In the present study we
In the present study, we showed that reduced neuron formation in vitro induced by IL-1β was ameliorated by activation of GPR55 in both human and mouse cultures of NSCs. Pre-treatment with GPR55 agonists also blocked upregulation of inflammatory cytokine receptor mRNA (IL-1R1, IL-6st) while increasing mRNA for anti-inflammatory or neuroprotective cytokine receptors (IL-10Rα, TNFR2) within NSCs. This was apparent in both human and mouse cells. We also show for the first time that GPR55 activation induces neuronal differentiation of murine primary hippocampal NSCs similar to results seen with human NSCs as reported previously(Hill et al., 2018). In vivo, we sought to determine if a chronic, low-level GSK-3 Inhibitor IX (systemic administration of LPS) would elicit negative effects on hippocampal neurogenesis similar to those seen after high level, acute infection. Interestingly, a dose of 0.2 mg/kg/day of LPS was sufficient to significantly reduce NSC survival (as measured by BrdU) and neuroblast formation within the SGZ of the hippocampus. Direct intrahippocampal administration of O-1602, a potent GPR55 agonist, protected against reduced NSC survival and neuroblast formation during insult with chronic, low-level infusion of LPS. We determined that neuroprotective effects were elicited by direct involvement on NSCs as microglial activation (detected by comprehensive image analysis) did not differ between LPS animals that received vehicle as compared to O-1602 treatment. To further assess GPR55 function during the immune response, we treated animals with a low-level, chronic LPS paradigm and assessed inflammatory mRNA levels within the hippocampus showing an altered immune response in GPR55−/− animals. The presented data suggest that GPR55 can protect NSCs against inflammatory insult and poses a potent target with therapeutic potential. Inflammatory insult with IL-1β on NSCs has been extensively studied showing reduced proliferation and neuronal differentiation rates both in vitro and in vivo (Wu et al., 2013, Zhang et al., 2013, Crampton et al., 2012). We chose to focus on IL-1β for in vitro studies since insult with IL-1β has also been studied in the context of cannabinoid signaling and neuroprotection (Garcia-Ovejero et al., 2013). Our results indicate that pre-treatment with the GPR55 agonists ML184 or O-1602 on human or mouse NSCs, respectively, is neuroprotective against reduced neuronal formation caused by insult with IL-1β. This effect was attenuated by concurrent treatment with GPR55 antagonist, suggesting that these effects are GPR55-dependent. These results are consistent with other studies of the effects of CB1 and CB2 receptors on protection against insult by IL-1β implicating GPR55 as another target for therapeutic intervention against NSC injury. O-1602 has also been implicated in protection of neuronal cell viability, yet the exact receptor necessary for this effect was not elucidated (Janefjord et al., 2014). The number of GFAP positive cells was also decreased in samples treated with GPR55 agonist in combination with IL-1β indicating that this was not simply a blunting of gliogenesis, but rather a protective effect of GPR55 activation. Interestingly, we found that treatment of human NSCs with IL-1β reduced the number of GFAP positive cells in comparison to vehicle- treated samples. This observation goes against the notion that gliogenesis is increased during insult with inflammatory mediators. We speculate that this may be due to slowing of proliferation rates, thus resulting in fewer positive cells. Our studies in primary murine NSCs did show increases in GFAP-positive cells with IL-1β treatment similar to findings from other studies suggesting to us that our methods for quantification of neurogenesis were not faulty (Green and Nolan, 2012, Crampton et al., 2012). Numerous mechanisms have also been linked to IL-1β-induced reductions in neurogenesis directly within NSCs, including regulation of the transcription factor TLX and p53-dependent apoptosis (Ryan et al., 2013, Guadagno et al., 2015). We did not examine directly intracellular mechanisms in which GPR55 activation protected against reduced neurogenesis facilitating a need for more in-depth study of the exact mechanisms necessary for the neuroprotective effects of GPR55.