Abstract DP26: Neuronal eSINE-RNAs modulate post-stroke outcomes in the mouse brain

Introduction: Short interspersed nuclear elements (SINEs) are an abundant class of noncoding retrotransposons that can be transcribed by RNA polymerase III. Some SINEs have also been shown to act as genomic enhancers, thus giving rise to what are known as enhancer SINE-RNAs (eSINE-RNAs). The function of these novel RNAs within the post-stroke brain remains virtually unexplored. In this study, we map the genome-wide cortical expression of eSINE-RNAs in response to stroke, characterize their cell-type&subcellular localization, and determine their role in affecting post-stroke infarct volumes. Methods: Ischemic stroke was induced in male C57BL/6N mice using 1h middle cerebral artery occlusion (MCAO) followed by 6h of reperfusion (or sham surgery). Genome-wide RNA-seq and H3K27ac ChIP-seq (n=3/group/experiment) were performed on ipsilateral cortices to identify stroke-responsive enhancer RNA transcripts, which were then overlaid with genomic SINEs to identify eSINE-RNAs. Immunohistochemistry and RNA-FISH were used in tandem to determine the cell-type and subcellular localization of several highly expressed eSINE-RNAs. Select eSINE-RNAs were ablated via intracerebroventricular injection of antisense oligonucleotides followed by 1h MCAO and 24h reperfusion. Infarct volumes following knockdown were evaluated using cresyl violet staining (n=5-7/group). Results: We discovered 46 eSINE-RNAs upregulated in the cortex following MCAO, 33 of which were unique to stroke and not detected in the sham group. These RNAs were derived from several families of SINEs, including 24 from the B1 family, 20 from the B2 family, and one each from the less ubiquitous ID and MIR families. The expression of several abundant eSINE-RNAs was specific to the nuclei of neurons within the prospective infarct and peri-infarct territories, including the sensorimotor cortex and hippocampus. Ablation of select eSINE-RNAs resulted in significantly higher infarct volumes versus controls, suggesting that these eSINE-RNAs serve a neuroprotective role in the brain following ischemic stroke. Conclusion: Our study is the first report to characterize the expression of eSINE-RNAs in the post-stroke brain and begin to elucidate their function as regulators of post-stroke pathophysiology. Our results suggest that the transcription of eSINE-RNAs represents a robust neuroprotective response to transient focal ischemia, thus positioning them as potential molecular targets for modulating post-stroke outcomes.