., 2007). The epidemiology of ischemic stroke is sexually dimorphic in that ischemic events occur with greater frequency in men vs. women until advanced age (Sudlow and Warlow, 1997, Sacco et al., 1998, Incidence and Prevalence, 2006). As the incidence of stroke increases dramatically in women after menopause, exposure to reproductive hormones may be a major contributor to this sex difference. Extensive preclinical evidence demonstrates that estrogen improves neuronal survival after injury both in vivo and in vitro (Garcia-Segura et al., 2001, McCullough and Hurn, 2003). However, recent clinical trials evaluating the efficacy of hormone replacement therapy in post-menopausal women for prevention of stroke (Rossouw, 2002, Wassertheil-Smoller, 2003) led to the surprising finding of increased vascular risk in hormone-treated women. In epidemiological studies, stroke rates do not climb until well after the menopause, suggesting that non-hormonal factors play a role in ischemic sexual dimorphism (Rosamond et al., 2007). In addition, sex differences in outcomes from stroke and hypoxic–ischemic encephalopathy are present in neonatal populations where levels of circulating hormones are equivalent between the sexes (Fullerton, 2003, Marlow, 2005, Zhu, 2006, Johnston and Hagberg, 2007, Renolleau, 2007).

Several distinct yet overlapping pathways contribute to cell death in experimental stroke. Mitochondrial damage results in the release of cytochrome c, triggering activation of the intrinsic pro-apoptotic caspase cascade and formation of the apoptosome (Ferrer and Planas, 2003, Chan, 2004, Stefanis, 2005). In addition, a distinct cell death pathway exists whereby DNA damage from rising levels of Nitric Oxide (NO) and peroxynitrite (ONOO) leads to the activation of the DNA repair enzyme poly ADP-ribose polymerase 1 (PARP1) and formation of poly (ADP-ribose) polymerase (PAR) polymers. This triggers the release of apoptosis inducing factor (AIF) from the mitochondria leading to caspase-independent cell death (Bredt, 1990, Susin, 2000, Yu, 2002). Key evidence in establishing NO toxicity/PARP1 activation as a major cytotoxic mechanism has accumulated from exclusively male animals or mixed sex primary neuronal cell cultures (Huang, 1994, Eliasson, 1997, Cao, 2003, Wang, 2004). In contrast, we have recently discovered that this pathway exhibits dramatic sexual dimorphism and that the integrity of nNOS/PARP1 signaling is paradoxically protective in the female (McCullough et al., 2005). Similar sex disparities exist in vitro, as female neurons are intrinsically protected after an oxidative challenge, eliminating differences in gonadal hormone levels as the sole explanation for these findings (Du, 2004, Li, 2005).

We propose that there are distinct sex-based cell death programs that involve the differential regulation of the NO/PARP pathway and its downstream mediator AIF. This pathway mediates cell death in the male, but not the female brain. We utilized PARP deficient and low AIF-expressing mice to determine if reductions in PARP/AIF activation would protect the female brain.

Section snippets

Experimental animals and groups

PARP1 −/−, Harlequin (HQ) mutant mice expressing low levels of AIF (Pdcd8Hq; Hq) and wild type mice (WT) used in this study were purchased from The Jackson Laboratory (Bar Harbor, Me; Culmsee et al., 2005). The present study was conducted in accordance with the NIH guidelines for the care and use of animals in research and under protocols approved by the Animal Care and Use Committee of the University of Connecticut Health Center.

Ischemic model

Cerebral ischemia was induced by 90 min of reversible middle

PAR accumulation after stroke

Due to our previous findings of striking sexual dimorphism in PARP-mediated cell death (McCullough et al., 2005), we hypothesized that PARP represents an important branch-point in determining the mechanism by which brain dies after an induced stroke. We began by evaluating PAR polymer formation in wild type (WT) mice after stroke. PAR polymers directly trigger mitochondrial AIF release and lead to direct neurotoxicity in vitro (Andrabi, 2006, Yu, 2006). Nuclear PAR levels began to increase

Discussion

The present study represents the initial steps in the investigation of sex differences in cell death pathways after ischemic injury. Stroke is now the third leading cause of death in the U.S. and the most common cause of disability (Rosamond et al., 2007). The economic burden of stroke is increasing, making the prevention and treatment of stroke a critical public health issue as our population ages. Data is emerging from both in vivo and in vitro studies that suggest that cell death pathways

Acknowledgments

This work was supported by NIH R01 NS050505 and NS055215 (LDM) and the AHA (LDM) and by 5T32NS041224-09 (Neuroscience Training at University of Connecticut Health Center).

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