Abstract T MP20: Histone Methylation Patterns in Astrocytes are Influenced by Age Following Ischemia

Introduction: In animal models, ischemia results in larger infarcts in middle-aged females as compared to adult females. This age difference in infarct severity is associated with reduced functional capacity of astrocytes, a critical support cell. Astrocytes from middle-aged females synthesize less IGF-1, a neuroprotective growth factor, and display reduced ability to clear glutamate. The impaired response of astrocytes following stroke in middle-aged females may be related to epigenetic alterations, including histone methylation, that occur during aging. Hypothesis: We evaluated the hypothesis that astrocytes from middle-aged females would have a distinctive pattern of histone methylation following ischemia as compared to astrocytes from younger females, resulting in differential protein expression. Methods: The middle cerebral artery was occluded in adult (6 month) and middle-aged (11+ month) female Sprague Dawley rats (n=9-15). Astrocytes were extracted from the ischemic hemisphere 48 hours after ischemia. ChIP-seq was used to measure H3K4 and H3K9 trimethylation (me3) across the genome. In a second set of astrocytes, the expression of genes/proteins with highly methylated promoters was measured with either qPCR or ELISA immunoassays (n=6-7). Results: Adult females had more H3K4me3 peaks (912 vs 72) and fewer H3K9me3 (4 vs 22) peaks than middle-aged females. Increased H3K4me3 was found at promoters for genes encoding the mir17-20 cluster and vascular endothelial growth factor (VEGF). In agreement with the methylation data, astrocytes from adult females had higher protein levels of VEGF and greater expression of mir20 than middle-aged females (p<.05). Conclusions: This study found more H3K4me3 and less H3K9me3 in astrocytes from adult females indicating a general increase in gene transcription following ischemia. Increased trimethylation of mir20 was accompanied with increased expression of mir20 in astrocytes from adult females. Importantly, trimethylation of lysine 4 on histone 3 results in increased gene transcription whereas mir20 negatively regulates translation. This complex interaction of epigenetic modifications provides insight into possible mechanisms for differences in stroke severity observed during aging.