Fig 2: The CRTH2 blockade effect on hippocampal neurogenesis and cognitive performance affected by CHI3L1.(A) Schematic diagram of assays for shRNA-mediated CRTH2 knockdown effect on NSC proliferation and differentiation affected by msAQP4-IgG. The lentiviruses expressing GFP together with a scrambled nontargeting control shRNA (Lenti-shNC) or an shRNA targeting CRTH2 (Lenti-shCRTH2_1). Scale bars, 100 μm. (B) CRTH2 knockdown effect on NSC proliferation in DG, quantified by the numbers of proliferating cells expressing an shRNA (GFP+EdU+) and radial glia–like NSCs (GFP+EdU+GFAP). n = 4 animals. Scale bars, 20 μm. (C) Quantification of shRNA-expressing (GFP+) NSC (BrdU+) differentiation into immature neurons (DCX+) in DG. n = 4. Scale bars, 20 μm. (D) Quantification of NSC differentiation into mature neurons (GFP+BrdU+NeuN+) in DG. n = 4. Scale bars, 20 μm. (E) Schematic diagram of in vitro assays for the effect of AZD1981, a selective CRTH2 antagonist, on neurogenesis suppressed by CHI3L1 signaling secondary to mini-pump infusion of msCtrl-IgG or msAQP4-IgG. (F) Quantification of NSC proliferation in DG, in the presence of CHI3L1 or PBS and with AZD1981 or DMSO. n = 4. Scale bars, 100 μm. (G) Quantification of NSC neuronal differentiation. n = 4. Scale bars, 100 μm. (H) Schematic diagram of in vivo assays for AZD1981 efficacy to rescue neurogenesis affected by msAQP4-IgG–induced CHI3L1 signaling. (I and J) Quantification of NSC proliferation by computing total proliferating cells (EdU+) and radial glia–like NSCs (EdU+GFAP+Sox2+) in DG. n = 4. (K and L) Quantification of NSC proliferation into immature (BrdU+DCX+) and mature (BrdU+NeuN+) neurons. n = 4 animals. All quantitative data presented as bar graphs in means ± SEM and evaluated by one-way ANOVA and Tukey’s post hoc multiple comparisons. Nonsignificant comparisons are not identified. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: The effects of astrocyte-specific CHI3L1 knockout on hippocampal neurogenesis and cognitive performance.(A) Schematic diagram of the astrocyte-specific CHI3L1 knockout by intra-hippocampus injections of AAVs expressing Cre recombinase plus eGFP by GFAP promoter in heterozygous (Chil1f/+) or homozygous (Chil1f/f) CHI3L1-floxed mice. The injected mice were sacrificed 2 weeks later for knockout efficiency or received the intra-hippocampal injections of msCtrl-IgG or msAQP4-IgG 4 weeks later. (B) Representative images of Chil1f/f hippocampal section expressing Cre. Scale bars, 100 μm. (C) Evaluation of CHI3L1 knockout efficiency by qPCR on hippocampal lysates from AAV-injected Chil1f/+ and Chil1f/f mice. n = 3 animals. (D) Representative MWM movement paths of CHI3L1-expressing (Chil1f/+) and CHI3L1-deficient (Chil1f/f) mice receiving stereotaxic injections of msCtrl-IgG or msAQP4-IgG. (E) Quantification of MWM crossing numbers. n = 9. (F) Quantification of MWM escape latencies to find the platform. n = 9. (G) Schematic diagram of assays for astrocyte-specific CHI3L1 knockout effect on adult hippocampal neurogenesis affected by mini-pump infusion of msCtrl-IgG or msAQP4-IgG. (H and I) Quantification of NSC proliferation plotted by total proliferating cells (EdU+) and radial glia–like NSCs (EdU+GFAP+). n = 4. (J and K) Quantification of NSC proliferation into immature (BrdU+DCX+) and mature (BrdU+NeuN+) neurons. n = 4. (L) Timeline of assays for NSC proliferation and neuronal differentiation in the msCtrl-IgG– or msAQP4-IgG–treated mice, receiving the coinjections of neutralizing antibody anti-CHI3L1 or PBS. (M to O) Quantification of total proliferating cells (EdU+), radial glia–like NSCs (EdU+GFAP+), and transiently amplifying progenitor-like cells (EdU+GFAP−). n = 4. (P and Q) Quantification of proliferating (BrdU+) cells at the time of IgG injection, newborn immature neurons (BrdU+DCX+), and mature neurons (BrdU+NeuN+). n = 4. All quantitative data presented in means ± SEM; (C), by Student’s t test; (F), by two-way ANOVA with Tukey’s post hoc analyses; the remaining bar graphs, by one-way ANOVA with Tukey’s post hoc analyses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4: Characterization of CHI3L1 induction and inflammatory features of primary astrocytes activated by the proinflammatory stimuli.(A) Schematic diagram showing the treatment of primary mouse astrocyte cultures with IL-1β (100 ng/ml) or PBS, the monoclonal mouse antibodies against AQP4 (msAQP4-IgG, 100 ng/ml) or the control mouse IgG (msCtrl-IgG, 100 ng/ml), and human anti-AQP4 autoantibodies purified from NMO patients (hsAQP4-IgG, 100 ng/ml) or control human IgG (hsCtrl-IgG, 100 ng/ml). (B) Relative levels of CHI3L1 mRNA in primary astrocytes treated with IL-1β, msAQP4-IgG, or hsAQP4-IgG. n = 3. (C) Representative images and quantification of astroglial marker expression in astrocyte cultures treated with PBS or IL-1β after 24 hours. n = 4. (D) Representative images and quantification of astroglial marker expression in astrocyte cultures treated with msAQP4-IgG or hsAQP4-IgG, alongside msCtrl-IgG or hsCtrl-IgG for 24 hours. n = 4. (E) RNA-seq of hsCtrl-IgG– and hsAQP4-IgG–treated astrocytes, with 91 DEGs being overlapped with the dataset of Walker-Caulfield et al. (37), shown in hierarchical clustering heatmap of gene fold changes (normalized to the untreated astrocytes). (F) Venn diagram of the overlapped DEGs in comparison to the genome-wide dataset of all secreted proteins. (G) GO enrichment analysis of the 91 overlapped DEGs. (H) Hierarchical clustering of 21 overlapped DEGs that were secreted proteins. (I) Analysis of the gene fold changes in the overlapped DEGs with the GO of neuroinflammation. (J) Analysis of the gene fold changes in the overlapped DEGs by the gene set of astrocyte activation [Zamanian et al. (42); “reactive”]. Scale bar, 100 μm. The bar graphs were presented as means ± SEM; the statistical evaluation of (C) and (D) was performed with Student’s t test for two conditions; for (B), with one-way analysis of variance (ANOVA) and Tukey’s post hoc multiple comparisons. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: The effects of CHI3L1 overexpression on hippocampal neurogenesis and cognitive performance.(A) Lentiviral vectors of control (Lenti-Ctrl) or CHI3L1expression (Lenti-CHI3L1), with eGFP coexpression. (B) Timeline of assays for NSC proliferation (by EdU labeling, green arrows) and neuronal differentiation (by BrdU labeling) and their behavioral consequences (red arrows) in control or CHI3L1-overexpressing mice. (C) CHI3L1 expression by immunoblotting of hippocampal lysates from control or CHI3L1-overexpressing mice. (D and E) Representative movement paths and quantification of MWM crossing number control for CHI3L1-overexpressing mice. Lenti-Ctrl, n = 8 animals. Lenti-CHI3L1, n = 12. (F) Escape latencies to find the platform in MWM. Lenti-Ctrl, n = 8. Lenti-CHI3L1, n = 12. (G) Representative images of Lenti-Ctrl– or Lenti-CHI3L1–injected hippocampus immunostained for NSC proliferation. Scale bar, 100 μm. (H to J) Quantification of total proliferating cells (EdU+), radial glia–like NSCs (EdU+GFAP+), and transiently amplifying progenitor-like cells. n = 4 animals. (K) Representative images and quantification of newborn immature neurons (BrdU+DCX+) in Lenti-Ctrl– or Lenti-CHI3L1–injected hippocampus. n = 4. Scale bar, 100 μm. (L) Representative images and quantification of newborn immature neurons (BrdU+NeuN+) in Lenti-Ctrl– or Lenti-CHI3L1–injected hippocampus. n = 4. Scale bar, 100 μm. All data presented in means ± SEM; bar graphs (E) and (H) to (L), by Student’s t test for two-group comparisons; (F), by two-way ANOVA with Tukey’s multiple comparisons test. Nonsignificant comparisons are not identified. *P < 0.05, **P < 0.01, ***P < 0.001.