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  • br Materials and methods br Results br Discussion The compou

    2021-09-22


    Materials and methods
    Results
    Discussion The compound P7C3 has been reported to activate NAMPT (Wang et al., 2014) and augment NAD+ levels, thereby promoting the survival of mature neurons and postnatal neurogenesis throughout the post-ischemic 2-D08 (Loris et al., 2017). However, it has been suggested that GSK-3 inhibition (by Ser9 phosphorylation) is the key effector in mediating the preservation of NAD+ content and limiting mitochondrial damage in the protection of ischemic postconditioning (Juhaszova et al., 2004). In addition, modulation of the expression of NAD+-dependent sirtuins proteins, such as SIRT1 and SIRT2, through the activation of the Akt/GSK-3 signaling pathway is required for the promotion of mouse embryonic stem cells (Si et al., 2013; Koga et al., 2015). Accordingly, we propose that the preservation of brain function, BBB integrity, and neuronal survival triggered by activating NAD+/NAMPT in ischemic mice may depend on the activation of Akt/GSK-3 signaling. Furthermore, researchers have demonstrated that the inhibition of GSK-3 activity and its downstream signaling is an effective neuroprotective strategy against ischemic stroke (Chuang et al., 2011; Chern et al., 2012). Conversely, the activation of GSK-3 mediates the generation of an inflammatory environment and damages adult neurogenesis (Lucas et al., 2001; Sirerol-Piquer et al., 2011). Thus, we propose GSK-3 activity as a core target in the treatment of ischemic stroke. In this study, we observed a slight increase in the number of cells involved in endogenous neurogenesis near the peri-infarct cortex and SVZ zone in CI/R mice; however, these effects were very limited within 24 h immediately after cerebral ischemic injury. These findings are consistent with those in our previous reports (Chern et al., 2012, Chern et al., 2014; Chien et al., 2016). Nonetheless, P7C3 treatment dramatically enhanced the number of DCX-positive and β-tub3-positive, two makers of neuroblasts/neuronal progenitor cells, within 24 h after cerebral ischemic stroke. This enhancement most likely occurred through the activation of cAMP/PKA and PI3K/Akt, two important signaling pathways following GLP-1R activation (Meng et al., 2016; Koole et al., 2013; Ying et al., 2015). This, in turn, would cause GSK-3 inhibition and β-catenin activation for the upregulation of its downstream cell protective proteins (e.g., Bcl-2, ki67, DCX, and β-tub3) as well as the neurodevelopmental proteins adam11 and adamts20 (Chuang et al., 2011; Chern et al., 2012, Chern et al., 2014; Gao et al., 2017; Chien et al., 2016; McEntee et al., 1999). However, treatment of P7C3 alone in mice without CI/R injury (sham) did not significantly activate the GLP-1R-associated signaling (activation of cAMP/PKA and PI3K/Akt) in this study. This lack of activation could be partially explained by the observation that GLP-1R is up-expressed (compared to sham control) in the ischemic region at 12–24 h after an ischemic insult (Lee et al., 2011), and we propose that the dose of P7C3 used in this model was not sufficiently potent to trigger the activation of GLP-1R in sham-operated mice, which display relative lower levels of GLP-1R expression (Lee et al., 2011). It has been reported that the activation of GLP-1R is neuroprotective in cases of stroke (Darsalia et al., 2016) and cerebral ischemia (Lee et al., 2011). The GLP-1R is particularly well documented for its role in Gαs coupling, favoring the production of cyclic adenosine monophosphate (cAMP) through an increase in the enzymatic activity of adenylate cyclase with consequent activation of PKA. This results in β-catenin phosphorylation/activation, leading to its nuclear translocation and initiation of Bcl-2 upregulation (Meng et al., 2016; Koga et al., 2015). In addition, activated PKA phosphorylates PI3K, followed by the activation of Akt and the subsequent phosphorylation/inactivation of GSK-3 (SpGSK-3) for β-catenin activation as well as the inhibition of caspase and NFκB activity (Chuang et al., 2011; Meng et al., 2016; Koga et al., 2015). In this study, we found that the protective effects of P7C3 were dramatically compromised by the GLP-1R antagonist (exendin 9–39 (E9)) and that application of a GLP-1R agonist (exendin-4) led to a survival rate comparable to that of P7C3. In addition, surface plasmon resonance (SPR) assays revealed that P7C3 and GLP-1 bind GLP-1R with a Kd value of 0.53 ± 0.04 (μM) and 2.73 ± 0.16 (μM), respectively. Furthermore, P7C3 ameliorated CI/R-mediated decline in animal survival and increased cyclic adenosine monophosphate (cAMP) production, both of which were antagonized by exendin9–39 co-treatment. Moreover, P7C3 promoted the secretion of insulin in a GLP-1R expressing beta cell line, but not in sham-operated and ischemic stroke mice (unpublished data). Finally, molecular modeling enabled us to predict that P7C3 would bind at the positive allosteric site of GLP-1R (Fig. 8). Although similar observations related to the P7C3-induced inhibition of GSK-3 for neuronal protection have previously been reported (Gu et al., 2017), here our results further revealed that activation of cAMP/PKA/β-catenin and Akt/GSK-3/β-catenin pathways is modulated by P7C3, and that the protective and neurogenesis-promoting effects were nearly completely reversed by a GLP-1P antagonist (exendin 9–39). Thus, we propose that GLP-1R positive allosteric activation is involved in the neuronal protective and neurogenesis-promoting effects of P7C3 against CI/R injury.