Inhibition of miR-181a protects female mice from transient focal cerebral ischemia by targeting astrocyte estrogen receptor-α☆
Graphical abstract
Introduction
Stroke remains a major source of mortality and morbidity worldwide, including in the United States (Mozaffarian et al., 2015). Despite advances in gene therapy techniques and promising results in pre-clinical animal models, treatment for the most common form of stroke (thrombotic/non-hemorrhagic) remains limited to early reperfusion with thrombolytics or clot retrieval. In the human population, sexual dimorphism in thrombotic stroke risk and outcome is well documented. The Framingham Heart Study demonstrated women overall have an lower risk of stroke than men (Petrea et al., 2009), however in old age, women have a higher incidence of stroke and poorer functional outcome (Towfighi et al., 2007, Petrea et al., 2009, Mozaffarian et al., 2015). This age-dependent sexual dimorphism in stroke risk and outcome may hold clues to the translation barrier that persists in the development of new stroke therapies. Importantly, a similar sexual dimorphism has been identified for injury outcome in rodent models of stroke. Overall, adult female rodents have smaller infarct volumes and improved neurobehavioral outcomes following transient cerebral ischemia than their age-matched male counterparts (Alkayed et al., 1998, Selvamani et al., 2014, Xiong et al., 2011), however this effect is reversed in aged female rodents (Manwani et al., 2014). Delineating the mechanisms that regulate the differences between males and females in risk and outcome following stroke may yield new insight and more effective therapeutics.
Although a full understanding of the mechanisms underlying sex differences in stroke remains to be determined, the ovarian hormone estrogen is known to play a role in ischemic protection (Manwani et al., 2015, Simpkins et al., 1997). Lower circulating levels of estrogen that occur with physiologic estrous cycling in female rats and mice are associated with a greater degree of injury from cerebral ischemia (Xiong et al., 2015, Liao et al., 2001). Estrogen exerts a protective effect via several mechanisms including antioxidant and immune modulation, as well as activation of cell survival signaling pathways (for review, Ritzel et al., 2013). The sex difference in infarct volume observed between intact adult males and females is abolished with ovariectomy (OVX), which depletes estrogen, resulting in larger infarcts in adult females (Alkayed et al., 1998). This effect is in part reversed by exogenous replacement of 17β-estradiol (E2, Dubal et al., 1998, Rusa et al., 1999) improving sensorimotor and spatial memory deficits in ovariectomized females (Li et al., 2004, Gulinello et al., 2006).
MicroRNAs (MiRs) are short (19–22 nucleotide), non-coding RNAs that modulate endogenous gene expression in all tissues during both normal physiological functioning and in response to stress. MiR expression patterns are organ- and cell type-specific, and in the brain miRs play a central role in the response to cerebral ischemia (for review (Ouyang et al., 2013). MiR expression patterns have also been observed to exhibit both sex- and age-related dysregulation in response to cerebral ischemia (Selvamani et al., 2014). MiRs most commonly function by binding the 3′ untranslated region (UTR) of target messenger RNAs (mRNAs) to inhibit protein translation. We have previously demonstrated a protective effect of miR-181a-5p inhibition in male rodent models of stroke (Ouyang et al., 2012) and forebrain ischemia (Moon et al., 2013) by targeting a stress protein and regulators of apoptosis. However, whether sex differences exist in the role of miR-181a-5p (hereafter miR-181a) in stroke has not been previously investigated. In endometrial cells, the miR-181 family has been shown to target estrogen receptor-α (ERα, Su et al., 2014), a transcription factor that activates pro-survival pathways (Yu et al., 2012), suggesting that sex differences in downstream targets of miR-181a may exist. Therefore, in the present study we examined the roles of miR-181a, E2, and ERα in female mice subjected to OVX with or without replacement of E2, and subjected to cerebral ischemia two weeks later.
Astrocytes are principle regulators of neuronal homeostasis and survival following injury and express greater levels of miR-181a than neurons (Hutchison et al., 2013). We have previously demonstrated that local astrocyte dysfunction precedes and contributes to neuronal cell death following forebrain ischemia (Ouyang et al., 2007), and that targeting astrocytes can be an effective therapeutic approach for protection (Xu et al., 2010). In focal ischemia as used here, astrocyte cell death is part of the core injury. We previously demonstrated that reducing levels of miR-181a in astrocytes helps preserve mitochondrial function and protect against astrocyte cell death from in vitro ischemia (Ouyang et al., 2012). Substantial previous work supports a role for E2 and estrogen receptors in astrocytes (see Ritzel et al., 2013 for review). Therefore, in the present study we followed up stroke studies in female mice with studies in female and male astrocyte cultures evaluating differences in the effect of miR-181a, E2 and ERα in the response to cell stress.
Section snippets
In vivo experimental protocols
All experimental protocols using animals were approved by the Stanford University Animal Care and Use Committee, and performed in accordance with NIH guidelines. Adult female C57/B6 mice (age 8–10 weeks, Charles River, Wilmington, MA) were surgically ovariectomized (OVX, described below) and randomly assigned by coin flip to subcutaneous placement of a capsule with or without E2. These two groups were further randomly subdivided (Fig. 1A) into groups receiving intracerebroventricular injection
Results
When measured before OVX, serum E2 levels were equivalent, while 7 and 13 days after OVX, E2 was significantly lower in animals replaced with vehicle, versus E2 replacement (Fig. 2A). Treatment with either miR-181a antagomir or E2 decreased MCAO infarct volume (Fig. 2B,C), while combined treatment resulted in significantly greater protection than either treatment alone. Similarly, miR-181a antagomir or E2 significantly improved neurological deficit score (Fig. 2D), with combined treatment
Discussion
The present study examined the efficacy of miR-181a antagomir in female stroke, with and without E2. We observed significant protection, similar in magnitude to the protection seen in male mice (about 48% reduction in infarct volume) (Ouyang et al., 2012) only with combined E2 and miR-181a antagomir (53% reduction), while antagomir alone provided a significant reduction of infarct volume in females of 24%. We identified ERα as a target for miR-181a in female brain and astrocyte culture, but not
Summary
This report is the first to demonstrate that decreasing brain levels of miR-181a in female mice is protective, and enhances the neuroprotective effects of E2. This combined effect was recapitulated in female primary astrocyte cultures but not male cultures. We identified ERα as a novel target of miR-181a in female cortical astrocytes.
Support
This study was funded in part by NIH grants NS084396 and NS080177 to RGG, American Heart Association grant FTF-19970029 to CMS.
Disclosures
None.
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This study was funded in part by NIH grants NS084396 and NS080177 to RGG, American Heart Association grant FTF-19970029 to CMS.
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These authors contributed equally to this work.