Role of Astrocytic miR-20a-3p as a Potential Therapeutic for Ischemic Stroke

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Project Summary The goal of this project is to test a therapeutic compound that has the potential to improve stroke outcomes in older females. It is critical to focus on this demographic because studies show that after menopause, women have greater risk and severity of ischemic stroke. Previous studies from the sponsor's lab have shown that reproductively senescent (acyclic) (RS) female rats have larger infarct and worse sensorimotor impairments than younger females. Moreover, estrogen replacement to RS females exacerbates stroke impairment. Thus this animal model replicates key features of stroke-related consequences seen in the human population, resulting in a model with translational potential, in which neurotherapeutic compounds can be tested. Prior research from the sponsor's lab has shown that astrocytes from reproductively senescent (RS) female rats exhibit age-related changes that contribute to poorer recovery after stroke. This includes a reduced capacity for glutamate clearance, decreased secretion of trophic factors, and increased release of inflammatory cytokines, compared to younger females. Furthermore, these aging astrocytes also display reduced histone methylation of the H3K4 chromatin region, resulting in less transcription of several genes including the microRNA mir-17-92 cluster. MicroRNAs have recently gained popularity as potential therapeutics for a variety of conditions, including ischemic stroke. Preliminary data for this proposal shows that miR-20a-3p, a member of the mir-17-92 cluster is dramatically down regulated in astrocytes of middle-aged females. Furthermore, iv injection of miR-20a-3p mimics four hours after middle cerebral artery occlusion (MCAo) decreased infarct volume and attenuated sensorimotor impairment in middle-aged females, suggesting that the miR-20a-3p could be mechanistically linked to stroke outcomes. This raises the intriguing possibility that in younger females, endogenous astrocyte-derived miR- 20a-3p may be `distributed' to other cells to improve stroke outcomes. This hypothesis will be tested in the following aims: Aim 1 will determine whether miR-20a-3p alters the composition of astrocytice extracellular vesicles (EVs) and that miR-20a-3p modified EVs will confer neuroprotection. Astrocytes have been shown to release neuroprotective factors, organelles and RNA translational machinery through EVs after stroke. In silico analysis predicts that miR-20a-3p may regulate inflammatory mediators and mitochondrial genes. Thus, miR- 20a-3p could drastically change the composition of the EVs. In aim 2, replication-deficient adenovirus constructs will be used to increase miR-20a-3p levels only in astrocytes and determine whether this recapitulates the neuroprotective effect of iv miR-20a-3p. Using clinically relevant stroke models, a battery of behavioral assays, and modern gene transfer techniques, this proposal will critically examine whether modification of the aging astrocyte will promote neuroprotection.