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. 2023 Sep 27;9(39):eadg8148. doi: 10.1126/sciadv.adg8148

Fig. 5. The CHI3L1 receptor and downstream pathway in NSCs to regulate neurogenesis in neuroinflammation.

Fig. 5.

(A) Summary of CHI3L1 receptors and downstream signaling pathways. (B) Expression of CRTH2 receptor in NSCs (GFAP+Sox2+) but not immature (DCX+) or mature (NeuN+) neurons in adult hippocampus. Scale bars, 20 μm. (C) CRTH2 expression in cultured NSCs (Sox2+Nestin+). (D) Activation of GSK-3β (reduced inhibitory serine-9 phosphorylation) and inhibition of β-catenin (increased degradation) in NSC cultures treated with CHI3L1 (100 ng/ml). n = 3. (E) Lentiviral vectors to express two shRNAs for CRTH2 knockdown (Lenti-shCRTH2, shCRTH2_1, and shCRTH2_2) and a control shRNA (Lenti-shNC). (F) shRNA-mediated CRTH2 knockdown efficiency. n = 3. (G) CRTH2 knockdown effect on GSK-3β/β-catenin pathway activation by CHI3L1 (2-hour treatment). n = 3. (H) CRTH2 knockdown effect on NSC proliferation in shRNA-expressing cultures, with or without CHI3L1 for 3 days. n = 4. Scale bars, 100 μm. (I) CRTH2 knockdown effect on NSC differentiation into neurons (Tuj1+) and glia (GFAP+) in shRNA-expressing cultures, with or without CHI3L1 for 3 days. n = 4. Scale bars, 100 μm. (J) Identified CHI3L1 signaling pathway inhibitory to neurogenesis: CHI3L1 binds to CRTH2 receptor and activates GSK-3β that in turn phosphorylates and destabilizes β-catenin and leads to reduced transcriptional activities for neurogenesis. (K) Timeline for assays of NSC proliferation and neuronal differentiation in msCtrl-IgG– or msAQP4-IgG–injected mice, receiving a potent GSK-3β inhibitor, TWS119 (30 mg/kg), intraperitoneally daily for 3 weeks. (L to N) Quantification of total proliferating cells (EdU+), radial glia–like NSCs (EdU+GFAP+), and transiently amplifying progenitor-like (EdU+GFAP−) cells. n = 4. (O to Q) Quantification of proliferating cells (BrdU+) at the time of hsAQP4-IgG injection, newborn immature neurons (BrdU+DCX+), and mature neurons (BrdU+NeuN+). n = 4. All bar graphs presented in means ± SEM and analyzed by one-way ANOVA and Tukey’s post hoc analyses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.