Abstract TMP120: Transcriptomic Analysis Reveals Common Age Independent Immunomodulatory Proteins As A Mode Of Neuroprotection In P2X4R KO Mice After Ischemic Stroke

Background and Purpose: Identification of a new potential drug target protein and their plausible mechanism for stroke treatment is critically needed. We earlier showed that genetic deletion as well as short term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides acute neuroprotection and thus can be potential drug targets to treat ischemic stroke. However, potential mechanisms remain unknown. Therefore, in this study, we employed RNA-seq technology to identify the gene expression profiles, pathways analysis and qPCR validation of differentially expressed genes (DEGs). This analysis will identify role of those DEGs in certain biological processes responsible for P2X4R dependent neuroprotection after stroke. Methods: We subjected young (8-12 weeks-old n= 4/group), and aged (12-18-month-old; n=8/group) male and female Global P2X4 KO and littermate WT mice to right middle cerebral artery occlusion MCAo for 60 min followed by 3 day of reperfusion. After 3 days, mice were sacrificed and prefrontal cortex tissue was isolated to extract total RNA using Trizol and used for RNA-seq sample preparations as well as for validation by Nanostring mediated qPCR technique. DESeq2 and Gene Ontology (GO) and /or Ingenuity Pathway Analysis (IPA) were used to identify mRNA transcript expression profiles and biological pathway. qPCR was analyzed with nSolver Data Analysis Support system. Results: We found 2246 DEGs in P2X4R KO vs WT tissue after stroke. Out of these DEGs 1920 gene were downregulated and 325 genes were upregulated in KO. GO/IPA analysis of top 300 DEGs suggests an enrichment of ion channel transport system, inflammation and extracellular matrix component genes. QPCR validation of top 30 DEGs revealed down regulation of two common age independent genes: Lnterleukin-6 ( IL-6) , an inflammatory cytokine and Cytotoxic T Lymphocyte-Associated Protein 2 alpha ( Ctla2a ), an immunosuppressive factor KO group. Conclusion: This data suggests P2X4R mediated neuroprotection after stroke is brought by attenuation of immune modulatory pathways in both young and aged mice of both sexes. Future studies will delineate the detailed role of IL-6 and Ctla2a in P2X4R mediated neuroprotection mechanisms after stroke.