Abstract WP247: Two Novel Interferon Signaling Proteins (Ifi27l2a And Ifitm3) Regulate Microglial Inflammation With Brain Aging And Following Stroke.

Abstract

Interferon signaling drives neuroinflammation in AD and stroke. Microglia (MG) play a critical role in initiating and spreading inflammation and are involved in the chronic recovery phase following stroke. We compared the MG transcriptomic response to stroke in young and aged mice by single-cell RNA sequencing (scRNAseq) to identify novel key players in inflammation. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) or sham surgery was performed in young and aged mice (3 & 20 months). scRNAseq was performed to define the transcriptional signature in brains at 14-days after stroke versus sham surgery. Primary MG culture was used to compare the mRNA levels of Ifi27l2a and Ifitm3 after cytokine treatment. We used qRT-PCR to validate scRNAseq findings and compare specific MG genes. Results: Using comprehensive scRNAseq analysis, our unbiased approach identified Ifi27l2a and Ifitm3 as the most highly up-regulated genes in aged MG following stroke. Further analysis showed a positive correlation between Ifi27l2a and Ifitm3 in MG ( R =0.4, p<0.001) as well as in all cells. Our qRT-PCR results validated scRNAseq findings and further showed that Ifi27l2a was significantly up-regulated in the thalamus of aged brains (p=0.04; n=4 young/aged). Expression was increased in cortex, but below significance level (p=0.23). We also found upregulation of Ifitm3 in brain at PSD 3 ( p =0.01), and trending at PSD 14 ( p =0.08) (n=5-6 stroke vs. sham). In contrast, Ifitm1 and Ifitm2 were not changed. Interestingly, Ifitm3 was recently proposed as an activator to γ-secretase. In vitro exposure of MG to inflammatory cytokines (TNFα and IFNγ, 6 hrs) caused significant increase in Ifi27l2a ( p =0.03, n=5-6) and Ifitm3 mRNA (p=0.02, n=5-6) compared to untreated MG. The in vitro co-expression of these genes was also well correlated ( R =0.5). Conclusions: We identified Ifi27l2a and Ifitm3 as highly up-regulated and correlated genes in MG in aging and following stroke. Our findings suggest that co-expression of Ifi27l2a and Ifitm3 may contribute to neuroinflammation. Further studies will be required to identify the specific functions of these genes and to determine the therapeutic potential of targeting these proteins to regulate neuroinflammation in aging and stroke.