Introduction: Astrocytes from middle-aged reproductively senescent rats, who typically have larger infarcts after stroke, have been shown to have a reduced functional capacity for glutamate clearance and production of trophic factors compared to astrocytes from adult rats, who typically have smaller infarcts. Epigenetic analysis revealed greater H3K4 trimethylation of the promoter region of the mir17-92 cluster in adult astrocytes, and qRT-PCR analysis confirmed increased expression of all members of this cluster including a 240-fold elevation of mir20a-3p. Intravenous injection of mir20a-3p mimics 4h after induction of ischemia improved stroke outcomes in middle-aged female rats. Bioinformatics indicate that mir20a-3p modulates many mitochondrial genes, including Glycerol-3-Phosphate Acyltransferase, Cytochrome B5 Type B, Acyl-coenzyme A synthetase. This study tested the effect of mir20a-3p on mitochondrial function in astrocytes in normoxic and ischemic conditions.
Methods: Male and female human astrocyte cultures were assigned to normoxic (21% O 2 , 25 mM glucose) or ischemic (1% O 2 , 0 mM glucose) conditions for 6h. Cells were treated with 50 nM mir20a-3p, 50 nM scrambled control miR or vehicle. Mitochondrial function was assessed by Fluorescent Recovery After Photobleaching (FRAP).
Results: Imaging of astrocytes indicates FAM-labeled mir20a-3p uptake, and qRT-PCR analysis indicates a significant sex difference in mir20a-3p expression after OGD that recapitulates the in vivo phenotype. Treatment with mir20a-3p accelerated fluorescent recovery compared to cells treated with scrambled miR or vehicle in normoxia and OGD for both sexes. A significant sex difference was observed in fluorescent recovery, with the male cells experiencing a dampened effect in response to mir20a-3p or scrambled treatment.
Conclusion: Since the rate of fluorescent recovery measures mitochondrial membranes continuity, these data suggest that mir20a-3p promotes mitochondrial fusion. If greater continuity of the mitochondrial membrane compensates for the greater energy demand after stroke, these data may explain why stroke recovery is better in young females compared to males.
Supported by NIH RF1 AG042189 to FS and a TAMU Graduate Merit Fellowship to TB