Neural stem cells at sleep: Selective inhibition of orexin 2 receptor in mouse cortical neural stem cells increases proliferation and differentiation toward Oligodendrocytes Neda Karami1,2,3 , Hadi Aligholi2, Tahereh Kalantari1,Elahe merajifar4 , Zahra Zeraatpisheh2, Fatemeh Zebardast5 1. Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran Objectives Results Conclusion In multiple sclerosis, oligodendrocytes are destroyed by the immune system[1]. neural stem/progenitor cells have the capacity to differentiate into mature myelinating oligodendrocytes. In the present study, By doing gene set enrichment analysis using DAVID[2] and Panther[3] websites, it was found that the expression level of Gpr37l1 was significantly higher in oligodendrocyte progenitor cells when compared with oligodendrocytes (more than 300 fold-change in expression). The selective Orexin 2 recptor (HCRTR2) antagonist jnj-10397049 has been shown to inhibit this orphan GPCR[4].Therefore, in this study we scrutinized embryonic cortical neural stem cells survival and differentiation behavior after inhibition of gpr37l1 and HCRTR2 by jnj-10397049. MTT analysis demonstrated that JNJ10397049 at 1 and 5 micromolar dramatically increases proliferation of neural stem cells by 2.62 and 2.43 respectively. Gpr37l1 and orexin2 receptor are more expressed in embryonic mouse cortex NS/PCs than embryonic mouse ganglionic eminence by 3.45 and 4.57. The inhibitor after 7 days at dose 15 micro molar increased the expression of Cnpase, PDGFRalpha and GFAP by 1 , 20.56 and 1.47 fold, respectively. The expression of Olig2 was prominent at 15 micro molar, yet neither beta III tubulin nor nestin presence was observed with this dosage after treatment. In our experiment it was outlined that selective inhibition of Orexin 2 receptor prominently increased not only the proliferation of Cortical neural stem cells which are enriched with oligodendrocyte progenitor cells but also enhances the differentiation of these cells toward Oligodendrocytes. Inhibition of Orexin 2 receptor can be a valid drug target in inducing remyelination. In an ongoing research by our department by CRISPR-mediated Knock-out of GPR37L1 we are sought to test whether JNJ10397049 exerts its effect only through inhibition of Orexin 2 receptor or also by inhibiting GPR37L1 as well. References Methods References 1. Dendrou, C.A., L. Fugger, and M.A. Friese, Immunopathology of multiple sclerosis. Nat Rev Immunol, 2015. 15(9): p. 545-58. 2. Huang, D.W., B.T. Sherman, and R.A. Lempicki, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols, 2008. 4: p. 44. 3. Mi, H., et al., Large-scale gene function analysis with the PANTHER classification system. Nature Protocols, 2013. 8: p. 1551. 4. Ngo, T., et al., Orphan receptor ligand discovery by pickpocketing pharmacological neighbors. Nat Chem Biol, 2017. 13(2): p. 235-242. Gene set enrichment and pathway analysis was done using Panther and DAVID Databases. Gpr37l1 was chosen out of Top 15 highly expressed membrane receptor genes enriched in Oligodendrocyte progenitor cells in comparison to mature myelinating Oligodendrocytes. Primary cortex neural stem cells were derived from embryonic mouse 13.5 and cultured. All assays were done after passage 2. cytotoxicity effects of various doses of JNJ10397049 was screened using MTT assay. The expression of gpr37l1, hcrtr2, PDGFRalpha and Cnpase expression was analyzed using real time PCR in cortical and ganglionic eminence neural stem cells. For differentiation analysis DMEM/F12 was supplemented with 1% Fetal Bovine Serum. GFAP, Cnpase and PDGFRalpha expression was analyzed using Real-time PCR on day 3 and 7 after treatment with 10, 15 and 20 micro-molar of JNJ10397049. The expression of Olig2, beta III tubulin, Myelin Basic Protein and nestin on day 7 after differentiation induction was shown by Immunocytochemistry.