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WO2024192765A1 - First in class use of the crth2 antagonist in treating neuromyelitis optica spectrum disorder (nmosd) and relevant autoimmune cns disorders - Google Patents

First in class use of the crth2 antagonist in treating neuromyelitis optica spectrum disorder (nmosd) and relevant autoimmune cns disorders Download PDF

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Publication number
WO2024192765A1
WO2024192765A1 PCT/CN2023/083374 CN2023083374W WO2024192765A1 WO 2024192765 A1 WO2024192765 A1 WO 2024192765A1 CN 2023083374 W CN2023083374 W CN 2023083374W WO 2024192765 A1 WO2024192765 A1 WO 2024192765A1
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chi3l1
subject
igg
crth2
nmosd
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French (fr)
Inventor
Changyong TANG
Yu-Wen Alvin HUANG
Wei Qiu
Yaxiong CUI
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Tsinghua University
Sun Yat Sen University
Brown University
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Tsinghua University
Sun Yat Sen University
Brown University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol

Definitions

  • NMOSD Neuromyelitis optica spectrum disorder
  • the present disclosure provides a method of treating neuroinflammation in a subject, the method comprising: administering to the subject an effective amount of a CRTH2 inhibitor, thereby treating the neuroinflammation in the subject.
  • the present disclosure provides a CRTH2 inhibitor for use in the treatment of neuroinflammation in a subject.
  • the present disclosure provides the use of a CRTH2 inhibitor for the manufacture of a medicament for the treatment of neuroinflammation in a subject.
  • the subject has a Neuromyelitis Optica Spectrum Disorder (NMOSD) .
  • NOSD Neuromyelitis Optica Spectrum Disorder
  • the subject has Alzheimer’s disease, glioblastoma, or an autoimmune disorder.
  • the autoimmune disorder is multiple sclerosis.
  • the subject has malignancy of glioblastoma.
  • the present disclosure provides a method of treating an autoimmune CNS disorder in a subject, the method comprising: administering to the subject an effective amount of a CRTH2 inhibitor, thereby treating the autoimmune CNS disorder in the subject.
  • the present disclosure provides a CRTH2 inhibitor for use in the treatment of autoimmune CNS disorder in a subject.
  • the present disclosure provides the use of a CRTH2 inhibitor for the manufacture of a medicament for the treatment of autoimmune CNS disorder in a subject.
  • the present disclosure provides a method of treating a Neuromyelitis Optica Spectrum Disorder (NMOSD) in a subject, the method comprising: administering to the subject an effective amount of a CRTH2 inhibitor, thereby treating the NMOSD in the subject.
  • the present disclosure provides a CRTH2 inhibitor for use in the treatment of a Neuromyelitis Optica Spectrum Disorder (NMOSD) in a subject.
  • the present disclosure provides the use of a CRTH2 inhibitor for the manufacture of a medicament for the treatment of a Neuromyelitis Optica Spectrum Disorder (NMOSD) in a subject.
  • the CRTH2 inhibitor comprises a small molecule, an antibody molecule, a nucleic acid, or a polypeptide.
  • the CRTH2 inhibitor comprises AZD1981, fevipiprant, OC00459, BI671800, AMG853, TQC3564, setipiprant, ASP5642, ARRY-502, AZD6430, ADC-3680, MK-1029, MK-7246, or BI1021958.
  • the CRTH2 inhibitor is administered at a dose of 25 to 1200mg.
  • the CRTH2 inhibitor is administered daily or twice daily.
  • the CRTH2 inhibitor is administered intravenously, orally, parenterally, or topically.
  • the NMO is monophasic NMO, relapsing NMO, acute NMOSD, chronic NMOSD, AQP4 antibody negative NMOSD, AQP4 antibody positive NMOSD, MOG antibody negative NMOSD, and/or MOG antibody positive NMOSD.
  • the subject is suffering from a neurological symptom, neuroinflammation, cognitive impairment, loss of spinal cord functions, loss of optic nerve functions, vision loss, and/or motor deficits.
  • treatment results in lessening of cognitive impairment in the subject. In some embodiments, treatment results in an increase in neurogenesis in the subject. In some embodiments, treatment results in an increase in neural stem cell proliferation in the subject. In some embodiments, treatment results in an increase in neural stem cell differentiation in the subject.
  • the subject has detectable levels of CHI3L1 prior to the administration. In some embodiments, the subject has an elevated level of CHI3L1 compared to a healthy subject prior to the administration. In some embodiments, the subject has an elevated level of astrocyte activation prior to the administration. In some embodiments, the subject has detectable levels of CRTH2 prior to the administration. In some embodiments, the subject is a human.
  • the present disclosure provides a method of treating neuroinflammation in a subject, the method comprising: administering to the subject an effective amount of a CHI3L1 inhibitor, thereby treating the neuroinflammation in the subject.
  • the present disclosure provides a CHI3L1 inhibitor for use in the treatment of neuroinflammation in a subject.
  • the present disclosure provides the use of a CHI3L1 inhibitor for the manufacture of a medicament for the treatment of neuroinflammation in a subject.
  • the CHI3L1 inhibitor comprises a small molecule, an antibody molecule, a nucleic acid, or a polypeptide.
  • the subject has a Neuromyelitis Optica Spectrum Disorder (NMOSD) .
  • NOSD Neuromyelitis Optica Spectrum Disorder
  • Figure 1A is a schematic diagram showing the treatment of primary cultures of wildtype mouse astrocytes with the classic pro-inflammatory cytokine IL-1 ⁇ (100 ng/ml) or the control of vehicle PBS, the monoclonal mouse antibodies against AQP4 (msAQP4-IgG, 100 ng/ml) or the control mouse IgG (msCtrl-IgG, 100 ng/ml) , and human anto-AQP4 autoantibodies purified from NMO patients (hsAQP4-IgG, 100 ng/ml) or control human IgG (hsCtrl-IgG, 100 ng/ml) for 6, 12 and 24 hours.
  • Figure 1B depicts relative levels of CHI3L1 mRNA induction in primary astrocytes treated with IL-1 ⁇ , msAQP4-IgG or hsAQP4-IgG at each time point assayed by qPCR and normalized to the levels of the individual control conditions (set as 1.0) .
  • n 3 per group.
  • the bar graphs are presented as the mean ⁇ SEM; the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 1C depicts representative confocal images of (top) and the quantification of (bottom) astroglial marker expression in mouse primary astrocytes treated with vehicle (PBS) or IL-1 ⁇ (100 ng/ml) after 24 hours.
  • Top immune-fluorescent labeling, from left to right, of GFAP, AQP4, CHI3L1 and DAPI.
  • the bar graphs of are presented as the mean ⁇ SEM; the statistical evaluation was performed with Student’s t test for 2 conditions. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 1D depicts representative immune-fluorescent images (left panels) and the quantification of fluorescence signals (right bar graphs) of, from left to right, GFAP, AQP4, CHI3L1 and DAPI in mouse primary astrocytes treated with msAQP4-IgG (100 ng/ml) or hsAQP4-IgG (100 ng/ml) alongside the individual controls (msCtrl-IgG or hsCtrl-IgG, 100 ng/ml, value set as 1 for normalization) for 24 hours.
  • the bar graphs of are presented as the mean ⁇ SEM; the statistical evaluation was performed with Student’s t test for 2 conditions. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 1E depicts transcriptomic analyses of mouse astrocytes activated by AQP4 autoantibodies (3 biological replicates with 3 different batches of human IgG purification) , with the list of the identified 152 differentially expressed genes (DEGs) from bulk RNA-sequencing being contrasted with the published dataset by Waler-Caulfield et al. (PMID: 26423139) .
  • DEGs differentially expressed genes
  • the 91 DEGs overlapped between two studies were analyzed by the hierarchical clustering heatmap of gene fold changes on a log2 scale.
  • Figure 1F depicts a Venn diagram of the DEGs identified by two independent transcriptomic studies of mouse astrocytes stimulated by human anti-AQP4 antibodies (hsAQP4-IgG vs. hsCtrl-IgG) , showing that 21 out of the 91 overlapped genes were secreted proteins.
  • Figure 1G depicts Gene Ontology (GO) enrichment analysis of the 91 overlapped DEGs.
  • Figure 1H depicts hierarchical clustering of 21 overlapped DEGs that were secreted proteins presented in the heatmap of gene fold on a log2 scale; 20 upregulated (including CHI3L1 among the top genes) and one, Igsf21, downregulated by immune-mediated astroglial activation.
  • Figure 1I depicts the analysis of the gene fold changes in the overlapped DEGs with the GO of Neuroinflammation, plotted on a log10 scale in the comparison of the untreated samples to unmask the effect of control IgG treatment (zero-fold change, white) .
  • Figure 1J depicts the analysis of the gene fold changes in the overlapped DEGs with a published transcriptional response pattern of astrocyte activation (PMID: 22553043; “reactive” ) .
  • Figure S1A depicts data from evaluation of CHI3L1 induction in neuroinflammation by transcriptomic analyses of wildtype mouse astrocytes in primary cultures treated with pro-inflammatory cytokines IL-1 ⁇ , TNF and C1q (ITC for 24h; ‘in vitro’ ) and acutely purified from mice stimulated by intraperitoneal injection of liposaccharide (LPS after 24h; ‘in vivo’ ) , based on the bulk RNA-Seq datasets from Guttenplan et al. (PMID: 32719333) and Hasel et al. (PMID: 34413515) .
  • Figure S1B depicts functional principal component analysis (PCA) of bulk RNA-Seq from this study on primary mouse astrocytes treated with hsCtrl-IgG or hsAQP4-IgG, 100 ng/ml for 24h, showing separation between control and hsAQP4-IgG treated conditions.
  • PCA functional principal component analysis
  • Figure S1C is a volcano plot of gene fold changes induced by hsAQP4-IgG as compared to the hsCtrl-IgG stimulation from this study. p value ⁇ 0.05 and a fold change cutoff of log2 ratio ⁇ 0.5 were used to detect the differentially expressed genes (DEGs) . There were110 upregulated genes and 42 downregulated genes. The exemplary secreted protein genes overlapped with the published dataset of Waler-Caulfield et al. (PMID: 26423139) were indicated.
  • Figure S1D shows GO enrichment analysis of DEGs between primary astrocytes treated with hsCtrl-IgG or hsAQP4-IgG from this study.
  • Figure S1E shows a pathway visualization of the NF ⁇ B canonical pathway, identified via pathway visualization of the 91 overlapped DEGs performed by extracting the Kyoto Encyclopedia of Genes and Genomes database (KEGG) with the data integration tool of Pathview.
  • KEGG Kyoto Encyclopedia of Genes and Genomes database
  • Figure 2A depicts measurements of CHI3L1 levels in serum and CSF samples from healthy controls (HCs) and NMO patients by ELISA presented as the mean ⁇ SEM.
  • Statistical significance was evaluated with Student’s t test for two-group comparisons; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 2B shows representative confocal images of biopsied human brains tissues from a control subject and a NMO patient, the characteristic astroglial features of decreased AQP4 expression (second panel from left) particularly within the end-feet covering the micro-vessels (labeled by CD31 for outlining endothelial cells, left panel) , together with the increased GFAP (middle panel) expression.
  • Scale bar 50 ⁇ m.
  • Figure 2C depicts the correlation between CHI3L1 CSF levels and neuropsychological assessments in NMO patients, presented in a bubble chart of the Pearson correlation coefficient.
  • CHI3L1 levels positively correlated with increased anxiety and depression-like symptoms assayed by HARS and HDRS tests, and negatively correlated with the cognitive performance of learning and memory functions assayed by tests of BVMT-R, multiple CLVT formats, SDMT and MMSE.
  • Figure 2D shows assessments of the hippocampal formation volumes from the head MRI scans of healthy controls (HCs, left boxes) and NMO patients (right boxes) presented as the mean ⁇ SEM.
  • Statistical significance was evaluated with Student’s t test for two-group comparisons; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 2E depicts a schematic diagram for experiments to demonstrate the effect of CHI3L1 on the proliferation and neuronal differentiation of neural stem cells (NSCs) in vitro.
  • the NSCs were first stimulated with recombinant CHI3L1 protein (100ng/mL) or vehicle control of PBS for 3 days in the proliferation media.
  • EdU was administered in half of the treated cultures for 2 hours before the fixation and immuno-staining to evaluate the proliferation.
  • the remaining half of the NSC cultures were switched into the differentiation media, being allowed to differentiate for additional 3 days with the incubation of PBS or CHI3L1 (100 ng/ml) , and were then fixed and subject to confocal analyses for neuronal differentiation.
  • Figure 2F shows imaging and quantification of NSC proliferation in the absence (PBS) or presence of CHI3L1 stimulation presented as the mean ⁇ SEM.
  • Left the nuclear fluorescence of the colocalized EdU and DAPI signals in PBS-or CHI3L1-treated NSC cultures.
  • Right the average numbers of the percentage of EdU+ nuclei in the DAPI-labeled ones were plotted.
  • n 4 independent experiments.
  • Scale bars 100 ⁇ m.
  • Statistical significance was evaluated with Student’s t test for two-group comparisons; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 2G shows representative confocal images and the analyses of NSC neuronal differentiation with or without CHI3L1 treatment presented as the mean ⁇ SEM.
  • Left Tuj1 to mark the differentiated neurons; immune-staining of GFAP and DAPI.
  • Right quantification of Tuj1+ and GFAP+ cells graphed in the average percentage of colocalization with DAPI+ cells from 4 independent experiments. Scale bars, 100 ⁇ m.
  • Statistical significance was evaluated with Student’s t test for two-group comparisons; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 2H depicts a schematic diagram for the experiments to test the effect of astrocyte-secreted CHI3L1 on proliferation and neuronal differentiation of NSCs.
  • the primary cultures of mouse astrocytes were first treated with hsCtrl-IgG or hsAQP4-IgG at 100 ng/ml for 24 hours.
  • the astrocyte-conditioned media (ACM) were harvested and treated with vehicle control or the neutralizing anti-CHI3L1 antibodies (100 ng/ml) to eliminate the secreted CHI3L1 after the removal of IgG contents.
  • the cleared ACM were then supplemented with factors to constitute the growth media for the proliferation or the differentiation of NSCs.
  • the purified mouse NSCs were cultured in ACM-based proliferation media for 3 days.
  • Figure 2J shows representative images of proliferating mouse NSCs (EdU+) grown in hsCtrl-IgG-treated or hsAQP4-IgG-treated astrocyte-conditioned media (ACM) , without (vehicle) or with the clearance of CHI3L1 (by anti-CHI3L1 neutralizing antibodies) .
  • Scale bars 100 ⁇ m.
  • Figure 2L shows representative images of newly differentiated neurons from NSCs of 4 conditions –grown in hsCtrl-IgG-ACM or hsAQP4-IgG-ACM, without (vehicle) or with CHI3L1 clearance (anti-CHI3L1) –immune-stained with nuclear marker DAPI and neural markers of Tuj 1 and GFAP. Scale bars, 100 ⁇ m. Statistics were computed with one-way ANOVA and Tukey’s post hoc multiple comparisons; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure S2A shows representative MRI images of brain, spinal cord and optic nerve from healthy control (HC) subjects and NMO patients. The typical lesions of neuromyelitis and optic neuritis were indicated by arrows.
  • Figure S2B shows the atlas-based segmentation of the hippocampus on the representative MRI head images with 3D rendering, to facilitate the analyses of the anatomical structures within the hippocampus region and complement the results in Fig. 2D.
  • Figure S2C shows the scoring of the neuropsychological tests in health control (HC, left boxes) subjects and NMO patients (right boxes) , plotted in the box chart of distribution for the test actual scores.
  • Data are presented as the mean ⁇ SEM; the statistical significance was evaluated with Student’s t test for two-group comparisons (HC vs. NMO) .
  • Figure S2D shows the 3D size quantification of the hippocampal subfields on the left side for healthy control (HC, left boxes) subjects and NMO patients (right boxes) .
  • Data are presented as the mean ⁇ SEM; the statistical significance was evaluated with Student’s t test for two-group comparisons (HC vs. NMO) .
  • Figure S2E shows the 3D size quantification of the hippocampal subfields on the right side for healthy control (HC, left boxes) subjects and NMO patients (right boxes) .
  • Data are presented as the mean ⁇ SEM; the statistical significance was evaluated with Student’s t-test for two-group comparisons (HC vs. NMO) .
  • Figure 3A depicts a schematic diagram and the timeline of the experiments to assay the hippocampal neurogenesis and cognitive performance in 8-week-old wildtype mice receiving the stereotaxic delivery of the control (msCtrl-IgG) or the monoclonal mouse anti-AQP4 antibodies (msAQP4-IgG) into the dentate gyrus within the hippocampal formation on both sides, at the amount of 1 ⁇ g IgG in 1 ⁇ l PBS for each side.
  • the control msCtrl-IgG
  • msAQP4-IgG monoclonal mouse anti-AQP4 antibodies
  • Figure 3C depicts immunostaining of the astrocyte marker GFAP for quantification of astrogliosis in response to msAQP4-IgG treatment, presented in the average numbers of GFAP+ cells within the volume of dentate gyrus (DG, determined by 3D rendering of confocal images) .
  • DG dentate gyrus
  • Figure 3D shows assessment of CHI3L1 expression in astrocytes induced by msAQP4-IgG, quantified by the intensity of CHI3L1 fluorescence that was colocalized with GFAP signals and presented as the relative levels after setting the value of msCtrl-IgG as 1.0.
  • n 3 animals per group.
  • Scale bar 20 ⁇ m.
  • Bar graphs were presented as the mean ⁇ SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 3E depicts representative movement paths of the mice receiving msCtrl-IgG or msAQP4-IgG stereotaxic injections in the standard Morris water maze (MWM) task.
  • MLM Morris water maze
  • Figure 3H depicts representative images and the confocal analyses of the total proliferating cells (labeled by EdU administration for 2 hours) in the sub-granular zone (SGZ) of the hippocampus after one week of IgG stereotaxic injections (see Fig. 3A) .
  • SGZ sub-granular zone
  • IgG stereotaxic injections see Fig. 3A.
  • DG dentate gyri
  • Figure 3I shows characterization of the proliferating NSCs by quantifying the populations of the radial glia-like cells (let bar graphs, triple-labeled by EdU+GFAP+Sox2+) and the transiently amplifying progenitor-like cells (right bar graphs, EdU+GFAP-Sox2+) , with the fluorescence signals of EdU, Sox2, GFAP, and DAPI.
  • Figure 3J shows analysis of newborn neurons differentiated from BrdU-labeled NSCs 4 weeks after the IgG stereotaxic injections (arrows in Fig. 3A) .
  • Representative confocal images showed the immunostaining of BrdU (left panel) and the DCX, marker for immature neurons (right panel, merge with BrdU channel) .
  • Left bar graphs the average numbers of total BrdU+ cells within the volume of dentate gyrus from msCtrl-IgG and msAQP4-IgG groups.
  • Right bar graphs the quantifications of immature neurons differentiated from NSCs after the IgG (BrdU+DCX+) inside the dentate gyri of the two groups.
  • n 4 animals per group. Scale bar, 100 ⁇ m. Bar graphs were presented as the mean ⁇ SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 3K shows imaging quantification of newly-differentiated mature neurons within the dentate gyrus regions from the BrdU-labeled mice (Fig. 3A) .
  • BrdU was immune-stained along with NeuN, the mature neuronal marker.
  • n 4 animals per group.
  • Scale bar 100 ⁇ m. Bar graphs were presented as the mean ⁇ SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 3L depicts a schematic diagram and the timeline of the experiments to assay the hippocampal neurogenesis and cognitive performance in 8-week-old wildtype mice receiving mini-pump infusion of msCtrl-IgG or msAQP4-IgG into the hippocampal regions on both sides, with a total of 1 ⁇ g in 100 ⁇ l PBS on each side and at the rate of 0.25 ⁇ l per hour for 2 weeks.
  • the EdU and BrdU were administrated i.p. to evaluate the proliferation and differentiation, respectively, similar as the design in Figure 3A.
  • Figure S3A depicts measurements of CHI3L1 levels by ELISA on the lysates of acutely dissected, unfixed hippocampal tissues from the mouse brains stereotaxically injected with msCtrl-IgG-or msAQP4-IgG-treated, complementing the immune-staining results of CHI3L1 induction by msAQP4-IgG in Fig. 3D.
  • n 3 animals per group. Data were presented as the mean ⁇ SEM in bar graphs, and the statistical significance was evaluated with Student’s t test for two-group comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure S3B depicts examinations of the pro-inflammatory genes induced upon astroglial activation by msAQP4-IgG in the hippocampal lysates as in fig. S3A.
  • Figure S3C shows the representative movement paths of the mice stereotaxically injected with msCtrl-IgG or msAQP4-IgG measured by the standardized open field test before the Morris water maze (Fig. 3E-G) , to evaluate their overall locomotor activities and anxiety-like behaviors.
  • Figure S3E shows quantification of the open field test results by measuring the average crossing numbers through the center area (E) .
  • n 9 mice per group.
  • Data were presented as the mean ⁇ SEM in bar graphs, and the statistical significance was evaluated with Student’s t test for two-group comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure S3F shows representative confocal images for the analyses of NSC proliferation, complementing the results in Fig. 3M-O.
  • the labeling of EdU, Sox2, GFAP, and DAPI was to identify the total proliferating cells (EdU+) , the radial glia-like cells (EdU+GFAP+Sox2+) , and the transiently amplifying progenitor-like cells (EdU+GFAP Sox2+) .
  • Scale bar 100 ⁇ m.
  • Figure S3G shows representative confocal images of labeling the newborn immature neurons in the hippocampus SGZ from mice injected with msCtrl-IgG or msAQP4-IgG, complementing the results in Fig. 3P.
  • BrdU was immune-stained (left panels) along with the immature neuronal marker DCX (right panels, merge with BrdU channel) .
  • Scale bar 100 ⁇ m.
  • Figure S3H shows representative confocal images of labeling the mature neurons newly differentiated from NSCs in the hippocampus SGZ as in fig. S3G, complementing the results in Fig. 3Q.
  • BrdU was immune-stained (left panels) along with the mature neuronal marker NeuN (right panels, merge with BrdU channel) .
  • Scale bar 100 ⁇ m.
  • Scale bar 100 ⁇ m.
  • Figure 4A is a schematic diagram of the lentiviral vector design to transduce eGFP alone as a control (Lenti-Ctrl) or CHI3L1 with co-expression of eGFP (Lenti-CHI3L1, or Lenti-GFP-2A CHI3L1) under the control of the CMV promoter.
  • the lentiviral particles were concentrated to transduction units (TU) per ml, and 1 ⁇ l was stereotaxically injected into the dentate gyrus within the hippocampus on either side of 8-week-old wildtype mice.
  • Figure 4B depicts the timeline for the experiments to assess NSC proliferation by EdU labeling, NSC differentiation and behavioral consequences in the mice that over expressed eGFP only (Lenti-Ctrl) or CHI3L1 plus eGFP (Lenti-CHI3L1) in their hippocampi.
  • the overall experiment design was similar as the described in Fig. 3A, with the BrdU being administered immediately after the intra-hippocampal lentiviral injections for assays of NSC differentiation at the Week 4-6 and EdU being given at the Week 2 for 2 hours for the evaluation of NSC proliferation.
  • the behavioral tests of the open field test (fig. S4A-C) and Morris water maze were carried out in tandem at the Week 4-6 right before the sacrifice and tissue harvest.
  • Figure 4C shows the validation of CHI3L1 over-expression in the acutely dissected, unfixed hippocampal tissues by immunoblotting of the hippocampal lysates harvested from mice brains injected with Lenti-Ctrl or Lenti-CHI3L1.
  • Figure 4D shows the representative movement paths for both Lenti-Ctrl and Lenti-CHI3L1 groups.
  • the task of Morris water maze (MWM) was performed at 6-Apr weeks after the lentiviral injections.
  • Figure 4E shows the analysis of MWM by quantification of the crossing numbers.
  • Figure 4F shows the analysis of MWM by quantification of the escape latencies to find the platform during the training.
  • Data were analyzed with two-way ANOVA with Tukey’s multiple comparisons test. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • the immune-staining of GFAP was used to further classify the proliferating cells of a proper NSC morphology (EdU+) into the radial glia-like cells (EdU+GFAP+) and the transiently amplifying progenitor-like cells (EdU+GFAP-) .
  • Scale bar 100 ⁇ m.
  • Figure 4H depicts quantification of totally proliferating (EdU+) cells plotted as the average cell numbers within the volume of dentate gyrus (decided by the imaging processing of 3D reconstruction) .
  • n 4 animals per group. Data in were presented as the mean ⁇ SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 4K shows representative images and confocal analyses of NPC differentiation into immature neurons in the mouse hippocampal sections, which were labeled with BrdU and transduced by the Lenti-Ctrl or Lenti-CHI3L1 lentiviruses (Fig. 4B) .
  • the fluorescence signals of BrdU were counterstained with the immature neuronal marker DCX to identify the newborn immature neurons (BrdU+DCX+) , the average numbers of which within the volume of dentate gyrus were plotted for both tested groups.
  • n 4 animals per group. Scale bar, 100 ⁇ m.
  • Figure 4L shows representative images and confocal analyses of NPC differentiation into mature neurons in the mouse hippocampal sections, which were labeled with BrdU and transduced by the Lenti-Ctrl or Lenti-CHI3L1 lentiviruses (Fig. 4B) .
  • the fluorescence signals of BrdU were counterstained with the mature neuronal marker NeuN to identify the newborn mature neurons (BrdU+NeuN+) , the average numbers of which within the volume of dentate gyrus were plotted for both tested groups.
  • n 4 animals per group. Scale bar, 100 ⁇ m. Data in were presented as the mean ⁇ SEM; statistical significance was evaluated with Student’s t test for two-group comparisons.
  • Figure S4A shows the results of the open field test that was performed prior to Morris water maze at week 4-6 (Fig. 4B) to evaluate the locomotor activities and anxiety-like behaviors in mice lentivirally transduced to over-express eGFP (Lenti-Ctrl) or CHI3L1 plus eGFP (Lenti-CHI3L1) . Representative movement paths of both groups were shown here.
  • Figure S4B depicts quantification of the open field test by measuring the total travel distances of both groups.
  • Figure S4C depicts quantification of the open field test by measuring the crossing numbers through the center area of both groups.
  • Data are presented as the mean ⁇ SEM; statistical significance was evaluated with unpaired one-tailed Student’s t test for two group comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 5A shows the summary of the CHI3L1 receptors and the downstream signaling pathways that could be activated by the CHI3L1-receptor binding.
  • Figure 5B depicts the expression of CRTH2 receptor on NSCs but not immature or mature neurons, assayed by the immune-staining of CRTH2 (left panels of all rows) and neural markers GFAP (center left panels of top two rows) , Sox2 (center right panels of top two rows) , DCX (center left panel of bottom row) , NeuN (center right panel of bottom row) and DAPI (shown in merge with other channels, right panels of all rows) .
  • GFAP center left panels of top two rows
  • Sox2 center right panels of top two rows
  • DCX center left panel of bottom row
  • NeuN center right panel of bottom row
  • DAPI shown in merge with other channels, right panels of all rows
  • the top 2 rows demonstrated the colocalization of fluorescence signals for CRTH2 and NSC (GFAP+SOX2+) ; the bottom raw showed little if any colocalization between CRTH2 and immature neuronal marker DCX markers (left 2 panels) or mature neuronal marker NeuN (right 2 panels) .
  • These hippocampal slices were from 8-week-old wildtype mice. Scale bars, 20 ⁇ m.
  • Figure 5C depicts the characterization of the CHI3L1 receptor signaling pathways activated by CHI3L1 binding, focusing on GSK-3 ⁇ and ⁇ -catenin that are most relevant to cell proliferation and neurogenesis.
  • the cultures of purified NSCs grown in proliferation medium were treated with the vehicle control PBS or recombinant CHI3L1 protein (100ng/mL) for 30 minutes, 1 hour and 2 hours, and then lysed and harvested at the same time.
  • the treated NSC lysates were then subject to immunoblotting for assaying the protein levels of total GSK-3 ⁇ kinase, phosphorylated GSK-3 ⁇ (p-GSK-3 ⁇ , phosphorylation of Serine 9, the inactive form unable to phosphorylate substrate ⁇ -catenin for degradation) , transcription factor ⁇ -catenin and the loading control ⁇ -actin.
  • n 3 experiments per treatment condition. Bar graphs were presented as the mean ⁇ SEM; the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 5D depicts a schematic diagram of the lentiviral vector designs to transduce the expression of 2 shRNAs targeting CRTH2 for knockdown (Lenti-shCRTH2, shCRTH2_1 and shCRTH2_2) and a scrambled non-targeting control shRNA (Lenti-shNC) by a U6 promoter and the co-expression of eGFP by a CMV promoter.
  • the shRNA design was also used to deliver shRNAs targeting 2 other CHI3L1 receptors, IL-13R ⁇ 2 and TMEM219 (fig. S5C-F) .
  • the eGFP signal was used to calculate the transducing unit (TU) in the lentiviral preparations for the desired multiplicity of infection (MOI) of 10- May in mouse NSC cultures.
  • Figure 5E shows the immunoblotting analyses to evaluate the knockdown efficiency of the 2 shRNAs against CRTH2 in cultured mouse NSCs that were transduced by lentiviruses for 3 days in proliferation medium before the lysing and sample collections.
  • a representative blot of NSC lysates immunoblotted with CRTH2 and the loading control, with the lentiviral transduction of the control shRNA (shNC) and 2 shRNAs against CRTH2 (shCRTH2_1 and shCRTH2_2) .
  • n 3 experiments per condition. Bar graphs were presented as the mean ⁇ SEM; the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons.
  • Figure 5F shows the evaluation of the CRTH2 knockdown effect on the activation of CHI3L1 signaling pathways in NSCs.
  • the mouse NSC cultures were transduced by lentiviruses to express the control shNC and shRNA against CRTH2 (the shCRTH2_1 from fig. S5E) , and then treated with PBS or recombinant CHI3L1 protein (100ng/mL) for 2 hours, followed by lysing for immunoblotting.
  • Figure 5G shows the investigation of the CRTH2 knockdown effect on NSC proliferation, by EdU labeling of the mouse NSCs grown in proliferation medium (as in Fig. 2E) and transduced via lentivirus to express shNC or shCRTH2_1 together with 3-day treatments of PBS or CHI3L1 (100 ng/ml) .
  • the immune-fluorescence signal of GFP co-expressed with either shRNA was positive for >99%of cells labeled by DAPI.
  • Figure 5H shows the examination of the CRTH2 knockdown effect on the neuronal proliferation of NSCs, by transducing the shNC or shCRTH2_1 to be expressed in mouse NSCs grown in the differentiation medium and treated with PBS or CHI3L1 (100 ng/ml) for 3 days (as in Fig. 2E) .
  • Figure 5I shows the identified CHI3L1 receptor signaling pathway inhibitory to neurogenesis, in which CHI3L1 binds to CRTH2 receptor and activates GSK-3 ⁇ that in turn destabilizes ⁇ -catenin and leads to reduced transcriptional activities for neurogenesis.
  • Figure S5A shows little expression of IL-13R ⁇ 2, a common CHI3L1 receptor for immune cells, in NSCs within the SGZ of dentate gyrus from 8-week-old wildtype mice, assayed by immunostaining of IL-13R ⁇ 2 (left panel) and the radial glia-like NSC markers Sox2 (center right panel) and GFAP (center left panel) plus the nuclear marker DAPI (right panel, shown in a merge with the other channels) .
  • This confocal imaging complemented Fig. 5B. Scale bars, 20 ⁇ m.
  • Figure S5B shows a very low level of CHI3L1 receptor TMEM219 in NSCs within the SGZ of dentate gyrus, assayed by immunostaining of TMEM219 (left panel) and the radial glia-like NSC markers Sox2 (center right panel) and GFAP (center left panel) plus the nuclear marker DAPI (right channel, shown in a merge with the other channels) .
  • This confocal imaging complemented Fig. 5B. Scale bars, 20 ⁇ m.
  • Figure S5C depicts the knockdown efficiency of 2 shRNAs targeting against IL-13R ⁇ 2, assayed by immunoblotting of cell lysates from NSCs transduced with lentiviruses to express the non-targeting control shRNA (shNC) , shIL-13R ⁇ 2_1 or shIL-13R ⁇ 2_2 as described in Fig. 5D-E.
  • shNC non-targeting control shRNA
  • shIL-13R ⁇ 2_1 shIL-13R ⁇ 2_1
  • shIL-13R ⁇ 2_2 as described in Fig. 5D-E.
  • ⁇ -actin served as the loading control.
  • Figure S5D shows, relevant to Fig. 5D-E, the evaluation of TMEM219 knockdown efficiency by 2 shRNAs, shTMEM219-1 and shTMEM219_2, transduced in NSC cultures and compared to the protein levels of TMEM219 and ⁇ -actin.
  • Figure S5E shows the knockdown of IL-13R ⁇ 2 or TMEM219 did not affect the inhibition of NSC proliferation by CHI3L1, in parallel to the experiments in Fig. 5G.
  • the cultured mouse NSCs grown in proliferation medium were transduced to express shNC, shIL-13R ⁇ 2_1 or shTMEM219_1 and then treated with PBS or CHI3L1 (100 ng/ml) .
  • the EdU was added into the cultures two hours before the fixation and immune-staining.
  • Left panels representative confocal images of the indicated 4 groups.
  • Figure S5F shows, in relation to Fig. 5H, the knockdown of IL-13R ⁇ 2 or TMEM219 did not affect the inhibition of NSC differentiation by CHI3L1.
  • the mouse primary NSCs were cultured in the differentiation medium and transduced to express shNC, shIL-13R ⁇ 2_1 or shTMEM219_1, with the incubation of PBS or CHI3L1 (100 ng/ml) for 3 days. After fixation, the immuno-staining was performed to label essentially all cells with GFP and DAPI, the newly differentiated neurons with Tuj1 and astroglial cells with GFAP, in the panels of the representative images.
  • the bar graphs were the quantification of neuronal differentiation, shown in the ratio of neurons and total cells (GFP+Tuj1+/GFP+, left) and the ratio of astroglia and total cells (GFP+GFAP+/GFP+, right) .
  • n 4 experiments.
  • Scale bars 100 ⁇ m.
  • Data were presented as the mean ⁇ SEM and their statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • FIG. 6A depicts a schematic diagram of the astrocyte-specific CHI3L1 knockout by stereotaxic injection of AAVs expressing Cre recombinase and eGFP under the control of the GFAP promoter (AAV-GFAP-GFP-2A-Cre, or AAV-Cre) into hippocampi of the 8-week-old transgenic mice carrying one (CHI3L1 f/+ ) or two (CHI3L1 f/f ) CHI3L1-floxed alleles.
  • the AAV preparations were at the concentration of TU/ml and stereotaxically injected into the dentate gyrus of 8-week-old transgenic mice with 1 ⁇ l on each side.
  • mice A subset of the injected mice was sacrificed two weeks after the AAV injections for the evaluation of knockout efficiency. The remainder of those AAV-injected mice were allowed to recover for 4 weeks before they received the intra-hippocampal injections of msCtrl-IgG or msAQP4-IgG as described in Fig. 3A-K to elicit the astroglial activation, and then went through behavioral tests at the Week 8 before the sacrifice.
  • Figure 6B shows representative confocal images of the hippocampal slices from the CHI3L1 f/f transduced to express Cre and GFP in astroglial cells labeled with GFAP.
  • the signals of CHI3L1 immuno-fluorescence were reduced in astrocytes with Cre-mediated CHI3L1 deletion (GFP+GFAP+) , as indicated by the dots (right panel) .
  • the DAPI signals were used to outline the dentate gyrus. Scale bars, 100 ⁇ m.
  • Figure 6C shows the evaluation of CHI3L1 knockout efficiency in the AAV-Cre-injected hippocampal tissues from CHI3L1 f/+ and CHI3L1 f/f mice, with qPCR assays to measure CHI3L1 mRNA levels in the hippocampal lysates harvested from the dissected fresh brain tissues.
  • the CHI3L1 f/+ mice still expressed CHI3L1 abundantly but the CHI3L1 f/f mice expressed a low level, ⁇ 10%of the CHI3L1 f/+ group.
  • n 3 animals per group. Data were presented as the mean ⁇ SEM; statistical significance was evaluated with Student’s t test for two-group comparisons.
  • Figure 6D depicts results from the task of Morris water maze (MWM) which was performed in both CHI3L1-expressing (CHI3L1 f/+ ) and CHI3L1-deficient (CHI3L1 f/f ) mice, 8 weeks after the stereotaxic injections of AAV-Cre and four weeks after the stereotaxic deliveries of msCtrl-IgG or msAQP4-IgG.
  • MLM Morris water maze
  • Figure 6G shows a schematic diagram and experiment timeline to test the effects of astrocyte-specific CHI3L1 knockout on adult hippocampal neurogenesis.
  • the stereotaxic injections of AAV-Cre rendered CHI3L1 expressed in CHI3L1 f/+ hippocampal tissues but depleted in the CHI3L1 f/f groups (as in Fig. 6A-C) .
  • the astroglial activation was carried out by the mini-pump method as described in Fig. 3L-Q to chronically infuse msCtrl-IgG or msAQP4-IgG into hippocampus for the Week 1 and 2.
  • the evaluation of NSC proliferation and neuronal differentiation was facilitated by the labeling of EdU and BrdU, respectively, administered in a manner as described in Fig. 3A.
  • Figure 7A shows a schematic diagram and experiment timeline for characterizing the effects of shRNA mediated CRTH2 knockdown on NSC proliferation and differentiation in astroglial activation triggered by msAQP4-IgG.
  • the lentiviruses expressing GFP together with a scrambled non-targeting control shRNA (Lenti-shNC) or a shRNA targeting CRTH2 (Lenti-shCRTH2_1) as described in Fig. 5D were prepared at the concentration of TU/ml and stereotaxically injected into the hippocampi of 8-week-old wildtype mice with 1 ⁇ l on each side.
  • mice One week after the shRNA lentivirus injections, a subset of mice was sacrificed by perfusion and their hippocampal sections were immune-stained with GFP and DAPI to show the adequate lentiviral transduction efficiency in the representative confocal images. Scale bars, 100 ⁇ m.
  • the msCtrl-IgG and msAQP4-IgG were then infused chronically by a mini-pump for 2 weeks, and EdU and BrdU were administered for the evaluation of NSC proliferation and neuronal differentiation, as described in Fig. 3L.
  • Figure 7B depicts the evaluation of CRTH2 knockdown effect on NSC proliferation that was done by the immune staining of GFP (top left) , EdU (top right) and GFAP (bottom left) , with representative images shown in left panels.
  • the quantifications (right) were plotted as the average numbers of proliferating cells transduced to express a shRNA (GFP+EdU+, the left bar graphs) and the average numbers of radial-glia-like NSCs (GFP+EdU+GFAP+, the right bar graphs) within the volume of dentate gyrus (DG) .
  • n 4 animals per group. Scale bars, 20 ⁇ m. Data were presented as the mean ⁇ SEM with the statistical significance evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 7C depicts the confocal assays for NSC differentiation into immature neurons, quantified by the average numbers of newborn immature neurons transduced to express a shRNA (with GFP co-expression) within the DG volume.
  • n 4 animals per group.
  • Scale bars 20 ⁇ m. Data were presented as the mean ⁇ SEM with the statistical significance evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 7E depicts a schematic diagram and timeline for the experiments to probe the efficacy of AZD1981, a selective CRTH2 antagonist, as a therapeutic compound to ameliorate the inhibition of neurogenesis by CHI3L1 signaling in astroglial activation.
  • the mini-pump infusion of msCtrl-IgG or msAQP4-IgG for 2 weeks, and the administration of EdU and BrdU were performed similarly as described in Fig. 3L.
  • the vehicle control DMSO or AZD1981 was given at the dosage of 1 mg/kg via i.p. injections every other day for 4 weeks.
  • Figure 7F depicts quantification of NSC proliferation by analyzing the immune-fluorescence of EdU, GFAP and SOX2, as described in Fig. 3H and I.
  • Figure 7G depicts quantification of NSC proliferation by analyzing the immune-fluorescence of EdU, GFAP and SOX2, as described in Fig. 3H and I.
  • Figure 7H shows quantification of NSC proliferation into immature neurons throughout the experiment timeline, assayed by immune-staining of BrdU, and DCX and plotted as the average numbers of newborn immature neurons (BrdU+DCX+) .
  • Data were presented as the mean ⁇ SEM with the statistical significance evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure S7A depicts a schematic diagram for the in vitro experiments to evaluate the effect of blocking CRTH2 by antagonist AZD1981 on the inhibition of NSC proliferation and differentiation by CHI3L1.
  • the overall design was essentially the same as the one described in Fig. 2E, other than the co-incubation with the vehicle control DMSO or AZD1981 (10 nM) in the proliferation medium or differentiation medium for 3 days.
  • Figure S7C depicts the assays of NSC differentiation into neurons as described in Fig. 2G to evaluate the effect of AZD191 in counteracting the CHI3L1-induced inhibition.
  • Representative images of the differentiating NSC cultures were analyzed for the differentiated neurons immune-labeled by Tuj1, astroglial cells by GFAP, and all the nuclei by DAPI.
  • the bar graphs showed the ratio of Tuj1+ cells to DAPI+ cells on the left and the ratio of GFAP+ cells to DAPI+ cells.
  • n 4 experiments per group. Scale bars, 100 ⁇ m. Data were presented as the mean ⁇ SEM and the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • the present disclosure provides, for example, methods of targeting the detrimental effects of neuroinflammation on cognitive performance by blocking the interaction of CHI3L1 with the CRTH2 receptor within the central nervous system.
  • the disclosure also provides a previously undescribed mechanism whereby the inflammatory signaling mediator CHI3L1 engages the CRTH2 receptor on neural stem cells in the brain and triggers a downstream inhibitory pathway to prevent the process of neurogenesis, which promotes proper cognitive performance.
  • the present disclosure provides, for instance, therapy for neuroinflammation comprising a CTHR2 antagonist.
  • a CTHR2 antagonist for instance, therapy for neuroinflammation comprising a CTHR2 antagonist.
  • Exemplary CTHR2 antagonists are described herein.
  • a reference to a cell comprises a combination of two or more cells.
  • the term “consisting essentially of” means the listed elements are required for a given embodiment. The term permits additional elements that do not materially affect the basic and functional characteristics of that embodiment of the invention.
  • an effective amount refers to the amount sufficient to cause beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. Many ways are known in the biomedical art to determine the effective amount for an application. For example, pharmacological methods for dosage determination can be used in the therapeutic context. In therapeutic or prophylactic applications, the amount of a composition administered to the subject depends on the type and severity of the disease and the characteristics of the individual, such as general health, age, sex, body weight, tolerance to drugs, and on the degree, severity, and type of disease. Persons having ordinary skill in the biomedical art can determine appropriate dosages depending on these and other factors. In some embodiments, an effective amount results in inhibition of a target protein by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • expression refers to the transcription or translation of a particular nucleic acid sequence driven by a promoter. In some embodiments, expression refers to the level of accumulation of an RNA. In some embodiments, expression refers to the accumulation of a protein.
  • CHI3L1 inhibitor refers to an agent that specifically binds and inhibits activity of a chitinase 3 like 1 (known as CHI3L1 or YKL-40) protein, or an agent that specifically reduces expression of the CHI3L1 protein.
  • the agent comprises a molecule or a complex.
  • the agent comprises a small molecule that binds CHI3L1.
  • the agent comprises an antibody molecule.
  • the agent that specifically reduces expression of the CHI3L1 protein comprises a nucleic acid that binds CHI3L1 mRNA and reduces the level or translation of the CHI3L1 mRNA.
  • CRTH2 inhibitor refers to an agent that specifically binds and inhibits activity of a Prostaglandin D 2 receptor 2 (known as CRTH2 or PDG2) protein, or an agent that specifically reduces expression of the CRTH2 protein.
  • the agent comprises a molecule or a complex.
  • the agent comprises a small molecule that binds CRTH2.
  • the agent comprises an antibody molecule.
  • the agent that specifically reduces expression of the CRTH2 protein comprises a nucleic acid that binds CRTH2 mRNA and reduces the level or translation of the CRTH2 mRNA.
  • the term “inhibitor” refers to an agent that causes a decrease of a certain parameter.
  • the parameter is an activity of a given molecule.
  • the decrease is a decrease by at least 10%as compared to a reference level (e.g., the absence of a treatment or agent) and can include more significant decreases, for example, a decrease by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more.
  • Activities for the inhibitors can be determined using any suitable assay, for instance, an assay as described herein or an assay known in the art.
  • the inhibitor specifically binds a target molecule.
  • the inhibitor prevents the activity of a bound target molecule.
  • the inhibitor reduces expression of a given molecule.
  • the terms “treat” , “treatment” and “treating” refer to the reversal, alleviation, amelioration, inhibition, reduction, slowing or halting of the progression, severity and/or duration of a disease, disorder, or medical condition, or the amelioration of one or more symptoms of a disease, disorder, or medical condition.
  • the terms “treat” , “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of the disease, disorder, or medical condition not necessarily discernible by the patient.
  • the terms “treat” , “treatment” and “treating” refer to the inhibition of the progression of disease, disorder, or medical condition, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both.
  • Treatment is effective, for example, if one or more symptoms or clinical markers are reduced or if the progression of a condition is reduced or halted.
  • Treatment includes not just the improvement of symptoms or markers but also a cessation or at least slowing of progress or worsening of symptoms expected absent treatment.
  • the term “subject” refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate.
  • Subjects can be house pets (e.g., dogs, cats) , agricultural stock animals (e.g., cows, horses, pigs, chickens, etc. ) , laboratory animals (e.g., mice, rats, rabbits, etc. ) , but are not so limited.
  • Subjects include human subjects.
  • the human subject may be a pediatric, adult, or geriatric subject.
  • the human subject may be of either sex.
  • the subject may have a condition or disease or be at risk of developing a condition or disease.
  • an antibody molecule refers to a naturally occurring antibody, an engineered antibody, or a fragment thereof.
  • an antibody molecule is an antigen binding portion of a naturally occurring antibody or an engineered antibody.
  • an antibody molecule includes an antibody or an antigen-binding fragments thereof (e.g., Fab, Fab’, F (ab’) 2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv) , Fd fragments consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH) , nanobodies, or camelid VHH domains) , an antigen-binding fibronectin type III (Fn3) scaffold such as a fibronectin polypeptide minibody, a ligand, a cytokine, a chemokine, or a T cell receptor (TCR) .
  • Fn3 antigen-binding fibronectin type III
  • an antibody molecule is a humanized antibody molecule. In some embodiments, an antibody molecule is an intact IgA, IgG, IgE or IgM antibody. In some embodiments, an antibody molecule is a bi-or multi-specific antibody (e.g., etc) . In some embodiments, antibody molecules are antibody fragments such as Fab fragments, Fab’ fragments, F (ab’) ⁇ fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof.
  • an antibody molecule is a single chain Fv (scFv) , a polypeptide-Fc fusion, a single domain antibody (e.g., shark single domain antibodies such as IgNAR or fragments thereof) , or a cameloid antibody.
  • antibody molecules are masked antibodies (e.g., ) , Small Modular ImmunoPharmaceuticals ( “SMIPsTM” ) , single chain or Tandem diabodies VHHs; minibodies, ankyrin repeat proteins or DARTs, TCR-like antibodies, MicroProteins, or
  • nucleic acid refers to a polymeric molecule incorporating units of ribonucleic acid, deoxyribonucleic acid, or an analog thereof.
  • the nucleic acid is in single stranded form.
  • the nucleic acid is in double stranded form.
  • the nucleic acid is genomic DNA, cDNA, or RNA (e.g. mRNA) .
  • the nucleic acid contains analogues of natural nucleotides that have similar binding properties as the reference nucleic acid.
  • nucleic acid containing analogues of natural nucleotides are metabolized in a manner similar to naturally occurring nucleotides.
  • the terms “peptide, ” “polypeptide, ” and “protein” are used interchangeably, and refer to a molecule comprised of two or more amino acid residues covalently linked by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • the polypeptide comprises a modified amino acid.
  • the polypeptide refers to a natural peptide, a recombinant peptide, or a combination thereof.
  • the polypeptide refers to short chains of amino acids.
  • the polypeptide refers to long chains of amino acids.
  • the polypeptide refers to a biologically active fragment, a substantially homologous polypeptide, an oligopeptide, a variant of a polypeptide, a modified polypeptide, a derivative, an analog, or a fusion protein.
  • a person having ordinary skill in the biomedical art recognizes that individual substitutions, deletions, or additions to a peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence are a conservatively modified variant where the alteration results in the substitution of amino acid with chemically similar amino acid and retains the desired activity of the polypeptide.
  • conservatively modified variants also do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.
  • the term “or” refers to and/or.
  • the term and/or as used in a phrase such as A and/or B herein includes both A and B; A or B; A (alone) ; and B (alone) .
  • the term and/or as used in a phrase such as A, B, and/or C encompasses each embodiment: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; Band C; A (alone) ; B (alone) ; and C (alone) .
  • the CRTH2 inhibitor comprises a shRNA. In some embodiments, the CRTH2 inhibitor comprises a small molecule. In some embodiments, the CRTH2 inhibitor comprises a noncompetitive antagonist of CRTH2.
  • the CRTH2 inhibitor comprises AZD1981 or a pharmaceutically acceptable salt thereof.
  • AZD1981 has a chemical structure according to Formula I:
  • the CRTH2 inhibitor comprises OC000459 or a pharmaceutically acceptable salt thereof.
  • OC000459 has a chemical structure according to Formula III:
  • the CRTH2 inhibitor comprises BI671800 or a pharmaceutically acceptable salt thereof.
  • BI671800 has a chemical structure according to Formula IV:
  • the CRTH2 inhibitor comprises AMG853 or a pharmaceutically acceptable salt thereof.
  • AMG853 has a chemical structure according to Formula V:
  • the CRTH2 inhibitor comprises setipiprant or a pharmaceutically acceptable salt thereof.
  • Setipiprant has a chemical structure according to Formula V:
  • the CRTH2 inhibitor comprises ARRY-502 or a pharmaceutically acceptable salt thereof.
  • ARRY-502 has a chemical structure according to Formula VI:
  • the CRTH2 inhibitor comprises MK-7246 or a pharmaceutically acceptable salt thereof.
  • MK-7246 has a chemical structure according to Formula VII:
  • the CRTH2 inhibitor comprises MK-1029 or a pharmaceutically acceptable salt thereof.
  • MK-1029 has a chemical structure according to Formula VIII:
  • the CRTH2 inhibitor comprises AZD6430 or a pharmaceutically acceptable salt thereof.
  • the CRTH2 inhibitor comprises ADC-3680 or a pharmaceutically acceptable salt thereof.
  • the CRTH2 inhibitor comprises ASP5642 or a pharmaceutically acceptable salt thereof.
  • the CRTH2 inhibitor comprises BI1021958 or a pharmaceutically acceptable salt thereof.
  • the CRTH2 inhibitor comprises TQC3564 or a pharmaceutically acceptable salt thereof.
  • the CHI3L1 inhibitor comprises 2- ( ⁇ 3- [2- (1-cyclohexen-1-yl) ethyl] -6, 7-dimethoxy-4-oxo-3, 4-dihydro-2-quinazolinyl ⁇ sulfanyl) -N- (4-ethylphenyl) butanamide (K284) .
  • the CHI3L1 inhibitor comprises kasugamycin.
  • the CHI3L1 inhibitor comprises a monoclonal anti-CHI3L1 antibody molecule.
  • compositions described herein are used to treat neuromyelitis optica spectrum disorder (NMOSD) .
  • NMOSD is an autoimmune disease of the central nervous system that affects the optic nerves and the spinal cord.
  • the brain is affected.
  • a primary astrocytopathy is caused by autoantibodies targeting AQP4.
  • autoantibodies targeting AQP4 are not present.
  • Example 1 CHI3L1 signaling impairs hippocampal neurogenesis and cognitive function in autoimmune-mediated neuroinflammation
  • This example describes the disruption of CHI3L1 signaling in in vitro and in vivo models of neuromyelitis optica spectrum disorder in order to promote neurogenesis, neuronal differentiation, and cognitive function.
  • the sample size was not pre-determined by any statistical methods as the effect sizes were unavailable before the experiments.
  • the samples sizes were all described in the figure legends.
  • the normality of the data distribution was routinely determined by a Shapiro-Wilk normality test (p ⁇ 0.05 indicating a non-normal distribution) .
  • For the data confirmed to be normally distributed data we used Student’s t test for pair-wise comparisons, one-way or two-way ANOVA followed by Tukey’s post-hoc test for 3 groups or more, as indicated in the figure legends.
  • non-parametric alternatives such as Mann-Whitney or Kruskal-Wallis tests. p ⁇ 0.05 is considered to be statistically significant.
  • the whole brains from neonatal mouse pups at postnatal day 0 were dissected out on ice. After removal of the meninges, the brains were washed in cold DMEM-F12 (C11330500BT, GIBCO) supplemented with 10%FBS (ST30-3302, PANTM SERATECH) and 1%penicillin streptomycin (15140-122, GIBCO) . The brains were transferred to 0.25%trypsin EDTA for 20 minutes, and then DMEM-F12 complete medium was used to stop the trypsinization. Cell debris and aggregates were removed by passing the single-cell suspension through a 40 ⁇ m nylon mesh.
  • the single-cell suspension was collected and cultured with astrocyte medium: DMEMF12 (C11330500BT, GIBCO) containing 10%FBS (ST30-3302, PANTM SERATECH) , 1%NEAA, 1%penicillin streptomycin (15140-122, GIBCO) , 1%glutamine (GIBCO, 25030-081) and 1 mM sodium pyruvate (S8636, Sigma) in T-75 flasks for 14 days, and the medium was replaced every 3 days.
  • DMEMF12 C11330500BT, GIBCO
  • 10%FBS ST30-3302, PANTM SERATECH
  • 1%NEAA 1%penicillin streptomycin
  • GIBCO 1%glutamine
  • S8636 1 mM sodium pyruvate
  • astrocytes were plated onto poly-D lysine-coated glass coverslips in 24-well plates at a density of 8x10 3 cells per well and allowed to settle at 37 °C in a 5%CO2 incubator for 24 hours.
  • the cultures were treated with controls or biological reagents at the indicated final concentration for the described experimental durations: Dulbecco's phosphate buffered saline (PBS, as the vehicle) , IL-1 ⁇ (10 ng/mL; 200-01B, PeproTech) , monoclonal mouse antibodies targeting AQP4 (msAQP4-IgG, 100 ng/mL) , control mouse IgG (100 ng/ml) , purified human autoantibodies against AQP4 (hsAQP4-IgG, 100 ng/ml) , and control human IgG (100 ng/ml) .
  • PBS Dulbecco's phosphate buffered saline
  • IL-1 ⁇
  • the acquisition of mouse and human antibodies were detailed in a following section.
  • the astrocyte-conditioned media were collected at the completion of individual reagent treatments and filtered with 0.22 ⁇ m nylon mesh (BS-QT-037, Biosharp) .
  • the filtered ACM were added with the anti-CHI3L1 monoclonal antibodies (rabbit, Proteintech) or msCtrl-IgG at 100 ng/ml plus Protein A beads (71149800, GE Healthcare) , tumbled for 4 hours at 4°C and centrifuged to clear the IgG contents.
  • the cleared ACM were then supplemented with factors to constitute the culture media for the proliferation or neuronal differentiation of cultured mouse neural stem cells (NSCs) .
  • the primary astrocyte cultures were harvested by fixation with 4%paraformaldehyde (PFA) for immune-staining and by lysis buffers for RNA-seq and qPCR assays.
  • PFA paraformaldehyde
  • NSCs Primary mouse neural stem cells
  • the mouse neural stem cells (NSCs) used in this study were purified from living dentate gyrus tissues acutely dissected from 8-week young adult wildtype mice.
  • NSCs mouse neural stem cells
  • the anatomical structure of the dentate gyrus was visualized by a dissection microscope, carefully removed out of the hippocampal regions, and then subject to the enzymatic digestion by using the MACS neural tissue dissociation kit according to the manufacturer’s protocol (130-092-628, MACS Miltenyi Biotec) .
  • trypsin inhibitor S10088, Yuan Ye Biology
  • the single-cell suspension was collected and cultured with the maintenance medium for proliferation (proliferation medium) , formulated with neurobasal (21103049, GIBCO) medium containing 2%B27 supplement (17504044, GIBCO) , 20 ng/mL basic fibroblast growth factor (FGF-2, K1606, PeproTech) , 20 ng/mL epidermal growth factor (EGF, A2306, PeproTech) , 1%penicillin streptomycin (15140-122, GIBCO) and 2 mM L-glutamine (25030-081, GIBCO) in a 5%CO2 incubator at 37 °C. Without further description, half of the medium was replaced every 3 days.
  • the maintenance medium for proliferation formulated with neurobasal (21103049, GIBCO) medium containing 2%B27 supplement (17504044, GIBCO) , 20 ng/mL basic fibroblast growth factor (FGF-2, K1606, PeproTech) , 20 ng/mL epidermal growth
  • the cells were incubated with the indicated vehicle controls or biological reagents for 3 days, and then the EdU from a Cell Proliferation Kit was administered according to the manufacturer’s protocol (C0078L, Beyotime) .
  • the assays of neuronal differentiation 48 hours after plating, the cells were changed to the differentiation medium composed of neurobasal medium containing 2%B27 supplement (17504044, GIBCO) , 1%penicillin streptomycin (15140-122, GIBCO) and 2 mM L-glutamine (25030-081, GIBCO) , with the co-incubation of the indicated vehicle controls or biological reagents.
  • the NSC cultures for proliferation and differentiation, were treated with recombinant CHI3L1 protein (100 ng/ml; 2599-CH-050, R&D Systems) or vehicle control for 3 days.
  • the treated cultures were harvested by fixation with 4%PFA and then subject to immune-staining for confocal imaging of the EdU or BrdU expression, in conjunction with neural or nuclear markers.
  • the hybridoma cell line producing monoclonal mouse IgG specifically recognizing the mouse AQP4 extracellular domain (clone E5415B; hereafter referred to as msAQP4-IgG) was kindly provided by Dr. Yoichiro Abe at the Keio University, Tokyo, Japan. These antibodies are commercially available (MABN2526-25UG, Sigma-Aldrich) .
  • the control mouse IgG was the normal mouse IgG validated for use in immunoprecipitation and immunoblotting (12-371, Sigma-Aldrich) .
  • the human autoantibodies against AQP4 were purified from the pooled plasma samples of anti-AQP4 seropositive NMO patients (hsAQP4-IgG) or healthy volunteers (hsCtrl-IgG) undergoing plasma exchange using Protein A beads (71149800, GE Healthcare) .
  • the beads were eluted with 100 mM glycine HCl (pH 2.5) , and then the eluent was concentrated using an Amicon Ultra 15 centrifugal filtration unit (100 KD, Millipore, Billerica, MA) .
  • the concentration of IgG was 0.22 ⁇ M, and the working portion was kept at -80 °C.
  • mice astrocytes or NSCs were fixed with 4%PFA for 40 minutes, followed by washing in PBS. Then, the cells were blocked with 300 ⁇ L/10 mL donkey serum and 250 ⁇ L/10 mL 10%Triton X-100 for 1 hour at room temperature and sequentially incubated with primary antibodies overnight at 4°C. The cells were washed with PBS three times, and then the cells were incubated with the secondary antibody at room temperature for 1 hour. The nuclei were then stained with DAPI and observed under a laser confocal microscope. Each group of immunohistochemical experiments was repeated at least 3 times.
  • the following primary antibodies were used: chicken anti-GFAP (1: 1000, Abcam) , mouse anti-Tuj1 (1: 1000, Abcam) , mouse anti-IL13R ⁇ 2 (1: 500, CST) , rabbit anti-CHI3L1 (1: 100, Proteintech) , rabbit anti-CRTH2 (1: 500, Invitrogen) , and rabbit anti-TMEM219 (1: 500, CST) .
  • the following fluorescent secondary antibodies were used: goat anti-mouse 488, goat anti-mouse 568, goat anti-mouse 647, goat anti-rabbit 488, goat anti-rabbit 568, goat anti-rabbit 647, and goat anti-chicken 647.
  • RNA isolation and quantitative real-time PCR qPCR
  • mRNA from mouse hippocampal tissue or primary astrocytes was extracted with a miRNeasy kit (Qiagen) . Then, mRNA was quantified and checked for purity using a Nanodrop spectrophotometer (ThermoFisher Scientific) . The cDNA was converted from 1 ⁇ g mRNA using the SureScript First-Strand cDNA Synthesis Kit (Genecopoeia Company) . RT-qPCR was performed using BlazeTaq SYBR Green qPCR Mix (Genecopoeia Company) with Applied Biosystems (Thermo Fisher Scientific) . Fold changes were calculated as 2 - ⁇ CT with GAPDH used as the endogenous control. The sequences of the primers for the individual genes of interest are listed in Table M1.
  • Cell lysates were derived from primary mouse astrocytes or NSCs using radioimmunoprecipitation assay (RIPA) lysis buffer.
  • the proteins were separated on 8-15%sodium dodecyl sulfate-polyacrylamide gels by electrophoresis and then transferred to methanol-activated polyvinylidene fluoride (PVDF) membranes.
  • PVDF polyvinylidene fluoride
  • the membranes were blocked in 5%defatted milk powder for 1 hour at 37 °C and incubated with primary antibodies overnight at 4 °C. The membranes were then washed three times with TBST and incubated with horseradish peroxidase (HRP) secondary antibodies for 1 hour.
  • HR horseradish peroxidase
  • Protein bands were visualized with Millipore ECL Plus reagent and imaged on a Tanon 5500 Imaging Analysis System.
  • the primary antibodies used were rabbit anti-CHI3L1 (1: 1000, Abcam) , rabbit anti-CRTH2 (1: 1000, Invitrogen) , mouse anti-TMEM219 (1: 1000, Abcam) , rabbit anti-IL13R ⁇ 2 (1: 1000, Biorbyt) , rabbit anti-GSK-3 ⁇ (1: 1000, Bioss) , rabbit anti-p-GSK-3 ⁇ (1: 1000, CST) , rabbit anti- ⁇ -catenin (1: 1000, CST) , and mouse anti- ⁇ -actin (1: 1000, Huabio) .
  • ⁇ -Actin was used as the loading control.
  • the intensity of the bands was quantified using ImageJ software.
  • the ELISA samples included human serum, human cerebrospinal fluid (CSF) , the astrocyte-conditioned media and the lysates of freshly dissected mouse hippocampal tissues. Blood samples with the anticoagulant heparin added were first spun for 15 minutes at 8,000 rpm at 4 °C and the supernatant serum was then collected. Levels of CHI3L1 in serum or CSF were measured with commercial ELISA kits (R&D Systems, MN, USA) based on the provided instructions, including the generation of a standard curve based on a series of dilution of CHI3L1 proteins. Levels of CHI3L1 were measured with commercial ELISA kits (MEIMIAN, MM-44752M2) . The absorbance of each standard and sample was measured at 450 nm.
  • the lentivirus and AAV preparations were routinely produced through the transfection of human embryonic kidney (HEK293T) cells with the desired vectors.
  • the sequences of the shRNAs targeting against the test CHI3L1 receptors are listed in the Table M2.
  • the HEK293T cells were maintained at 37°C in a CO2 cell incubator (Thermo Fisher 371) in Dulbecco’s Modification of Eagle’s Medium (DMEM) 1 ⁇ (MOD.
  • lentivirus particles (319-005-CL, MULTICELL) containing 10%fetal bovine serum (ST30-3302, PANTM SERATECH) , 1%penicillin streptomycin (15140-122, GIBCO) , and 1%glutamine (GIBCO, 25030-081) and passaged every 2-3 days with 0.05%trypsin EDTA.
  • the designed and cloned lentiviral vectors were co-transfected with helper plasmids psPAX and pMD2G into HEK293T cells using polyethyleneimine (PEI, PR40001, Proteintech Group) . The culture medium was changed within 4 hours.
  • the medium containing lentivirus was collected at 2-4 days post transfection, filtered, and concentrated by ultracentrifugation (Beckman SW32 Ti) .
  • the viruses were washed once with PBS and then resuspended in 200 ⁇ L PBS. Each virus tube was labeled and stored at -80°C.
  • the package with AAV-DJ capsids were used for the intended high efficiency of in vivo infection.
  • AAV vectors with pHelper and pRC-DJ were transfected into HEK293 cells, which were collected and lysed 72 hr later.
  • the virus preparations were concentrated from cell lysates by fractioning with iodixanol gradient (40%) and filtering with 100,000 MWCO tube filter.
  • the virus titer was measured by transducing HEK293 with a series of dilutions and analyzing the fluorescent signal from the co-expressed eGFP. Before the concentration, the typical titer was among the range of transduction units (TU) ml before the concentration.
  • TU transduction units
  • For the in vitro lentiviral transductions we calculated the amount of virus preparations to deliver the multiplicity of infection (MOI) of 5-10 in primary cultures of mouse astrocytes and NSCs.
  • MOI multiplicity of infection
  • the virus preparations were concentrated by ultracentrifugation to achieve the concentration of 10 8-9 as indicated in figure legends.
  • mice were used for the described experiments, and maintained for additional four to eight weeks according to the individual study design. These mice were placed in the animal facility of Guangdong Laboratory Animals Monitoring Institute under a 12-hour light/dark cycle constantly under 22-26°C and 50%–60%humidity. The mice could obtain food and water in the cage ad libitum.
  • CHI3L1 f/f mice were created by Cyagen Biosciences (NCBI: 12654) . All mice in the study were backcrossed to the C57BL/6 background for at least six generations. The animals were randomly assigned to the experimental group. Before the experiment was completed, the experimenter did not know the identities of the animals.
  • mice were isolated from eight-to twelve-week-old WT mice. All procedures and feeding were carried out in accordance with the scheme approved by the Laboratory Animals Monitoring Institute and the animal experiment ethics committee of the Third affiliated Hospital of Sun Yat-Sen University. No mice were used in other research projects. The influence of sex was not evaluated in this study.
  • mouse tails were digested overnight in 200 mL lysis solution (containing 1 mg/mL proteinase K, 50 mM Tris-HCl pH 8.0, 100 mM EDTA, 100 mM NaCl and 1%SDS) at 55 °C. Four hundred milliliters of NaCl (6 M) were added to each sample and the solution was mixed well.
  • the solution was incubated on ice for 10 minutes.
  • the samples were centrifuged at 13,000 rpm for 10 minutes at room temperature.
  • the supernatant was transferred to a new 1.5 mL centrifuge tube with 800 mL ethanol (70%in ddH2O) and mixed well.
  • the samples were centrifuged at 13,000 rpm for 10 minutes.
  • the genomic DNA pellet was dissolved in 200 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) . Genotyping was performed with PCR based assays using genomic DNA.
  • the sequences of the primers used were: the forward primer (5’-3’) , GCTACCCAACATGTCAATAGCTCA; the reverse primer (5’-3’) , CATATGGTGGGCAATAATCTTGGA.
  • mice For analysis of NSC proliferation and neuronal differentiation in the adult mouse hippocampal dentate gyrus, adult mice were intraperitoneally (i.p. ) injected with EdU (100 mg/kg) , sacrificed 2 hours after injection and then an EdU Cell Proliferation Kit was used according to the manufacturer’s protocol (C0078L, Beyotime) .
  • EdU 100 mg/kg
  • an EdU Cell Proliferation Kit was used according to the manufacturer’s protocol (C0078L, Beyotime) .
  • C0078L the manufacturer’s protocol
  • BrdU 100 mg/kg
  • AZD1981 (M04757, BJBALB Co. Ltd, Beijing, China) was administered by i.p. injections at the dosage of 1 mg/kg every other day for 4 weeks.
  • AZD1981 was documented to exhibit cross-species binding activity to CRTH2 and block the activation of eosinophils in mice, rats, guinea pigs, rabbits and dogs. In clinical trials, it was well tolerated and no safety concerns were identified.
  • the components of the osmotic pumps were first assembled according to the manufacturer’s instructions (ALZET, Model 1002) .
  • the mini-osmotic pump was then filled with msCtrl-IgG or msAQP4-IgG (1 ⁇ g/100 ⁇ l in PBS) and then positioned at the following coordinates relative to bregma, caudal side: -2.0 mm; transverse direction: +/-1.7 mm; ventral: -1.9 mm.
  • the infusion flow rate was set at 0.25 ⁇ l per hour for 14 days.
  • mice were euthanized by intraperitoneal injection of 2%pentobarbital sodium and given an initial flush with 0.01 M phosphate buffered saline (PBS) . Then, the mice were transcardially perfused with 4%PFA. Mouse brains were fixed in 4%PFA at 4°C overnight and then placed in 30%sucrose until submerged. The brains were cut into 40- ⁇ m-thick sections coronally using a microtome and placed in a 96-well plate. The slices were kept in cryoprotectant solutions (glycerol, ethylene glycol and 0.1 M phosphate buffer, pH 7.4, 1: 1: 2 by volume) at 4 °C.
  • cryoprotectant solutions glycerol, ethylene glycol and 0.1 M phosphate buffer, pH 7.4, 1: 1: 2 by volume
  • mice anti-GFAP (1: 1000, CST)
  • rabbit anti-GFAP (1: 1000, CST)
  • chicken anti-GFAP (1: 1000, Abcam)
  • rabbit anti-NeuN (1: 500, Abcam)
  • mouse anti-DCX (1: 200, Santa)
  • rabbit anti-Sox2 (1: 500, Abcam)
  • mouse anti-Sox2 (1: 500, Abcam)
  • rat anti-BrdU (1: 1000, Abcam)
  • mouse anti-Tuj 1 (1: 1000, Abcam)
  • rabbit anti-IIL13R ⁇ 2 (1: 500, CST)
  • rabbit anti-CHI3L1 (1: 500, Abcam)
  • goat anti-CHI3L1 (1: 500, R&D Systems
  • rabbit anti-CHI3L1 (1: 500, Solarbio)
  • rabbit anti-CRTH2 (1: 500, Invitrogen)
  • mouse anti-CRTH2 (1: 500, Invitrogen)
  • mouse anti-CRTH2 (1: 500, Invitrogen)
  • Antigen retrieval was needed. Antigen retrievals were performed in citrate buffer (pH 9.0) with a microwave for 10 minutes at 95 °C followed by 20 minutes of cooling at room temperature. The following fluorescent secondary antibodies were used: goat anti-mouse 488, goat anti-mouse 568, goat anti-mouse 647, goat anti-rabbit 488, goat anti-rabbit 568, goat anti-rabbit 647, goat anti-rat 568, and goat anti-chicken 647. Confocal single plane images and z stacks were taken with a laser confocal microscope (Leica, TCS SP8) equipped with four laser lines (405, 488, 568 and 647 nm) and 63x, 40x and 20x objective lenses.
  • mice of different experimental groups were placed in a 50 cm x 50 cm x 50 cm arena and allowed to explore the site freely for 10 minutes. After the test, they were placed back in their cage. The distance traveled was recorded to evaluate the motor ability of the mice, and the time spent in the central area was measured to detect the anxiety of the mice. Experimenters were blinded to the genotypes and mice.
  • the MWM test consisted of a water-filled pool (diameter 120 cm) with a hidden escape platform under the water surface.
  • the platform (10 cm wide) was located approximately 1 cm below the water level and was divided into four quadrants.
  • the MWM is divided into training and testing phases.
  • mice were randomly placed in the water maze from four different starting positions (NE, NW, SE and SW) .
  • the mice were trained for five days to find the hidden platform.
  • the water on the mouse was wiped with a towel and then the mouse was placed back in the cage.
  • a detection test was conducted, during which the platform was removed and times crossing the platform area were measured by camera to test short-term spatial memory. All tests were performed at the same time every day. Experimenters were blinded to the genotypes and mice.
  • Neuromyelitis optica (NMO) patients were diagnosed based on the 2015 International Panel for NMO Diagnosis (IPND) criteria and received treatments and follow-up cares at the Third affiliated Hospital of Sun Yat-Sen University and the Second affiliated Hospital of Guangzhou Medical University. Blood and CNS samples, MRI scans and neuropsychological assessments were acquired during a clinically stable stage when the patients were recovering from the attack or relapse for at least 3 months did not show symptomatic deterioration. For all the individuals participated in our clinical study, the informed consent was obtained after the nature and possible consequences of the study was explained.
  • IPND International Panel for NMO Diagnosis
  • Frozen sections of human brain were obtained from biopsy brain tissues of one AQP4-IgG serum-positive NMO patient and one control.
  • the clinical characteristic data of the pathological specimen providers are shown in Table S1.
  • Table S Information regarding the biopsied brain samples from the control and NMO patient (complementary to Fig. 2B) .
  • the hippocampus was accurately segmented into the following subfields: parasubiculum, presubiculum, subiculum, CA1, CA2/3, CA4, GC-DG, hippocampus-amygdala transition area (HATA) , fimbria, molecular layer, fissure, hippocampal tail.
  • HATA hippocampus-amygdala transition area
  • MMSE Mini-Mental State Examination Scale
  • SDMT Symbol Digit Modalities Test
  • BVMT-R Brief Visuospatial Memory Test Revised
  • the CVLT assessed the subjects’ learning and memory abilities, including immediate recall (Trials 1-5, T1-5) , short delayed free recall (SDFR) , short delayed cue recall (SDCR) , long delayed cue recall (LDCR) and long delayed free recall (LDFR) .
  • General anxiety and depression-relevant behaviors were assessed using the Hamilton Anxiety Rating Scale (HARS) and Hamilton Depression Rating Scale (HDRS) , respectively. Behavioral symptoms were assessed via interview with the informant and quantified using the Frontal Behavioral Inventory.
  • HAS Hamilton Anxiety Rating Scale
  • HDRS Hamilton Depression Rating Scale
  • CHI3L1 is induced in astrocytes activated by pro-inflammatory stimuli of cytokines and antibody-mediated immune response
  • CHI3L1 in activated astrocytes by analyzing two independent transcriptome datasets of mouse astrocytes undergoing neurotoxic activation after inflammatory stimulation in vivo and in vitro.
  • astrocytes acutely purified from young adult mice subject to intraperitoneal injection of lipopolysaccharide (LPS) CHI3L1 level was dramatically increased (fig. S1A in vivo; Hasel, P., et al. Neuroinflammatory astrocyte subtypes in the mouse brain. Nature neuroscience24, 1475-1587 (2021) ) ; the increase was even more obvious in the cultures of primary astrocytes treated with pro-inflammatory cytokines of IL-1, TNF and C1q (ITC; Fig.
  • CHI3L1 is induced in astrocytes activated by immune-mediated inflammatory stimuli and is an important secreted protein in neuroinflammation associated with the astroglial autoimmune response of hsAQP4-IgG expression.
  • CHI3L1 secretion correlates with neurocognitive impairment and hippocampal dysfunction in human brains affected by autoimmune-mediated astrocyte activation
  • Astrocyte-secreted CHI3L1 reduces proliferation and differentiation of neural stem cells in immune-mediated neuroinflammation
  • CHI3L1 secreted by activated astrocytes contributes to the hippocampal dysfunction underlying the neuroinflammation-related cognitive impairment
  • NSC neural stem cell
  • CHI3L1 treatment 100 ng/ml for 3 days
  • Fig. 2F EdU labeling
  • Our assays of multiple fluorescence immunostaining for neural markers showed that CHI3L1 treatment significantly shrank the population of the newly differentiated neurons (Fig. 2G; labeled by Tuj1) , implying a hampered neurogenesis process.
  • CHI3L1 secreted by astrocytes improves adult hippocampal neurogenesis and function in immune-mediated neuroinflammation
  • CHI3L1-CRTH2-GSK-3 ⁇ / ⁇ -catenin signaling cascade could be modulated to modify the inhibitory effect of CHI3L1 on hippocampal function and neurogenesis in vivo.
  • CHI3L1 is primarily secreted by activated astrocytes
  • the msCtrl-IgG or msAQP4-IgG was chronically perfused into the dentate gyrus for a period of two weeks by a minipump to elicit astrocyte activation and immune-mediated neuroinflammation.
  • the treated mice were then sacrificed at the indicated times to prepare the hippocampal slices for our confocal microscopy (Fig. 6G) .
  • Fig. 6G confocal microscopy
  • CHI3L1 depletion in astrocytes counteracted the inhibitory effect from msAQP4-IgG and made no obvious alternation in the pools of immature (BrdU+DCX+) and mature (BrdU+NeuN+) neurons as compared to the control msCtrl-IgG group (Fig. 6J and K) .
  • CRTH2 knockdown abolished such repressive influence from msAQP4-IgG and led to little or no apparent change in NSC proliferation (Fig. 7B) .
  • CRTH2 knockdown also preserved the numbers of immature (GFP+BrdU+DCX+) and mature (GFP+BrdU+NeuN+) newborn neurons, which were both reduced in the presence of CRTH2 to convey CHI3L1 signaling in neuroinflammation (Fig. 7C and 7D) .
  • AZD1981 relieved the repression of neurogenesis in the neuroinflammatory condition initiated by msAQP4-IgG perfusion: in msAQP4-IgG groups (msAQP4-IgG alone and msAQP4-IgG+AZD1981) , AZD1981-treated mice had significantly more proliferating cells (Fig. 7F, EdU+) that were radial glia-like NSCs (Fig. 7G, EdU+GFAP+Sox2+) and had also more newborn neurons that were immature (Fig. 7H, BrdU+DCX+) or mature (Fig. 7I, BrdU+NeuN+) at the time of examination.

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Abstract

The present disclosure provides, for instance, a method of treating neuroinflammation including NMOSD in a subject, the method comprising administering to the subject a CRTH2 antagonist, thereby treating the neuroinflammation in the subject.

Description

FIRST IN CLASS USE OF THE CRTH2 ANTAGONIST IN TREATING NEUROMYELITIS OPTICA SPECTRUM DISORDER (NMOSD) AND RELEVANT AUTOIMMUNE CNS DISORDERS BACKGROUND
Neuromyelitis optica spectrum disorder (NMOSD) is a neurological disorder characterized by neuroinflammation. One type of NMOSD results from primary astrocytopathy caused by autoantibodies targeting the astroglial protein aquaporin 4 (AQP4) , and results in severe neurological sequelae of vision loss, motor deficits and cognitive decline. There is a need in the art for new therapies that treat the cognitive deficits in NMOSD.
SUMMARY OF THE INVENTION
In some aspects, the present disclosure provides a method of treating neuroinflammation in a subject, the method comprising: administering to the subject an effective amount of a CRTH2 inhibitor, thereby treating the neuroinflammation in the subject. In some aspects, the present disclosure provides a CRTH2 inhibitor for use in the treatment of neuroinflammation in a subject. In some aspects, the present disclosure provides the use of a CRTH2 inhibitor for the manufacture of a medicament for the treatment of neuroinflammation in a subject.
In some embodiments, the subject has a Neuromyelitis Optica Spectrum Disorder (NMOSD) . In some embodiments, the subject has Alzheimer’s disease, glioblastoma, or an autoimmune disorder. In some embodiments, the autoimmune disorder is multiple sclerosis. In some embodiments, the subject has malignancy of glioblastoma.
In some aspects, the present disclosure provides a method of treating an autoimmune CNS disorder in a subject, the method comprising: administering to the subject an effective amount of a CRTH2 inhibitor, thereby treating the autoimmune CNS disorder in the subject. In some aspects, the present disclosure provides a CRTH2 inhibitor for use in the treatment of autoimmune CNS disorder in a subject. In some aspects, the present disclosure provides the use of a CRTH2 inhibitor for the manufacture of a medicament for the treatment of autoimmune CNS disorder in a subject.
In some aspects, the present disclosure provides a method of treating a Neuromyelitis Optica Spectrum Disorder (NMOSD) in a subject, the method comprising: administering to the subject an effective amount of a CRTH2 inhibitor, thereby treating the NMOSD in the subject. In some aspects, the present disclosure provides a CRTH2 inhibitor for use in the treatment of a Neuromyelitis Optica Spectrum Disorder (NMOSD) in a subject. In some aspects, the present disclosure provides the use of a CRTH2 inhibitor for the manufacture of a medicament for the treatment of a Neuromyelitis Optica Spectrum Disorder (NMOSD) in a subject.
In some embodiments, the CRTH2 inhibitor comprises a small molecule, an antibody molecule, a nucleic acid, or a polypeptide. In some embodiments, the CRTH2 inhibitor comprises AZD1981, fevipiprant, OC00459, BI671800, AMG853, TQC3564, setipiprant, ASP5642, ARRY-502, AZD6430, ADC-3680, MK-1029, MK-7246, or BI1021958. In some embodiments, the CRTH2 inhibitor is administered at a dose of 25 to 1200mg. In some embodiments the CRTH2 inhibitor is administered daily or twice daily. In some embodiments the CRTH2 inhibitor is administered intravenously, orally, parenterally, or topically.
In some embodiments, the NMO is monophasic NMO, relapsing NMO, acute NMOSD, chronic NMOSD, AQP4 antibody negative NMOSD, AQP4 antibody positive NMOSD, MOG antibody negative NMOSD, and/or MOG antibody positive NMOSD. In some embodiments, the subject is suffering from a neurological symptom, neuroinflammation, cognitive impairment, loss of spinal cord functions, loss of optic nerve functions, vision loss, and/or motor deficits.
In some embodiments, treatment results in lessening of cognitive impairment in the subject. In some embodiments, treatment results in an increase in neurogenesis in the subject. In some embodiments, treatment results in an increase in neural stem cell proliferation in the subject. In some embodiments, treatment results in an increase in neural stem cell differentiation in the subject.
In some embodiments, the subject has detectable levels of CHI3L1 prior to the administration. In some embodiments, the subject has an elevated level of CHI3L1 compared to a healthy subject prior to the administration. In some embodiments, the subject has an elevated level of astrocyte activation prior to the administration. In some embodiments, the subject has detectable levels of CRTH2 prior to the administration. In some embodiments, the subject is a human.
In some aspects, the present disclosure provides a method of treating neuroinflammation in a subject, the method comprising: administering to the subject an effective amount of a CHI3L1 inhibitor, thereby treating the neuroinflammation in the subject. In some aspects, the present disclosure provides a CHI3L1 inhibitor for use in the treatment of neuroinflammation in a subject. In some aspects, the present disclosure provides the use of a CHI3L1 inhibitor for the manufacture of a medicament for the treatment of neuroinflammation in a subject.
In some embodiments, the CHI3L1 inhibitor comprises a small molecule, an antibody molecule, a nucleic acid, or a polypeptide. In some embodiments, the subject has a Neuromyelitis Optica Spectrum Disorder (NMOSD) .
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A is a schematic diagram showing the treatment of primary cultures of wildtype mouse astrocytes with the classic pro-inflammatory cytokine IL-1β (100 ng/ml) or the control of vehicle PBS, the monoclonal mouse antibodies against AQP4 (msAQP4-IgG, 100 ng/ml) or the control mouse IgG (msCtrl-IgG, 100 ng/ml) , and human anto-AQP4 autoantibodies purified from  NMO patients (hsAQP4-IgG, 100 ng/ml) or control human IgG (hsCtrl-IgG, 100 ng/ml) for 6, 12 and 24 hours.
Figure 1B depicts relative levels of CHI3L1 mRNA induction in primary astrocytes treated with IL-1β, msAQP4-IgG or hsAQP4-IgG at each time point assayed by qPCR and normalized to the levels of the individual control conditions (set as 1.0) . n=3 per group. The bar graphs are presented as the mean ± SEM; the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 1C depicts representative confocal images of (top) and the quantification of (bottom) astroglial marker expression in mouse primary astrocytes treated with vehicle (PBS) or IL-1β (100 ng/ml) after 24 hours. Top, immune-fluorescent labeling, from left to right, of GFAP, AQP4, CHI3L1 and DAPI. Bottom, the bar graphs of the relative fluorescence intensity of GFAP (left) , AQP4 (middle) , CHI3L1 (right) after the normalization to the level of control (=1.0) . Scale bar, 100 μm. n=4 per group. The bar graphs of are presented as the mean ± SEM; the statistical evaluation was performed with Student’s t test for 2 conditions. *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 1D depicts representative immune-fluorescent images (left panels) and the quantification of fluorescence signals (right bar graphs) of, from left to right, GFAP, AQP4, CHI3L1 and DAPI in mouse primary astrocytes treated with msAQP4-IgG (100 ng/ml) or hsAQP4-IgG (100 ng/ml) alongside the individual controls (msCtrl-IgG or hsCtrl-IgG, 100 ng/ml, value set as 1 for normalization) for 24 hours. Scale bar, 100 μm. n=4 per group. The bar graphs of are presented as the mean ± SEM; the statistical evaluation was performed with Student’s t test for 2 conditions. *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 1E depicts transcriptomic analyses of mouse astrocytes activated by AQP4 autoantibodies (3 biological replicates with 3 different batches of human IgG purification) , with the list of the identified 152 differentially expressed genes (DEGs) from bulk RNA-sequencing being contrasted with the published dataset by Waler-Caulfield et al. (PMID: 26423139) . The 91 DEGs overlapped between two studies were analyzed by the hierarchical clustering heatmap of gene fold changes on a log2 scale.
Figure 1F depicts a Venn diagram of the DEGs identified by two independent transcriptomic studies of mouse astrocytes stimulated by human anti-AQP4 antibodies (hsAQP4-IgG vs. hsCtrl-IgG) , showing that 21 out of the 91 overlapped genes were secreted proteins.
Figure 1G depicts Gene Ontology (GO) enrichment analysis of the 91 overlapped DEGs.
Figure 1H depicts hierarchical clustering of 21 overlapped DEGs that were secreted proteins presented in the heatmap of gene fold on a log2 scale; 20 upregulated (including CHI3L1 among the top genes) and one, Igsf21, downregulated by immune-mediated astroglial activation.
Figure 1I depicts the analysis of the gene fold changes in the overlapped DEGs with the GO of Neuroinflammation, plotted on a log10 scale in the comparison of the untreated samples to unmask the effect of control IgG treatment (zero-fold change, white) .
Figure 1J depicts the analysis of the gene fold changes in the overlapped DEGs with a published transcriptional response pattern of astrocyte activation (PMID: 22553043; “reactive” ) . The changes induced by hsCtrl-IgG or hsAQP4-IgG were normalized to untreated samples (=0 in white) and mapped on a log10 scale.
Figure S1A depicts data from evaluation of CHI3L1 induction in neuroinflammation by transcriptomic analyses of wildtype mouse astrocytes in primary cultures treated with pro-inflammatory cytokines IL-1α, TNF and C1q (ITC for 24h; ‘in vitro’ ) and acutely purified from mice stimulated by intraperitoneal injection of liposaccharide (LPS after 24h; ‘in vivo’ ) , based on the bulk RNA-Seq datasets from Guttenplan et al. (PMID: 32719333) and Hasel et al. (PMID: 34413515) .
Figure S1B depicts functional principal component analysis (PCA) of bulk RNA-Seq from this study on primary mouse astrocytes treated with hsCtrl-IgG or hsAQP4-IgG, 100 ng/ml for 24h, showing separation between control and hsAQP4-IgG treated conditions.
Figure S1C is a volcano plot of gene fold changes induced by hsAQP4-IgG as compared to the hsCtrl-IgG stimulation from this study. p value<0.05 and a fold change cutoff of log2 ratio≥0.5 were used to detect the differentially expressed genes (DEGs) . There were110 upregulated genes and 42 downregulated genes. The exemplary secreted protein genes overlapped with the published dataset of Waler-Caulfield et al. (PMID: 26423139) were indicated.
Figure S1D shows GO enrichment analysis of DEGs between primary astrocytes treated with hsCtrl-IgG or hsAQP4-IgG from this study.
Figure S1E shows a pathway visualization of the NFκB canonical pathway, identified via pathway visualization of the 91 overlapped DEGs performed by extracting the Kyoto Encyclopedia of Genes and Genomes database (KEGG) with the data integration tool of Pathview.
Figure 2A depicts measurements of CHI3L1 levels in serum and CSF samples from healthy controls (HCs) and NMO patients by ELISA presented as the mean ± SEM. The sample sizes: HC-serum, n=20; NMO-serum, n=44; HC5 CSF, n=26; NMO-CSF, n=29. Statistical significance was evaluated with Student’s t test for two-group comparisons; *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 2B shows representative confocal images of biopsied human brains tissues from a control subject and a NMO patient, the characteristic astroglial features of decreased AQP4 expression (second panel from left) particularly within the end-feet covering the micro-vessels  (labeled by CD31 for outlining endothelial cells, left panel) , together with the increased GFAP (middle panel) expression. Scale bar, 50 μm.
Figure 2C depicts the correlation between CHI3L1 CSF levels and neuropsychological assessments in NMO patients, presented in a bubble chart of the Pearson correlation coefficient. CHI3L1 levels positively correlated with increased anxiety and depression-like symptoms assayed by HARS and HDRS tests, and negatively correlated with the cognitive performance of learning and memory functions assayed by tests of BVMT-R, multiple CLVT formats, SDMT and MMSE.
Figure 2D shows assessments of the hippocampal formation volumes from the head MRI scans of healthy controls (HCs, left boxes) and NMO patients (right boxes) presented as the mean ±SEM. HC: n=15; NMOSD patients: n=23. Statistical significance was evaluated with Student’s t test for two-group comparisons; *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 2E depicts a schematic diagram for experiments to demonstrate the effect of CHI3L1 on the proliferation and neuronal differentiation of neural stem cells (NSCs) in vitro. The NSCs were first stimulated with recombinant CHI3L1 protein (100ng/mL) or vehicle control of PBS for 3 days in the proliferation media. EdU was administered in half of the treated cultures for 2 hours before the fixation and immuno-staining to evaluate the proliferation. The remaining half of the NSC cultures were switched into the differentiation media, being allowed to differentiate for additional 3 days with the incubation of PBS or CHI3L1 (100 ng/ml) , and were then fixed and subject to confocal analyses for neuronal differentiation.
Figure 2F shows imaging and quantification of NSC proliferation in the absence (PBS) or presence of CHI3L1 stimulation presented as the mean ± SEM. Left, the nuclear fluorescence of the colocalized EdU and DAPI signals in PBS-or CHI3L1-treated NSC cultures. Right, the average numbers of the percentage of EdU+ nuclei in the DAPI-labeled ones were plotted. n=4 independent experiments. Scale bars, 100 μm. Statistical significance was evaluated with Student’s t test for two-group comparisons; *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 2G shows representative confocal images and the analyses of NSC neuronal differentiation with or without CHI3L1 treatment presented as the mean ± SEM. Left, Tuj1 to mark the differentiated neurons; immune-staining of GFAP and DAPI. Right, quantification of Tuj1+ and GFAP+ cells graphed in the average percentage of colocalization with DAPI+ cells from 4 independent experiments. Scale bars, 100 μm. Statistical significance was evaluated with Student’s t test for two-group comparisons; *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 2H depicts a schematic diagram for the experiments to test the effect of astrocyte-secreted CHI3L1 on proliferation and neuronal differentiation of NSCs. The primary cultures of mouse astrocytes were first treated with hsCtrl-IgG or hsAQP4-IgG at 100 ng/ml for 24 hours. The  astrocyte-conditioned media (ACM) were harvested and treated with vehicle control or the neutralizing anti-CHI3L1 antibodies (100 ng/ml) to eliminate the secreted CHI3L1 after the removal of IgG contents. The cleared ACM were then supplemented with factors to constitute the growth media for the proliferation or the differentiation of NSCs. The purified mouse NSCs were cultured in ACM-based proliferation media for 3 days. On Day 3, half of the cultures were subject to EdU labeling for 2 hours before the fixation and immune-staining to evaluate the proliferation. Half of the cultures were changed with the ACM-based differentiation media to initiate the neuronal differentiation, and 3 days later were fixed and immune-stained for confocal analyses of neuronal differentiation. Pearson correlation coefficient was used to assess the correlation between the neuropsychological testing and the CHI3L1 CSF levels. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 2I shows the ELISA measurements of the secreted CHI3L1 levels in astrocyte-conditioned-media (ACM) harvested from primary cultures of mouse astrocytes treated with hsCtrl-IgG or hsAQP4-IgG presented as the mean ± SEM. n=3 experiments per group. Statistical significance was evaluated with Student’s t test for two-group comparisons; *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 2J shows representative images of proliferating mouse NSCs (EdU+) grown in hsCtrl-IgG-treated or hsAQP4-IgG-treated astrocyte-conditioned media (ACM) , without (vehicle) or with the clearance of CHI3L1 (by anti-CHI3L1 neutralizing antibodies) . Scale bars, 100 μm.
Figure 2K depicts a bar graph showing quantification of NSC proliferation as in Fig. 3J by plotting the percentage of DAPI+EdU+ in all DAPI+ nuclei in each indicated condition presented as the mean ± SEM. n=4 experiments per group. Statistics were computed with one-way ANOVA and Tukey’s post hoc multiple comparisons; *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 2L shows representative images of newly differentiated neurons from NSCs of 4 conditions –grown in hsCtrl-IgG-ACM or hsAQP4-IgG-ACM, without (vehicle) or with CHI3L1 clearance (anti-CHI3L1) –immune-stained with nuclear marker DAPI and neural markers of Tuj 1 and GFAP. Scale bars, 100 μm. Statistics were computed with one-way ANOVA and Tukey’s post hoc multiple comparisons; *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 2M shows quantification presented as the mean ± SEM of neuronal differentiation of NSCs by calculating the numbers of Tuj1+ and GFAP+ cells astrocytes in the indicated 4 groups. n=4 experiments per group. Statistics were computed with one-way ANOVA and Tukey’s post hoc multiple comparisons; *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure S2A shows representative MRI images of brain, spinal cord and optic nerve from healthy control (HC) subjects and NMO patients. The typical lesions of neuromyelitis and optic neuritis were indicated by arrows.
Figure S2B shows the atlas-based segmentation of the hippocampus on the representative MRI head images with 3D rendering, to facilitate the analyses of the anatomical structures within the hippocampus region and complement the results in Fig. 2D.
Figure S2C shows the scoring of the neuropsychological tests in health control (HC, left boxes) subjects and NMO patients (right boxes) , plotted in the box chart of distribution for the test actual scores. HC, n=20; NMO, n=44. Data are presented as the mean ± SEM; the statistical significance was evaluated with Student’s t test for two-group comparisons (HC vs. NMO) . *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure S2D shows the 3D size quantification of the hippocampal subfields on the left side for healthy control (HC, left boxes) subjects and NMO patients (right boxes) . HC, n=15; NMO patients: n=23. Data are presented as the mean ± SEM; the statistical significance was evaluated with Student’s t test for two-group comparisons (HC vs. NMO) . *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure S2E shows the 3D size quantification of the hippocampal subfields on the right side for healthy control (HC, left boxes) subjects and NMO patients (right boxes) . HC, n=15; NMO patients: n=23. Data are presented as the mean ± SEM; the statistical significance was evaluated with Student’s t-test for two-group comparisons (HC vs. NMO) . *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 3A depicts a schematic diagram and the timeline of the experiments to assay the hippocampal neurogenesis and cognitive performance in 8-week-old wildtype mice receiving the stereotaxic delivery of the control (msCtrl-IgG) or the monoclonal mouse anti-AQP4 antibodies (msAQP4-IgG) into the dentate gyrus within the hippocampal formation on both sides, at the amount of 1 μg IgG in 1 μl PBS for each side. For the evaluation of NSC proliferation, in half of the injected mice, 5-Ethynyl-2’-deoxyuridine (EdU) we readministered intraperitoneally (i.p., 100 mg/kg) one week after the stereotaxic IgG injections, followed by the sacrifice 2 hours later. For the examination of NSC differentiation, the remaining half of the IgG-injected mice were given 5-bromo-2’-deoxyuridine (BrdU) i.p. at 100 mg/kg for 4 consecutive days starting from the next day of stereotaxic injections, and were sacrificed at around the 4th week. The standardized behavioral tests of the open field test (fig. S3C-E) and Morris water maze were performed in tandem at the Week 3-5 before the sacrifice.
Figure 3B shows analysis of AQP4 expression enriched within the astrocytic end-feet along the micro vessels in the hippocampal regions from mouse brains injected by msCtrl-IgG or msAQP4- IgG. The quantification was plotted by the calculated percentage of AQP4 coverage along the length of micro-vessels labeled by the endothelial marker CD31. n=3 animals per group. Scale bar, 20 μm. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3C depicts immunostaining of the astrocyte marker GFAP for quantification of astrogliosis in response to msAQP4-IgG treatment, presented in the average numbers of GFAP+ cells within the volume of dentate gyrus (DG, determined by 3D rendering of confocal images) . n=3 animals per group. Scale bar, 100 μm. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3D shows assessment of CHI3L1 expression in astrocytes induced by msAQP4-IgG, quantified by the intensity of CHI3L1 fluorescence that was colocalized with GFAP signals and presented as the relative levels after setting the value of msCtrl-IgG as 1.0. n=3 animals per group. Scale bar, 20 μm. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3E depicts representative movement paths of the mice receiving msCtrl-IgG or msAQP4-IgG stereotaxic injections in the standard Morris water maze (MWM) task.
Figure 3F shows quantification of the platform crossing numbers in the MWM task for both tested groups. n=9 animals per group. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3G depicts the escape latencies to find the platform during the training sessions in the MWM task for the msCtrl-IgG and msAQP4-IgG groups. n=9 animals per group. Data was analyzed with two-way ANOVA with Tukey’s multiple comparisons test. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3H depicts representative images and the confocal analyses of the total proliferating cells (labeled by EdU administration for 2 hours) in the sub-granular zone (SGZ) of the hippocampus after one week of IgG stereotaxic injections (see Fig. 3A) . Left bar graphs, the sizes of the dentate gyri (DG) from the msCtrl-IgG-injected and msAQP4-IgG-injected mice were not different after the 3D reconstruction of confocal imaging, following the standardized quantification method to assay neurogenesis (PMID: 32848155) . Right bar graphs, the average numbers of EdU+ cells within the volume of dentate gyrus (DG) for both the msCtrl-IgG and msAQP4-IgG groups. n=5 animals per group. Scale bar, 100μm. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3I shows characterization of the proliferating NSCs by quantifying the populations of the radial glia-like cells (let bar graphs, triple-labeled by EdU+GFAP+Sox2+) and the transiently amplifying progenitor-like cells (right bar graphs, EdU+GFAP-Sox2+) , with the fluorescence signals of EdU, Sox2, GFAP, and DAPI. The values were plotted as the average numbers of each cell types within the volume of the dentate gyrus. n=4 animals per group. Scale bar, 100 μm. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3J shows analysis of newborn neurons differentiated from BrdU-labeled NSCs 4 weeks after the IgG stereotaxic injections (arrows in Fig. 3A) . Representative confocal images showed the immunostaining of BrdU (left panel) and the DCX, marker for immature neurons (right panel, merge with BrdU channel) . Left bar graphs, the average numbers of total BrdU+ cells within the volume of dentate gyrus from msCtrl-IgG and msAQP4-IgG groups. Right bar graphs, the quantifications of immature neurons differentiated from NSCs after the IgG (BrdU+DCX+) inside the dentate gyri of the two groups. n=4 animals per group. Scale bar, 100 μm. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3K shows imaging quantification of newly-differentiated mature neurons within the dentate gyrus regions from the BrdU-labeled mice (Fig. 3A) . BrdU was immune-stained along with NeuN, the mature neuronal marker. n=4 animals per group. Scale bar, 100 μm. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3L depicts a schematic diagram and the timeline of the experiments to assay the hippocampal neurogenesis and cognitive performance in 8-week-old wildtype mice receiving mini-pump infusion of msCtrl-IgG or msAQP4-IgG into the hippocampal regions on both sides, with a total of 1 μg in 100 μl PBS on each side and at the rate of 0.25 μl per hour for 2 weeks. The EdU and BrdU were administrated i.p. to evaluate the proliferation and differentiation, respectively, similar as the design in Figure 3A.
Figure 3M shows quantification of total proliferating (EdU+) cells for both msCtrl-IgG and msAQP4-IgG groups, presented as the average cell numbers within the volume of the dentate gyrus. n=4 animals per group. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3N shows quantification of the radial glia-like (EdU+GFAP+Sox2+) cellsfor both msCtrl-IgG and msAQP4-IgG groups, presented as the average cell numbers within the volume of the dentate gyrus. n=4 animals per group. Bar graphs were presented as the mean ± SEM; statistical  significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3O shows quantification of the transiently amplifying progenitor-like (EdU+GFAP-Sox2+) cells for both msCtrl-IgG and msAQP4-IgG groups, presented as the average cell numbers within the volume of the dentate gyrus. n=4 animals per group. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3P shows quantification of the newborn immature (BrdU+DCX+) neurons within the dentate gyrus regions for both groups. n=4 animals per group. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure 3Q shows quantification of the mature (BrdU+NeuN+) neurons within the dentate gyrus regions for both groups. n=4 animals per group. Bar graphs were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p <0.05, **p<0.01, ***p < 0.001.
Figure S3A depicts measurements of CHI3L1 levels by ELISA on the lysates of acutely dissected, unfixed hippocampal tissues from the mouse brains stereotaxically injected with msCtrl-IgG-or msAQP4-IgG-treated, complementing the immune-staining results of CHI3L1 induction by msAQP4-IgG in Fig. 3D. n=3 animals per group. Data were presented as the mean ± SEM in bar graphs, and the statistical significance was evaluated with Student’s t test for two-group comparisons. *p < 0.05, **p<0.01, ***p < 0.001.
Figure S3B depicts examinations of the pro-inflammatory genes induced upon astroglial activation by msAQP4-IgG in the hippocampal lysates as in fig. S3A. The data of qPCR assays on the cytokines and chemokines including those identified by the RNA-seq (Fig. 1H-J) were plotted as the heatmap of gene fold change. Black indicated no change and white color indicated >10-fold chance. n=3 animals per group.
Figure S3C shows the representative movement paths of the mice stereotaxically injected with msCtrl-IgG or msAQP4-IgG measured by the standardized open field test before the Morris water maze (Fig. 3E-G) , to evaluate their overall locomotor activities and anxiety-like behaviors.
Figure S3D shows quantification of the open field test results by measuring the average total distance travelled. n=9 mice per group. Data were presented as the mean ± SEM in bar graphs, and the statistical significance was evaluated with Student’s t test for two-group comparisons. *p < 0.05, **p<0.01, ***p < 0.001.
Figure S3E shows quantification of the open field test results by measuring the average crossing numbers through the center area (E) . n=9 mice per group. Data were presented as the mean ±SEM in bar graphs, and the statistical significance was evaluated with Student’s t test for two-group comparisons. *p < 0.05, **p<0.01, ***p < 0.001.
Figure S3F shows representative confocal images for the analyses of NSC proliferation, complementing the results in Fig. 3M-O. The labeling of EdU, Sox2, GFAP, and DAPI was to identify the total proliferating cells (EdU+) , the radial glia-like cells (EdU+GFAP+Sox2+) , and the transiently amplifying progenitor-like cells (EdU+GFAP Sox2+) . Scale bar, 100 μm.
Figure S3G shows representative confocal images of labeling the newborn immature neurons in the hippocampus SGZ from mice injected with msCtrl-IgG or msAQP4-IgG, complementing the results in Fig. 3P. BrdU was immune-stained (left panels) along with the immature neuronal marker DCX (right panels, merge with BrdU channel) . Scale bar, 100 μm.
Figure S3H shows representative confocal images of labeling the mature neurons newly differentiated from NSCs in the hippocampus SGZ as in fig. S3G, complementing the results in Fig. 3Q. BrdU was immune-stained (left panels) along with the mature neuronal marker NeuN (right panels, merge with BrdU channel) . Scale bar, 100 μm. Scale bar, 100 μm.
Figure 4A is a schematic diagram of the lentiviral vector design to transduce eGFP alone as a control (Lenti-Ctrl) or CHI3L1 with co-expression of eGFP (Lenti-CHI3L1, or Lenti-GFP-2A CHI3L1) under the control of the CMV promoter. The lentiviral particles were concentrated to transduction units (TU) per ml, and 1 μl was stereotaxically injected into the dentate gyrus within the hippocampus on either side of 8-week-old wildtype mice.
Figure 4B depicts the timeline for the experiments to assess NSC proliferation by EdU labeling, NSC differentiation and behavioral consequences in the mice that over expressed eGFP only (Lenti-Ctrl) or CHI3L1 plus eGFP (Lenti-CHI3L1) in their hippocampi. The overall experiment design was similar as the described in Fig. 3A, with the BrdU being administered immediately after the intra-hippocampal lentiviral injections for assays of NSC differentiation at the Week 4-6 and EdU being given at the Week 2 for 2 hours for the evaluation of NSC proliferation. The behavioral tests of the open field test (fig. S4A-C) and Morris water maze were carried out in tandem at the Week 4-6 right before the sacrifice and tissue harvest.
Figure 4C shows the validation of CHI3L1 over-expression in the acutely dissected, unfixed hippocampal tissues by immunoblotting of the hippocampal lysates harvested from mice brains injected with Lenti-Ctrl or Lenti-CHI3L1.
Figure 4D shows the representative movement paths for both Lenti-Ctrl and Lenti-CHI3L1 groups. The task of Morris water maze (MWM) was performed at 6-Apr weeks after the lentiviral injections.
Figure 4E shows the analysis of MWM by quantification of the crossing numbers. Lenti-Ctrl, n=8 mice; Lenti-CHI3L1, n=12. Data in were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 4F shows the analysis of MWM by quantification of the escape latencies to find the platform during the training. Lenti-Ctrl, n=8; Lenti-CHI3L1, n=12. Data were analyzed with two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p<0.01, ***p < 0.001.
Figure 4G depicts representative images of the mouse hippocampal sections labeled with EdU and transduced by the Lenti-Ctrl or Lenti-CHI3L1 lentiviruses (Fig. 4B) . The immune staining of eGFP showed efficient lentiviral transduction within the dentate gyral regions in both groups. The EdU was immune-stained to label the proliferating cells within the SGZ. The immune-staining of GFAP was used to further classify the proliferating cells of a proper NSC morphology (EdU+) into the radial glia-like cells (EdU+GFAP+) and the transiently amplifying progenitor-like cells (EdU+GFAP-) . Scale bar, 100 μm.
Figure 4H depicts quantification of totally proliferating (EdU+) cells plotted as the average cell numbers within the volume of dentate gyrus (decided by the imaging processing of 3D reconstruction) . n=4 animals per group. Data in were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure 4I depicts quantification of radial glia-like (EdU+GFAP+) cells, plotted as the average cell numbers within the volume of dentate gyrus (decided by the imaging processing of 3D reconstruction) . n=4 animals per group.
Figure 4J depicts quantification of transiently amplifying progenitor-like (EdU+GFAP-) cells, plotted as the average cell numbers within the volume of dentate gyrus (decided by the imaging processing of 3D reconstruction) . n=4 animals per group.
Figure 4K shows representative images and confocal analyses of NPC differentiation into immature neurons in the mouse hippocampal sections, which were labeled with BrdU and transduced by the Lenti-Ctrl or Lenti-CHI3L1 lentiviruses (Fig. 4B) . The fluorescence signals of BrdU were counterstained with the immature neuronal marker DCX to identify the newborn immature neurons  (BrdU+DCX+) , the average numbers of which within the volume of dentate gyrus were plotted for both tested groups. n=4 animals per group. Scale bar, 100 μm.
Figure 4L shows representative images and confocal analyses of NPC differentiation into mature neurons in the mouse hippocampal sections, which were labeled with BrdU and transduced by the Lenti-Ctrl or Lenti-CHI3L1 lentiviruses (Fig. 4B) . The fluorescence signals of BrdU were counterstained with the mature neuronal marker NeuN to identify the newborn mature neurons (BrdU+NeuN+) , the average numbers of which within the volume of dentate gyrus were plotted for both tested groups. n=4 animals per group. Scale bar, 100 μm. Data in were presented as the mean ±SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p < 0.0001.
Figure S4A shows the results of the open field test that was performed prior to Morris water maze at week 4-6 (Fig. 4B) to evaluate the locomotor activities and anxiety-like behaviors in mice lentivirally transduced to over-express eGFP (Lenti-Ctrl) or CHI3L1 plus eGFP (Lenti-CHI3L1) . Representative movement paths of both groups were shown here.
Figure S4B depicts quantification of the open field test by measuring the total travel distances of both groups. Lenti-Ctrl, n=8 mice; Lenti-CHI3L1, n=12. Data are presented as the mean ± SEM; statistical significance was evaluated with unpaired one-tailed Student’s t test for two group comparisons. *p < 0.05, **p<0.01, ***p < 0.001.
Figure S4C depicts quantification of the open field test by measuring the crossing numbers through the center area of both groups. Lenti-Ctrl, n=8 mice; Lenti-CHI3L1, n=12. Data are presented as the mean ± SEM; statistical significance was evaluated with unpaired one-tailed Student’s t test for two group comparisons. *p < 0.05, **p<0.01, ***p < 0.001.
Figure 5A shows the summary of the CHI3L1 receptors and the downstream signaling pathways that could be activated by the CHI3L1-receptor binding.
Figure 5B depicts the expression of CRTH2 receptor on NSCs but not immature or mature neurons, assayed by the immune-staining of CRTH2 (left panels of all rows) and neural markers GFAP (center left panels of top two rows) , Sox2 (center right panels of top two rows) , DCX (center left panel of bottom row) , NeuN (center right panel of bottom row) and DAPI (shown in merge with other channels, right panels of all rows) . The top 2 rows demonstrated the colocalization of fluorescence signals for CRTH2 and NSC (GFAP+SOX2+) ; the bottom raw showed little if any colocalization between CRTH2 and immature neuronal marker DCX markers (left 2 panels) or mature neuronal marker NeuN (right 2 panels) . These hippocampal slices were from 8-week-old wildtype mice. Scale bars, 20 μm.
Figure 5C depicts the characterization of the CHI3L1 receptor signaling pathways activated by CHI3L1 binding, focusing on GSK-3β and β-catenin that are most relevant to cell proliferation and neurogenesis. The cultures of purified NSCs grown in proliferation medium were treated with the vehicle control PBS or recombinant CHI3L1 protein (100ng/mL) for 30 minutes, 1 hour and 2 hours, and then lysed and harvested at the same time. The treated NSC lysates were then subject to immunoblotting for assaying the protein levels of total GSK-3β kinase, phosphorylated GSK-3β (p-GSK-3β, phosphorylation of Serine 9, the inactive form unable to phosphorylate substrate β-catenin for degradation) , transcription factor β-catenin and the loading control β-actin. The densitometric quantifications revealed CHI3L1 treatment induced a rapid accumulation of active GSK-3β with a reduction of the inhibitory Serine 9 phosphorylation (within 30 minutes, reduced ratio of p-GSK-3β/β-actin in left bar graphs) and a prolonged decrease in β-catenin (2 hours, β-catenin/β-actin ratio in right bar graphs) that was phosphorylated by GSK-3β and targeted for proteosome degradation. n=3 experiments per treatment condition. Bar graphs were presented as the mean ± SEM; the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p <0.05, **p<0.01, ***p< 0.001, ****p < 0.0001.
Figure 5D depicts a schematic diagram of the lentiviral vector designs to transduce the expression of 2 shRNAs targeting CRTH2 for knockdown (Lenti-shCRTH2, shCRTH2_1 and shCRTH2_2) and a scrambled non-targeting control shRNA (Lenti-shNC) by a U6 promoter and the co-expression of eGFP by a CMV promoter. The shRNA design was also used to deliver shRNAs targeting 2 other CHI3L1 receptors, IL-13Rα2 and TMEM219 (fig. S5C-F) . The eGFP signal was used to calculate the transducing unit (TU) in the lentiviral preparations for the desired multiplicity of infection (MOI) of 10-May in mouse NSC cultures.
Figure 5E shows the immunoblotting analyses to evaluate the knockdown efficiency of the 2 shRNAs against CRTH2 in cultured mouse NSCs that were transduced by lentiviruses for 3 days in proliferation medium before the lysing and sample collections. Left, a representative blot of NSC lysates immunoblotted with CRTH2 and the loading control, with the lentiviral transduction of the control shRNA (shNC) and 2 shRNAs against CRTH2 (shCRTH2_1 and shCRTH2_2) . Right, the densitometric quantifications for the CRTH2 protein levels in relation to the loading control β-actin (CRTH2/β-actin ratio) . n=3 experiments per condition. Bar graphs were presented as the mean ± SEM; the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p< 0.001, ****p < 0.0001.
Figure 5F shows the evaluation of the CRTH2 knockdown effect on the activation of CHI3L1 signaling pathways in NSCs. The mouse NSC cultures were transduced by lentiviruses to express the control shNC and shRNA against CRTH2 (the shCRTH2_1 from fig. S5E) , and then treated with PBS or recombinant CHI3L1 protein (100ng/mL) for 2 hours, followed by lysing for immunoblotting. The  quantification of the densitometric analysis for the protein levels of the phosphorylated GSK-3β (p-GSK-3β Ser9, inactive) and β-catenin (β-catenin/β-actin) were plotted in the ratio to the signal intensity of the loading control β-actin in left and right bar graphs, respectively. n=3 experiments per group. Bar graphs were presented as the mean ± SEM; the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p< 0.001, ****p < 0.0001.
Figure 5G shows the investigation of the CRTH2 knockdown effect on NSC proliferation, by EdU labeling of the mouse NSCs grown in proliferation medium (as in Fig. 2E) and transduced via lentivirus to express shNC or shCRTH2_1 together with 3-day treatments of PBS or CHI3L1 (100 ng/ml) . The immune-fluorescence signal of GFP co-expressed with either shRNA was positive for >99%of cells labeled by DAPI. The immunostaining of EdU was performed to identify the proliferating cells, and the proliferation was quantified as the percentage of EdU+ cells in the population of GFP+ cells. n=4 experiments per group. Scale bars, 100 μm. Bar graphs were presented as the mean ± SEM; the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p< 0.001, ****p < 0.0001.
Figure 5H shows the examination of the CRTH2 knockdown effect on the neuronal proliferation of NSCs, by transducing the shNC or shCRTH2_1 to be expressed in mouse NSCs grown in the differentiation medium and treated with PBS or CHI3L1 (100 ng/ml) for 3 days (as in Fig. 2E) . The immune-staining of GFP was used to outline all the cultured cells; the neural markers of Tuj1 and GFAP were used to identify and quantify the newly differentiated neurons (GFP+Tuj1+) and astroglial cells (GFP+GFAP+) out of all the cultured cells (GFP+) , with the quantifications plotted in the ratio of GFP+Tuj1+/GFP+ (left bar graphs) and GFP+GFAP+/GFP+ (right bar graphs) . n=4 experiments per group. Scale bars, 100 μm. Bar graphs were presented as the mean ± SEM; the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p< 0.001, ****p < 0.0001.
Figure 5I shows the identified CHI3L1 receptor signaling pathway inhibitory to neurogenesis, in which CHI3L1 binds to CRTH2 receptor and activates GSK-3β that in turn destabilizes β-catenin and leads to reduced transcriptional activities for neurogenesis.
Figure S5A shows little expression of IL-13Rα2, a common CHI3L1 receptor for immune cells, in NSCs within the SGZ of dentate gyrus from 8-week-old wildtype mice, assayed by immunostaining of IL-13Rα2 (left panel) and the radial glia-like NSC markers Sox2 (center right panel) and GFAP (center left panel) plus the nuclear marker DAPI (right panel, shown in a merge with the other channels) . This confocal imaging complemented Fig. 5B. Scale bars, 20 μm.
Figure S5B shows a very low level of CHI3L1 receptor TMEM219 in NSCs within the SGZ of dentate gyrus, assayed by immunostaining of TMEM219 (left panel) and the radial glia-like NSC markers Sox2 (center right panel) and GFAP (center left panel) plus the nuclear marker DAPI (right channel, shown in a merge with the other channels) . This confocal imaging complemented Fig. 5B. Scale bars, 20 μm.
Figure S5C depicts the knockdown efficiency of 2 shRNAs targeting against IL-13Rα2, assayed by immunoblotting of cell lysates from NSCs transduced with lentiviruses to express the non-targeting control shRNA (shNC) , shIL-13Rα2_1 or shIL-13Rα2_2 as described in Fig. 5D-E. β-actin served as the loading control.
Figure S5D shows, relevant to Fig. 5D-E, the evaluation of TMEM219 knockdown efficiency by 2 shRNAs, shTMEM219-1 and shTMEM219_2, transduced in NSC cultures and compared to the protein levels of TMEM219 and β-actin.
Figure S5E shows the knockdown of IL-13Rα2 or TMEM219 did not affect the inhibition of NSC proliferation by CHI3L1, in parallel to the experiments in Fig. 5G. The cultured mouse NSCs grown in proliferation medium were transduced to express shNC, shIL-13Rα2_1 or shTMEM219_1 and then treated with PBS or CHI3L1 (100 ng/ml) . The EdU was added into the cultures two hours before the fixation and immune-staining. Left panels, representative confocal images of the indicated 4 groups. Right bar graphs, the quantification of NSC proliferation was plotted as the ratio of the proliferating cells (GFP+ EdU+) and total cells (GFP+, co-expressed with shRNAs; ～100%colocalization with DAPI+ cells) in cultures. n=4 experiments per group. Scale bars, 100 μm. Data were presented as the mean ± SEM and their statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure S5F shows, in relation to Fig. 5H, the knockdown of IL-13Rα2 or TMEM219 did not affect the inhibition of NSC differentiation by CHI3L1. The mouse primary NSCs were cultured in the differentiation medium and transduced to express shNC, shIL-13Rα2_1 or shTMEM219_1, with the incubation of PBS or CHI3L1 (100 ng/ml) for 3 days. After fixation, the immuno-staining was performed to label essentially all cells with GFP and DAPI, the newly differentiated neurons with Tuj1 and astroglial cells with GFAP, in the panels of the representative images. The bar graphs were the quantification of neuronal differentiation, shown in the ratio of neurons and total cells (GFP+Tuj1+/GFP+, left) and the ratio of astroglia and total cells (GFP+GFAP+/GFP+, right) . n=4 experiments. Scale bars, 100 μm. Data were presented as the mean ± SEM and their statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p <0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 6A depicts a schematic diagram of the astrocyte-specific CHI3L1 knockout by stereotaxic injection of AAVs expressing Cre recombinase and eGFP under the control of the GFAP promoter (AAV-GFAP-GFP-2A-Cre, or AAV-Cre) into hippocampi of the 8-week-old transgenic mice carrying one (CHI3L1f/+) or two (CHI3L1f/f) CHI3L1-floxed alleles. The AAV preparations were at the concentration of TU/ml and stereotaxically injected into the dentate gyrus of 8-week-old transgenic mice with 1 μl on each side. A subset of the injected mice was sacrificed two weeks after the AAV injections for the evaluation of knockout efficiency. The remainder of those AAV-injected mice were allowed to recover for 4 weeks before they received the intra-hippocampal injections of msCtrl-IgG or msAQP4-IgG as described in Fig. 3A-K to elicit the astroglial activation, and then went through behavioral tests at the Week 8 before the sacrifice.
Figure 6B shows representative confocal images of the hippocampal slices from the CHI3L1f/f transduced to express Cre and GFP in astroglial cells labeled with GFAP. The signals of CHI3L1 immuno-fluorescence were reduced in astrocytes with Cre-mediated CHI3L1 deletion (GFP+GFAP+) , as indicated by the dots (right panel) . The DAPI signals were used to outline the dentate gyrus. Scale bars, 100 μm.
Figure 6C shows the evaluation of CHI3L1 knockout efficiency in the AAV-Cre-injected hippocampal tissues from CHI3L1f/+ and CHI3L1f/f mice, with qPCR assays to measure CHI3L1 mRNA levels in the hippocampal lysates harvested from the dissected fresh brain tissues. After the Cre recombination, the CHI3L1f/+ mice still expressed CHI3L1 abundantly but the CHI3L1f/f mice expressed a low level, ～10%of the CHI3L1f/+ group. n=3 animals per group. Data were presented as the mean ± SEM; statistical significance was evaluated with Student’s t test for two-group comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 6D depicts results from the task of Morris water maze (MWM) which was performed in both CHI3L1-expressing (CHI3L1f/+) and CHI3L1-deficient (CHI3L1f/f) mice, 8 weeks after the stereotaxic injections of AAV-Cre and four weeks after the stereotaxic deliveries of msCtrl-IgG or msAQP4-IgG. Representative movement paths from the 4 tested groups were shown here: CHI3L1f/++msCtrl-IgG, CHI3L1f/++msAQP4-IgG, CHI3L1f/f+msCtrl-IgG and CHI3L1f/f+msAQP4-IgG.
Figure 6E depicts quantification of the MWM results plotted by the crossing numbers of the 4 tested groups. n=9 animals per group. Data were presented as the mean ± SEM; statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 6F depicts quantification of the MWM results plotted by the escape latencies for the 4 tested groups. to find the platform during the training sessions. n=9 animals per group. Data were  presented as the mean ± SEM; statistical significance was analyzed by two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 6G shows a schematic diagram and experiment timeline to test the effects of astrocyte-specific CHI3L1 knockout on adult hippocampal neurogenesis. The stereotaxic injections of AAV-Cre rendered CHI3L1 expressed in CHI3L1f/+ hippocampal tissues but depleted in the CHI3L1f/f groups (as in Fig. 6A-C) . The astroglial activation was carried out by the mini-pump method as described in Fig. 3L-Q to chronically infuse msCtrl-IgG or msAQP4-IgG into hippocampus for the Week 1 and 2. The evaluation of NSC proliferation and neuronal differentiation was facilitated by the labeling of EdU and BrdU, respectively, administered in a manner as described in Fig. 3A.
Figure 6H depicts quantification of NSC proliferation plotted in the average numbers of total proliferating (EdU+) cells within the volume of dentate gyrus, by confocal analyses after a 3D reconstruction process. n=4 animals per group. Data were presented as the mean ± SEM; statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p <0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 6I depicts quantification of NSC proliferation plotted in the average numbers of total radial glia-like (EdU+GFAP+) proliferating NSCs within the volume of dentate gyrus, by confocal analyses after a 3D reconstruction process. n=4 animals per group. Data were presented as the mean ±SEM; statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 6J shows quantification of NSC proliferation into immature and mature neurons throughout experimentation started by BrdU labeling, plotted by the average numbers of newborn immature (BrdU+DCX+) neurons within the volume of dentate gyrus for the 4 tested groups. n=4 experiments per group. Data were presented as the mean ± SEM; statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p <0.001, ****p <0.0001.
Figure 6K shows quantification of NSC proliferation into immature and mature neurons throughout experimentation started by BrdU labeling, plotted by the average numbers of newly differentiated mature (BrdU+NeuN+) neurons within the volume of dentate gyrus for the 4 tested groups. n=4 experiments per group. Data were presented as the mean ± SEM; statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure S6A shows data related to Fig. 6 D-F, the open filed test (OFT) was performed before the MWM experiments to assess the general locomotor activities and anxiety-like behaviors, with the  representative movement paths from the t tested groups shown here: CHI3L1f/++msCtrl-IgG, CHI3L1f/++msAQP4-IgG, CHI3L1f/f+msCtrl-IgG and CHI3L1f/f+msAQP4-IgG.
Figure S6B depicts Quantification of the OPT results plotted as the total travelled distances for the 4 groups. n=9 animal per group. Bar graphs were presented as the mean ± SEM and the statistical analyses were carried out with two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p<0.01, ***p < 0.001.
Figure S6C depicts quantification of the OPT results plotted as the numbers of crossing over the center area for the 4 groups. n=9 animal per group. Bar graphs were presented as the mean ± SEM and the statistical analyses were carried out with two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p<0.01, ***p < 0.001.
Figure 7A shows a schematic diagram and experiment timeline for characterizing the effects of shRNA mediated CRTH2 knockdown on NSC proliferation and differentiation in astroglial activation triggered by msAQP4-IgG. The lentiviruses expressing GFP together with a scrambled non-targeting control shRNA (Lenti-shNC) or a shRNA targeting CRTH2 (Lenti-shCRTH2_1) as described in Fig. 5D were prepared at the concentration of TU/ml and stereotaxically injected into the hippocampi of 8-week-old wildtype mice with 1 μl on each side. One week after the shRNA lentivirus injections, a subset of mice was sacrificed by perfusion and their hippocampal sections were immune-stained with GFP and DAPI to show the adequate lentiviral transduction efficiency in the representative confocal images. Scale bars, 100 μm. The msCtrl-IgG and msAQP4-IgG were then infused chronically by a mini-pump for 2 weeks, and EdU and BrdU were administered for the evaluation of NSC proliferation and neuronal differentiation, as described in Fig. 3L. There were 4 groups tested here: shNC+msCtrl-IgG, shNC+msAQP4-IgG, shCRTH2_1+msCtrl-IgG and shCRTH2_1+msAQP4-IgG.
Figure 7B depicts the evaluation of CRTH2 knockdown effect on NSC proliferation that was done by the immune staining of GFP (top left) , EdU (top right) and GFAP (bottom left) , with representative images shown in left panels. The quantifications (right) were plotted as the average numbers of proliferating cells transduced to express a shRNA (GFP+EdU+, the left bar graphs) and the average numbers of radial-glia-like NSCs (GFP+EdU+GFAP+, the right bar graphs) within the volume of dentate gyrus (DG) . n=4 animals per group. Scale bars, 20 μm. Data were presented as the mean ± SEM with the statistical significance evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 7C depicts the confocal assays for NSC differentiation into immature neurons, quantified by the average numbers of newborn immature neurons transduced to express a shRNA (with GFP co-expression) within the DG volume. The immunostaining of GFP (top left) , BrdU (top  right) and the immature neuronal marker DCX (bottom left) . n=4 animals per group. Scale bars, 20 μm. Data were presented as the mean ± SEM with the statistical significance evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 7D shows the assessment of NSC differentiation into mature neurons, by plotting the average numbers of newly-differentiated neurons (GFP+BrdU+NeuN+) within the DG volume from the 4 tested groups with the immune-staining of GFP (top left) , BrdU (top right) , and NeuN (bottom left) . n=4 per group. Scale bars, 20 μm. Data were presented as the mean ± SEM with the statistical significance evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 7E depicts a schematic diagram and timeline for the experiments to probe the efficacy of AZD1981, a selective CRTH2 antagonist, as a therapeutic compound to ameliorate the inhibition of neurogenesis by CHI3L1 signaling in astroglial activation. The mini-pump infusion of msCtrl-IgG or msAQP4-IgG for 2 weeks, and the administration of EdU and BrdU were performed similarly as described in Fig. 3L. On top of that, the vehicle control DMSO or AZD1981 was given at the dosage of 1 mg/kg via i.p. injections every other day for 4 weeks. There were 4 groups tested here: msCtrl- IgG+DMSO, msAQP4+DMSO, msCtrl-IgG+AZD1981 and msAQP4-IgG+AZD1981.
Figure 7F depicts quantification of NSC proliferation by analyzing the immune-fluorescence of EdU, GFAP and SOX2, as described in Fig. 3H and I. The bar graphs were plotted after calculating the average numbers of total proliferating cells (EdU+) within the DG volume. n=4 per group. Data were presented as the mean ± SEM with the statistical significance evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 7G depicts quantification of NSC proliferation by analyzing the immune-fluorescence of EdU, GFAP and SOX2, as described in Fig. 3H and I. The bar graphs were plotted after calculating the average numbers of radial glia-like NSCs (EdU+GFAP+Sox2+) within the DG volume. n=4 per group. Data were presented as the mean ± SEM with the statistical significance evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 7H shows quantification of NSC proliferation into immature neurons throughout the experiment timeline, assayed by immune-staining of BrdU, and DCX and plotted as the average numbers of newborn immature neurons (BrdU+DCX+) . Data were presented as the mean ± SEM with the statistical significance evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure 7I shows quantification of NSC proliferation mature neurons throughout the experiment timeline, assayed by immune-staining of BrdU and NeuN and plotted as the average numbers of newly differentiated mature neurons (BrdU+NeuN+) within the DG volume. n=4 animals per group. Data were presented as the mean ± SEM with the statistical significance evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001, ****p <0.0001.
Figure S7A depicts a schematic diagram for the in vitro experiments to evaluate the effect of blocking CRTH2 by antagonist AZD1981 on the inhibition of NSC proliferation and differentiation by CHI3L1. The overall design was essentially the same as the one described in Fig. 2E, other than the co-incubation with the vehicle control DMSO or AZD1981 (10 nM) in the proliferation medium or differentiation medium for 3 days.
Figure S7B shows the confocal analyses for NSC proliferation by quantifying the proliferating cells in all the cultured NSCs with immune-staining of EdU and DAPI, as described in Fig. 2F. Representative images were shown in left panels. The bar graphs on the right were plotted with the average ratio of DAPI+EdU+ cells and DAPI+ cells, in the presence of CHI3L1 or PBS and with AZD1981 or DMSO. n=4 experiments per group. Scale bars, 100 μm. Data were presented as the mean ± SEM and the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001.
Figure S7C depicts the assays of NSC differentiation into neurons as described in Fig. 2G to evaluate the effect of AZD191 in counteracting the CHI3L1-induced inhibition. Representative images of the differentiating NSC cultures were analyzed for the differentiated neurons immune-labeled by Tuj1, astroglial cells by GFAP, and all the nuclei by DAPI. The bar graphs showed the ratio of Tuj1+ cells to DAPI+ cells on the left and the ratio of GFAP+ cells to DAPI+ cells. n=4 experiments per group. Scale bars, 100 μm. Data were presented as the mean ± SEM and the statistical significance was evaluated with one-way ANOVA and Tukey’s post hoc multiple comparisons. *p < 0.05, **p<0.01, ***p < 0.001.
DETAILED DESCRIPTION OF THE INVENTION
The present disclosure provides, for example, methods of targeting the detrimental effects of neuroinflammation on cognitive performance by blocking the interaction of CHI3L1 with the CRTH2 receptor within the central nervous system. The disclosure also provides a previously undescribed mechanism whereby the inflammatory signaling mediator CHI3L1 engages the CRTH2 receptor on neural stem cells in the brain and triggers a downstream inhibitory pathway to prevent the process of neurogenesis, which promotes proper cognitive performance.
Accordingly, the present disclosure provides, for instance, therapy for neuroinflammation comprising a CTHR2 antagonist. Exemplary CTHR2 antagonists are described herein.
In the following description, for an explanation, numerous specific details provide a thorough understanding of the compositions and methods disclosed herein. However, it may be evident that the compositions and methods may be practiced without these specific details. Aspects, modes, embodiments, variations, and features of the compositions and methods are described below in various levels of detail to provide a substantial understanding of the present disclosure.
Definitions
For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are listed below. Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by a person having ordinary skill in the biomedical art to which this invention belongs. A term's meaning provided in this specification shall prevail if any apparent discrepancy arises between the meaning of a definition provided in this specification and the term's use in the biomedical art.
The singular forms a, an, and the like include plural referents unless the context dictates otherwise. For example, a reference to a cell comprises a combination of two or more cells.
As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. Using comprising indicates inclusion rather than limitation.
As used herein, the term “consisting essentially of” means the listed elements are required for a given embodiment. The term permits additional elements that do not materially affect the basic and functional characteristics of that embodiment of the invention.
As used herein, the term “consisting of” means compositions, methods, and respective components thereof, exclusive of any element not recited in that description of the embodiment.
As used herein, the term “effective amount” refers to the amount sufficient to cause beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. Many ways are known in the biomedical art to determine the effective amount for an application. For example, pharmacological methods for dosage determination can be used in the therapeutic context. In therapeutic or prophylactic applications, the amount of a composition administered to the subject depends on the type and severity of the disease and the characteristics of the individual, such as general health, age, sex, body weight, tolerance to drugs, and on the degree, severity, and type of disease. Persons having ordinary skill in the biomedical art can determine appropriate dosages depending on these and other factors. In some embodiments, an  effective amount results in inhibition of a target protein by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
As used herein, the term “expression” refers to the transcription or translation of a particular nucleic acid sequence driven by a promoter. In some embodiments, expression refers to the level of accumulation of an RNA. In some embodiments, expression refers to the accumulation of a protein.
As used herein, the term “CHI3L1 inhibitor” refers to an agent that specifically binds and inhibits activity of a chitinase 3 like 1 (known as CHI3L1 or YKL-40) protein, or an agent that specifically reduces expression of the CHI3L1 protein. In some embodiments, the agent comprises a molecule or a complex. In some embodiments the agent comprises a small molecule that binds CHI3L1. In some embodiments the agent comprises an antibody molecule. In some embodiments, the agent that specifically reduces expression of the CHI3L1 protein comprises a nucleic acid that binds CHI3L1 mRNA and reduces the level or translation of the CHI3L1 mRNA.
As used herein, the term “CRTH2 inhibitor” refers to an agent that specifically binds and inhibits activity of a Prostaglandin D2 receptor 2 (known as CRTH2 or PDG2) protein, or an agent that specifically reduces expression of the CRTH2 protein. In some embodiments, the agent comprises a molecule or a complex. In some embodiments the agent comprises a small molecule that binds CRTH2. In some embodiments the agent comprises an antibody molecule. In some embodiments, the agent that specifically reduces expression of the CRTH2 protein comprises a nucleic acid that binds CRTH2 mRNA and reduces the level or translation of the CRTH2 mRNA.
As used herein, the term “inhibitor” refers to an agent that causes a decrease of a certain parameter. In some embodiments, the parameter is an activity of a given molecule. In some embodiments, the decrease is a decrease by at least 10%as compared to a reference level (e.g., the absence of a treatment or agent) and can include more significant decreases, for example, a decrease by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. Activities for the inhibitors can be determined using any suitable assay, for instance, an assay as described herein or an assay known in the art. In some embodiments, the inhibitor specifically binds a target molecule. In some embodiments, the inhibitor prevents the activity of a bound target molecule. In some embodiments, the inhibitor reduces expression of a given molecule.
As used herein, the terms “treat” , “treatment” and “treating” refer to the reversal, alleviation, amelioration, inhibition, reduction, slowing or halting of the progression, severity and/or duration of a disease, disorder, or medical condition, or the amelioration of one or more symptoms of a disease,  disorder, or medical condition. In some embodiments, the terms “treat” , “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of the disease, disorder, or medical condition not necessarily discernible by the patient. In some embodiments, the terms “treat” , “treatment” and “treating” refer to the inhibition of the progression of disease, disorder, or medical condition, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. Treatment is effective, for example, if one or more symptoms or clinical markers are reduced or if the progression of a condition is reduced or halted. Treatment includes not just the improvement of symptoms or markers but also a cessation or at least slowing of progress or worsening of symptoms expected absent treatment.
As used herein, the term “subject” refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats) , agricultural stock animals (e.g., cows, horses, pigs, chickens, etc. ) , laboratory animals (e.g., mice, rats, rabbits, etc. ) , but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or geriatric subject. The human subject may be of either sex. In some embodiments, the subject may have a condition or disease or be at risk of developing a condition or disease.
As used herein, the term “antibody molecule” refers to a naturally occurring antibody, an engineered antibody, or a fragment thereof. In some embodiments, an antibody molecule is an antigen binding portion of a naturally occurring antibody or an engineered antibody. In some embodiments, an antibody molecule includes an antibody or an antigen-binding fragments thereof (e.g., Fab, Fab’, F (ab’) 2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv) , Fd fragments consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH) , nanobodies, or camelid VHH domains) , an antigen-binding fibronectin type III (Fn3) scaffold such as a fibronectin polypeptide minibody, a ligand, a cytokine, a chemokine, or a T cell receptor (TCR) . In some embodiments, an antibody molecule is a humanized antibody molecule. In some embodiments, an antibody molecule is an intact IgA, IgG, IgE or IgM antibody. In some embodiments, an antibody molecule is a bi-or multi-specific antibody (e.g., etc) . In some embodiments, antibody molecules are antibody fragments such as Fab fragments, Fab’ fragments, F (ab’) β fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof. In some embodiments, an antibody molecule is a single chain Fv (scFv) , a polypeptide-Fc fusion, a single domain antibody (e.g., shark single domain antibodies such as IgNAR or fragments thereof) , or a cameloid antibody. In some embodiments, antibody molecules are masked antibodies (e.g., ) , Small Modular ImmunoPharmaceuticals ( “SMIPsTM” ) , single chain or Tandem diabodiesVHHs; minibodies, ankyrin repeat proteins orDARTs, TCR-like antibodies, MicroProteins, or
As used herein, the term “nucleic acid” refers to a polymeric molecule incorporating units of ribonucleic acid, deoxyribonucleic acid, or an analog thereof. In some embodiments, the nucleic acid is in single stranded form. In some embodiments, the nucleic acid is in double stranded form. In some embodiments, the nucleic acid is genomic DNA, cDNA, or RNA (e.g. mRNA) . In some embodiments, the nucleic acid contains analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. In some embodiments, the nucleic acid containing analogues of natural nucleotides are metabolized in a manner similar to naturally occurring nucleotides.
As used herein, the terms “peptide, ” “polypeptide, ” and “protein” are used interchangeably, and refer to a molecule comprised of two or more amino acid residues covalently linked by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. In some embodiments, the polypeptide comprises a modified amino acid. In some embodiments, the polypeptide refers to a natural peptide, a recombinant peptide, or a combination thereof. In some embodiments, the polypeptide refers to short chains of amino acids. In some embodiments, the polypeptide refers to long chains of amino acids. In some embodiments, the polypeptide refers to a biologically active fragment, a substantially homologous polypeptide, an oligopeptide, a variant of a polypeptide, a modified polypeptide, a derivative, an analog, or a fusion protein. A person having ordinary skill in the biomedical art recognizes that individual substitutions, deletions, or additions to a peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence are a conservatively modified variant where the alteration results in the substitution of amino acid with chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants also do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.
As used herein, the term “or” refers to and/or. The term and/or as used in a phrase such as A and/or B herein includes both A and B; A or B; A (alone) ; and B (alone) . Likewise, the term and/or as used in a phrase such as A, B, and/or C encompasses each embodiment: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; Band C; A (alone) ; B (alone) ; and C (alone) .
This invention is not limited to the particular methodology, protocols, reagents, etc., described herein and as such can vary.
The disclosure described herein does not concern a process for cloning humans, processes for modifying the germ line genetic identity of humans, uses of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals likely to cause them suffering with no substantial medical benefit to man or animal, and animals resulting from such processes.
CRTH2 inhibitors
In some embodiments, the CRTH2 inhibitor comprises a shRNA. In some embodiments, the CRTH2 inhibitor comprises a small molecule. In some embodiments, the CRTH2 inhibitor comprises a noncompetitive antagonist of CRTH2.
AZD1981
In some embodiments, the CRTH2 inhibitor comprises AZD1981 or a pharmaceutically acceptable salt thereof. AZD1981 has a chemical structure according to Formula I:
Fevipiprant
In some embodiments, the CRTH2 inhibitor comprises Fevipiprant or a pharmaceutically acceptable salt thereof. Fevipiprant has a chemical structure according to Formula II:
OC000459
In some embodiments, the CRTH2 inhibitor comprises OC000459 or a pharmaceutically acceptable salt thereof. OC000459 has a chemical structure according to Formula III:
BI671800
In some embodiments, the CRTH2 inhibitor comprises BI671800 or a pharmaceutically acceptable salt thereof. BI671800 has a chemical structure according to Formula IV:
AMG853
In some embodiments, the CRTH2 inhibitor comprises AMG853 or a pharmaceutically acceptable salt thereof. AMG853 has a chemical structure according to Formula V:
Setipiprant
In some embodiments, the CRTH2 inhibitor comprises setipiprant or a pharmaceutically acceptable salt thereof. Setipiprant has a chemical structure according to Formula V:
ARRY-502
In some embodiments, the CRTH2 inhibitor comprises ARRY-502 or a pharmaceutically acceptable salt thereof. ARRY-502 has a chemical structure according to Formula VI:
MK-7246
In some embodiments, the CRTH2 inhibitor comprises MK-7246 or a pharmaceutically acceptable salt thereof. MK-7246 has a chemical structure according to Formula VII:
MK-1029
In some embodiments, the CRTH2 inhibitor comprises MK-1029 or a pharmaceutically acceptable salt thereof. MK-1029 has a chemical structure according to Formula VIII:
AZD6430
In some embodiments, the CRTH2 inhibitor comprises AZD6430 or a pharmaceutically acceptable salt thereof.
ADC-3680
In some embodiments, the CRTH2 inhibitor comprises ADC-3680 or a pharmaceutically acceptable salt thereof.
ASP5642
In some embodiments, the CRTH2 inhibitor comprises ASP5642 or a pharmaceutically acceptable salt thereof.
BI1021958
In some embodiments, the CRTH2 inhibitor comprises BI1021958 or a pharmaceutically acceptable salt thereof.
TQC3564
In some embodiments, the CRTH2 inhibitor comprises TQC3564 or a pharmaceutically acceptable salt thereof.
CHI3L1 inhibitors
In some embodiments, the CHI3L1 inhibitor comprises 2- ( {3- [2- (1-cyclohexen-1-yl) ethyl] -6, 7-dimethoxy-4-oxo-3, 4-dihydro-2-quinazolinyl} sulfanyl) -N- (4-ethylphenyl) butanamide (K284) .
In some embodiments, the CHI3L1 inhibitor comprises kasugamycin.
In some embodiments, the CHI3L1 inhibitor comprises a monoclonal anti-CHI3L1 antibody molecule.
Methods of use
In some embodiments, the compositions described herein are used to treat neuromyelitis optica spectrum disorder (NMOSD) . NMOSD is an autoimmune disease of the central nervous system that affects the optic nerves and the spinal cord. In some embodiments, the brain is affected. In some embodiments, a primary astrocytopathy is caused by autoantibodies targeting AQP4. In some embodiments, autoantibodies targeting AQP4 are not present.
EXAMPLES
Example 1: CHI3L1 signaling impairs hippocampal neurogenesis and cognitive function in autoimmune-mediated neuroinflammation
This example describes the disruption of CHI3L1 signaling in in vitro and in vivo models of neuromyelitis optica spectrum disorder in order to promote neurogenesis, neuronal differentiation, and cognitive function.
Materials and Methods
Study design
For all the individuals participated in our clinical study, the informed consent was obtained after the nature and possible consequences of the study was explained. There were 44 patients (40 women and 4 men; mean age, 43.2 ± 13.4 years) who met the 2015 International Panel for NMO Diagnosis criteria and were positive for AQP4-IgG. The clinical characteristics and demography of patients with NMO were summarized in Table 2. The blood and CSF samples, MRI scans, neurological and psychological assessments were acquired at the recovery stage with no clinical deterioration, at least three months from the end of attack or relapse.
Statistical analysis
The sample size was not pre-determined by any statistical methods as the effect sizes were unavailable before the experiments. The samples sizes were all described in the figure legends. The normality of the data distribution was routinely determined by a Shapiro-Wilk normality test (p<0.05 indicating a non-normal distribution) . For the data confirmed to be normally distributed data, we used Student’s t test for pair-wise comparisons, one-way or two-way ANOVA followed by Tukey’s post-hoc test for 3 groups or more, as indicated in the figure legends. For data that is not normally distributed, non-parametric alternatives, such as Mann-Whitney or Kruskal-Wallis tests. p<0.05 is considered to be statistically significant. All data in bar graphs and summary plots were shown as means ± standard error (SEM) of at least three independent biological replicates in all figures. Descriptive statistics were used to examine the demographic characteristics, information and patient neuropsychological testing of patients. In addition, the correlation between the neuropsychological testing and the CHI3L1 level was performed using the Pearson correlation coefficient. Significance was reported as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All statistical analyses were performed in GraphPad Prism 9 (GraphPad Software) .
Primary astrocyte culturing and treatments
In brief, the whole brains from neonatal mouse pups at postnatal day 0 (P0) were dissected out on ice. After removal of the meninges, the brains were washed in cold DMEM-F12 (C11330500BT, GIBCO) supplemented with 10%FBS (ST30-3302, PANTM SERATECH) and 1%penicillin streptomycin (15140-122, GIBCO) . The brains were transferred to 0.25%trypsin EDTA for 20 minutes, and then DMEM-F12 complete medium was used to stop the trypsinization. Cell debris and aggregates were removed by passing the single-cell suspension through a 40 μm nylon mesh. After 5 minutes of centrifugation at 1000 g, the single-cell suspension was collected and cultured with astrocyte medium: DMEMF12 (C11330500BT, GIBCO) containing 10%FBS (ST30-3302, PANTM SERATECH) , 1%NEAA, 1%penicillin streptomycin (15140-122, GIBCO) , 1%glutamine (GIBCO, 25030-081) and 1 mM sodium pyruvate (S8636, Sigma) in T-75 flasks for 14 days, and the medium was replaced every 3 days. Primary astrocytes were plated onto poly-D lysine-coated glass coverslips in 24-well plates at a density of 8x103 cells per well and allowed to settle at 37 ℃ in a 5%CO2 incubator for 24 hours. The cultures were treated with controls or biological reagents at the indicated final concentration for the described experimental durations: Dulbecco's phosphate buffered saline (PBS, as the vehicle) , IL-1β (10 ng/mL; 200-01B, PeproTech) , monoclonal mouse antibodies targeting AQP4 (msAQP4-IgG, 100 ng/mL) , control mouse IgG (100 ng/ml) , purified human autoantibodies against AQP4 (hsAQP4-IgG, 100 ng/ml) , and control human IgG (100 ng/ml) . The acquisition of mouse and human antibodies were detailed in a following section. The astrocyte-conditioned media were collected at the completion of individual reagent treatments and filtered with  0.22 μm nylon mesh (BS-QT-037, Biosharp) . To remove the astrocyte-secreted CHI3L1, the filtered ACM were added with the anti-CHI3L1 monoclonal antibodies (rabbit, Proteintech) or msCtrl-IgG at 100 ng/ml plus Protein A beads (71149800, GE Healthcare) , tumbled for 4 hours at 4℃ and centrifuged to clear the IgG contents. The cleared ACM were then supplemented with factors to constitute the culture media for the proliferation or neuronal differentiation of cultured mouse neural stem cells (NSCs) . The primary astrocyte cultures were harvested by fixation with 4%paraformaldehyde (PFA) for immune-staining and by lysis buffers for RNA-seq and qPCR assays.
Primary mouse neural stem cells (NSCs) isolation and culturing
The mouse neural stem cells (NSCs) used in this study were purified from living dentate gyrus tissues acutely dissected from 8-week young adult wildtype mice. In short, the anatomical structure of the dentate gyrus was visualized by a dissection microscope, carefully removed out of the hippocampal regions, and then subject to the enzymatic digestion by using the MACS neural tissue dissociation kit according to the manufacturer’s protocol (130-092-628, MACS Miltenyi Biotec) . One milliliter of trypsin inhibitor (S10088, Yuan Ye Biology) was added to each sample to stop digestion. After 5 minutes of centrifugation at 1000 g, the single-cell suspension was collected and cultured with the maintenance medium for proliferation (proliferation medium) , formulated with neurobasal (21103049, GIBCO) medium containing 2%B27 supplement (17504044, GIBCO) , 20 ng/mL basic fibroblast growth factor (FGF-2, K1606, PeproTech) , 20 ng/mL epidermal growth factor (EGF, A2306, PeproTech) , 1%penicillin streptomycin (15140-122, GIBCO) and 2 mM L-glutamine (25030-081, GIBCO) in a 5%CO2 incubator at 37 ℃. Without further description, half of the medium was replaced every 3 days. To assay the NSC proliferation, 24 hours after plating, the cells were incubated with the indicated vehicle controls or biological reagents for 3 days, and then the EdU from a Cell Proliferation Kit was administered according to the manufacturer’s protocol (C0078L, Beyotime) . For the assays of neuronal differentiation, 48 hours after plating, the cells were changed to the differentiation medium composed of neurobasal medium containing 2%B27 supplement (17504044, GIBCO) , 1%penicillin streptomycin (15140-122, GIBCO) and 2 mM L-glutamine (25030-081, GIBCO) , with the co-incubation of the indicated vehicle controls or biological reagents. The NSC cultures, for proliferation and differentiation, were treated with recombinant CHI3L1 protein (100 ng/ml; 2599-CH-050, R&D Systems) or vehicle control for 3 days. The treated cultures were harvested by fixation with 4%PFA and then subject to immune-staining for confocal imaging of the EdU or BrdU expression, in conjunction with neural or nuclear markers.
Human and mouse IgG acquisition
The hybridoma cell line producing monoclonal mouse IgG specifically recognizing the mouse AQP4 extracellular domain (clone E5415B; hereafter referred to as msAQP4-IgG) was kindly provided by Dr. Yoichiro Abe at the Keio University, Tokyo, Japan. These antibodies are  commercially available (MABN2526-25UG, Sigma-Aldrich) . The control mouse IgG was the normal mouse IgG validated for use in immunoprecipitation and immunoblotting (12-371, Sigma-Aldrich) . The human autoantibodies against AQP4 (hsAQP4-IgG) were purified from the pooled plasma samples of anti-AQP4 seropositive NMO patients (hsAQP4-IgG) or healthy volunteers (hsCtrl-IgG) undergoing plasma exchange using Protein A beads (71149800, GE Healthcare) . The beads were eluted with 100 mM glycine HCl (pH 2.5) , and then the eluent was concentrated using an Amicon Ultra 15 centrifugal filtration unit (100 KD, Millipore, Billerica, MA) . The concentration of IgG was 0.22 μM, and the working portion was kept at -80 ℃.
Immunofluorescence staining of NSC proliferation and differentiation
For in vitro experiments, primary cultures of mouse astrocytes or NSCs were fixed with 4%PFA for 40 minutes, followed by washing in PBS. Then, the cells were blocked with 300 μL/10 mL donkey serum and 250 μL/10 mL 10%Triton X-100 for 1 hour at room temperature and sequentially incubated with primary antibodies overnight at 4℃. The cells were washed with PBS three times, and then the cells were incubated with the secondary antibody at room temperature for 1 hour. The nuclei were then stained with DAPI and observed under a laser confocal microscope. Each group of immunohistochemical experiments was repeated at least 3 times. The following primary antibodies were used: chicken anti-GFAP (1: 1000, Abcam) , mouse anti-Tuj1 (1: 1000, Abcam) , mouse anti-IL13Rα2 (1: 500, CST) , rabbit anti-CHI3L1 (1: 100, Proteintech) , rabbit anti-CRTH2 (1: 500, Invitrogen) , and rabbit anti-TMEM219 (1: 500, CST) . The following fluorescent secondary antibodies were used: goat anti-mouse 488, goat anti-mouse 568, goat anti-mouse 647, goat anti-rabbit 488, goat anti-rabbit 568, goat anti-rabbit 647, and goat anti-chicken 647.
RNA isolation and quantitative real-time PCR (qPCR)
mRNA from mouse hippocampal tissue or primary astrocytes was extracted with a miRNeasy kit (Qiagen) . Then, mRNA was quantified and checked for purity using a Nanodrop spectrophotometer (ThermoFisher Scientific) . The cDNA was converted from 1 μg mRNA using the SureScript First-Strand cDNA Synthesis Kit (Genecopoeia Company) . RT-qPCR was performed using BlazeTaq SYBR Green qPCR Mix (Genecopoeia Company) with Applied Biosystems (Thermo Fisher Scientific) . Fold changes were calculated as 2-ΔΔCT with GAPDH used as the endogenous control. The sequences of the primers for the individual genes of interest are listed in Table M1.
Table M1. RT-qPCR primers for quantification of expression of genes related to inflammation

Immunoblotting and the densitometric analysis
Cell lysates were derived from primary mouse astrocytes or NSCs using radioimmunoprecipitation assay (RIPA) lysis buffer. The proteins were separated on 8-15%sodium  dodecyl sulfate-polyacrylamide gels by electrophoresis and then transferred to methanol-activated polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5%defatted milk powder for 1 hour at 37 ℃ and incubated with primary antibodies overnight at 4 ℃. The membranes were then washed three times with TBST and incubated with horseradish peroxidase (HRP) secondary antibodies for 1 hour. Protein bands were visualized with Millipore ECL Plus reagent and imaged on a Tanon 5500 Imaging Analysis System. The primary antibodies used were rabbit anti-CHI3L1 (1: 1000, Abcam) , rabbit anti-CRTH2 (1: 1000, Invitrogen) , mouse anti-TMEM219 (1: 1000, Abcam) , rabbit anti-IL13Rα2 (1: 1000, Biorbyt) , rabbit anti-GSK-3β (1: 1000, Bioss) , rabbit anti-p-GSK-3β (1: 1000, CST) , rabbit anti-β-catenin (1: 1000, CST) , and mouse anti-β-actin (1: 1000, Huabio) . β-Actin was used as the loading control. The intensity of the bands was quantified using ImageJ software.
The enzyme-linked immunosorbent assays (ELISA) for CHI3L1 levels
The ELISA samples included human serum, human cerebrospinal fluid (CSF) , the astrocyte-conditioned media and the lysates of freshly dissected mouse hippocampal tissues. Blood samples with the anticoagulant heparin added were first spun for 15 minutes at 8,000 rpm at 4 ℃ and the supernatant serum was then collected. Levels of CHI3L1 in serum or CSF were measured with commercial ELISA kits (R&D Systems, MN, USA) based on the provided instructions, including the generation of a standard curve based on a series of dilution of CHI3L1 proteins. Levels of CHI3L1 were measured with commercial ELISA kits (MEIMIAN, MM-44752M2) . The absorbance of each standard and sample was measured at 450 nm.
Production of lentivirus and adeno-associated virus (AAV) particles
The lentivirus and AAV preparations were routinely produced through the transfection of human embryonic kidney (HEK293T) cells with the desired vectors. The sequences of the shRNAs targeting against the test CHI3L1 receptors are listed in the Table M2. In summary, the HEK293T cells were maintained at 37℃ in a CO2 cell incubator (Thermo Fisher 371) in Dulbecco’s Modification of Eagle’s Medium (DMEM) 1× (MOD. ) (319-005-CL, MULTICELL) containing 10%fetal bovine serum (ST30-3302, PANTM SERATECH) , 1%penicillin streptomycin (15140-122, GIBCO) , and 1%glutamine (GIBCO, 25030-081) and passaged every 2-3 days with 0.05%trypsin EDTA. For the production of lentivirus particles, the designed and cloned lentiviral vectors were co-transfected with helper plasmids psPAX and pMD2G into HEK293T cells using polyethyleneimine (PEI, PR40001, Proteintech Group) . The culture medium was changed within 4 hours. The medium containing lentivirus was collected at 2-4 days post transfection, filtered, and concentrated by ultracentrifugation (Beckman SW32 Ti) . The viruses were washed once with PBS and then resuspended in 200 μL PBS. Each virus tube was labeled and stored at -80℃. As for the AAV, the package with AAV-DJ capsids were used for the intended high efficiency of in vivo infection. AAV vectors with pHelper and pRC-DJ were transfected into HEK293 cells, which were collected and  lysed 72 hr later. The virus preparations were concentrated from cell lysates by fractioning with iodixanol gradient (40%) and filtering with 100,000 MWCO tube filter. For both lentivirus and AAV production, the virus titer was measured by transducing HEK293 with a series of dilutions and analyzing the fluorescent signal from the co-expressed eGFP. Before the concentration, the typical titer was among the range of transduction units (TU) ml before the concentration. For the in vitro lentiviral transductions, we calculated the amount of virus preparations to deliver the multiplicity of infection (MOI) of 5-10 in primary cultures of mouse astrocytes and NSCs. For the stereotaxic injections in vivo, the virus preparations were concentrated by ultracentrifugation to achieve the concentration of 108-9 as indicated in figure legends.
Animal care and genotyping by the genomic DNA extraction and PCR
Overall, the 8-week-old wildtype and the CHI3L1 transgenic mice were used for the described experiments, and maintained for additional four to eight weeks according to the individual study design. These mice were placed in the animal facility of Guangdong Laboratory Animals Monitoring Institute under a 12-hour light/dark cycle constantly under 22-26℃ and 50%–60%humidity. The mice could obtain food and water in the cage ad libitum. CHI3L1f/f mice were created by Cyagen Biosciences (NCBI: 12654) . All mice in the study were backcrossed to the C57BL/6 background for at least six generations. The animals were randomly assigned to the experimental group. Before the experiment was completed, the experimenter did not know the identities of the animals. Adult neural stem cells were isolated from eight-to twelve-week-old WT mice. All procedures and feeding were carried out in accordance with the scheme approved by the Laboratory Animals Monitoring Institute and the animal experiment ethics committee of the Third Affiliated Hospital of Sun Yat-Sen University. No mice were used in other research projects. The influence of sex was not evaluated in this study. For the extraction of genomic DNA, mouse tails were digested overnight in 200 mL lysis solution (containing 1 mg/mL proteinase K, 50 mM Tris-HCl pH 8.0, 100 mM EDTA, 100 mM NaCl and 1%SDS) at 55 ℃. Four hundred milliliters of NaCl (6 M) were added to each sample and the solution was mixed well. The solution was incubated on ice for 10 minutes. The samples were centrifuged at 13,000 rpm for 10 minutes at room temperature. The supernatant was transferred to a new 1.5 mL centrifuge tube with 800 mL ethanol (70%in ddH2O) and mixed well. The samples were centrifuged at 13,000 rpm for 10 minutes. The genomic DNA pellet was dissolved in 200 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) . Genotyping was performed with PCR based assays using genomic DNA. The sequences of the primers used were: the forward primer (5’-3’) , GCTACCCAACATGTCAATAGCTCA; the reverse primer (5’-3’) , CATATGGTGGGCAATAATCTTGGA.
5-Ethynyl-2’-deoxyuridine (EdU) and 5-bromo-2’-deoxyuridine (BrdU) administration for confocal analyses of NSC proliferation and differentiation
For analysis of NSC proliferation and neuronal differentiation in the adult mouse hippocampal dentate gyrus, adult mice were intraperitoneally (i.p. ) injected with EdU (100 mg/kg) , sacrificed 2 hours after injection and then an EdU Cell Proliferation Kit was used according to the manufacturer’s protocol (C0078L, Beyotime) . For analysis of cell differentiation in the adult mouse hippocampal dentate gyrus, adult mice were i.p. injected with BrdU (100 mg/kg) for 4 days and sacrificed at the 4th week after injection. Immunofluorescence staining and quantification of BrdU+cells in the hippocampus subgranular zone (SGZ) and granulosa cell layers were performed largely as the standardized method (described by Zhao, X. and van Praag, H. Steps towards standardized quantification of adult neurogenesis. Nature communications11, 4275 (2020) ) , in which 2D confocal  images were reconstructed into a 3D rendition to calculate the volume of dentate gyrus for a more stable spatial reference for the quantification. For quantification of cells expressing stage-specific markers, 1 in 12 serial sections starting at the beginning of the hippocampus (relative to bregma, 1.5 mm) to the end of the hippocampus (relative to bregma, 3.5 mm) was used. Quantification of EdU+ or BrdU+ cells and phenotypic quantification of EdU+ or BrdU+ cells (double labeling with Sox2, GFAP, DCX and NeuN) in the granule layer were performed using a Leica confocal microscope in at least 3 sections containing the DG from at least 3 different animals. The value of n is described in the figure legends.
AZD1981 administration
AZD1981 (M04757, BJBALB Co. Ltd, Beijing, China) was administered by i.p. injections at the dosage of 1 mg/kg every other day for 4 weeks. AZD1981 was documented to exhibit cross-species binding activity to CRTH2 and block the activation of eosinophils in mice, rats, guinea pigs, rabbits and dogs. In clinical trials, it was well tolerated and no safety concerns were identified.
Stereotaxic injections of IgG, lentivirus and AAV into the hippocampus
Roughly eight-week-old C57BL/6 or CHI3L1f/f mice were anesthetized by the inhalation of isoflurane delivered by a mask (2%gas in air mixture) and fixed on a stereotaxic frame (RWD Life Science, Shenzhen, China) . One microliter of virus preparation with a titer greater than 5x108/mL was injected into the dentate gyrus of the hippocampus. The infusion needle was stereotaxically implanted into the DG using the following coordinates relative to bregma: caudal side: -2.0 mm; transverse direction: +/-1.7 mm; ventral: -1.9 mm. All mice were ambulating normally after awakening from anesthesia.
Mini-osmotic pump (mini-pump) implementation and IgG infusion
In short, the components of the osmotic pumps were first assembled according to the manufacturer’s instructions (ALZET, Model 1002) . The mini-osmotic pump was then filled with msCtrl-IgG or msAQP4-IgG (1 μg/100 μl in PBS) and then positioned at the following coordinates relative to bregma, caudal side: -2.0 mm; transverse direction: +/-1.7 mm; ventral: -1.9 mm. The infusion flow rate was set at 0.25 μl per hour for 14 days.
Immunohistochemistry for confocal imaging of mouse hippocampal slices
Mice were euthanized by intraperitoneal injection of 2%pentobarbital sodium and given an initial flush with 0.01 M phosphate buffered saline (PBS) . Then, the mice were transcardially perfused with 4%PFA. Mouse brains were fixed in 4%PFA at 4℃ overnight and then placed in 30%sucrose until submerged. The brains were cut into 40-μm-thick sections coronally using a microtome and placed in a 96-well plate. The slices were kept in cryoprotectant solutions (glycerol, ethylene glycol  and 0.1 M phosphate buffer, pH 7.4, 1: 1: 2 by volume) at 4 ℃. For immunofluorescence staining, floating sections were blocked with 300 μL donkey or goat serum and 250 μL 10%Triton X-100 per 10 mL PBS for 1 hour at room temperature and then sequentially incubated with primary antibodies overnight at 4℃. The sections were washed with PBS three times, and then incubated with the secondary antibody at room temperature for 1 hour. The nuclei were then stained with 4’, 6-dimethyl-2’-phenylindole dihydrochloride (DAPI, 2261b, Sigma Aldrich) . The images were observed under a laser confocal microscope. Each group of immunohistochemical experiments was repeated at least 3 times. The primary antibodies used were mouse anti-GFAP (1: 1000, CST) , rabbit anti-GFAP (1: 1000, CST) , chicken anti-GFAP (1: 1000, Abcam) , rabbit anti-NeuN (1: 500, Abcam) , mouse anti-DCX (1: 200, Santa) , rabbit anti-Sox2 (1: 500, Abcam) , mouse anti-Sox2 (1: 500, Abcam) , rat anti-BrdU (1: 1000, Abcam) , mouse anti-Tuj 1 (1: 1000, Abcam) , rabbit anti-IIL13Rα2 (1: 500, CST) , rabbit anti-CHI3L1 (1: 500, Abcam) , goat anti-CHI3L1 (1: 500, R&D Systems) , rabbit anti-CHI3L1 (1: 500, Solarbio) , rabbit anti-CRTH2 (1: 500, Invitrogen) , mouse anti-CRTH2 (1: 500, Invitrogen) , and mouse anti-TMEM219 (1: 500, CST) . For DCX or Sox2 staining, antigen retrieval was needed. Antigen retrievals were performed in citrate buffer (pH 9.0) with a microwave for 10 minutes at 95 ℃ followed by 20 minutes of cooling at room temperature. The following fluorescent secondary antibodies were used: goat anti-mouse 488, goat anti-mouse 568, goat anti-mouse 647, goat anti-rabbit 488, goat anti-rabbit 568, goat anti-rabbit 647, goat anti-rat 568, and goat anti-chicken 647. Confocal single plane images and z stacks were taken with a laser confocal microscope (Leica, TCS SP8) equipped with four laser lines (405, 488, 568 and 647 nm) and 63x, 40x and 20x objective lenses.
Open field test
Mice of different experimental groups were placed in a 50 cm x 50 cm x 50 cm arena and allowed to explore the site freely for 10 minutes. After the test, they were placed back in their cage. The distance traveled was recorded to evaluate the motor ability of the mice, and the time spent in the central area was measured to detect the anxiety of the mice. Experimenters were blinded to the genotypes and mice.
Morris water maze test
Briefly, the MWM test consisted of a water-filled pool (diameter 120 cm) with a hidden escape platform under the water surface. The platform (10 cm wide) was located approximately 1 cm below the water level and was divided into four quadrants. The MWM is divided into training and testing phases. In the training phase, mice were randomly placed in the water maze from four different starting positions (NE, NW, SE and SW) . The mice were trained for five days to find the hidden platform. After taking the mouse out of the maze, the water on the mouse was wiped with a towel and then the mouse was placed back in the cage. After 24 hours of visual platform training, a detection test was conducted, during which the platform was removed and times crossing the platform area  were measured by camera to test short-term spatial memory. All tests were performed at the same time every day. Experimenters were blinded to the genotypes and mice.
Clinical data collection
Neuromyelitis optica (NMO) patients were diagnosed based on the 2015 International Panel for NMO Diagnosis (IPND) criteria and received treatments and follow-up cares at the Third Affiliated Hospital of Sun Yat-Sen University and the Second Affiliated Hospital of Guangzhou Medical University. Blood and CNS samples, MRI scans and neuropsychological assessments were acquired during a clinically stable stage when the patients were recovering from the attack or relapse for at least 3 months did not show symptomatic deterioration. For all the individuals participated in our clinical study, the informed consent was obtained after the nature and possible consequences of the study was explained.
Human brain samples
Frozen sections of human brain were obtained from biopsy brain tissues of one AQP4-IgG serum-positive NMO patient and one control. The clinical characteristic data of the pathological specimen providers are shown in Table S1.
Table S1. Information regarding the biopsied brain samples from the control and NMO patient (complementary to Fig. 2B) .
MRI acquisition and hippocampal subfield volumes analysis.
All MRI data were obtained on a 3.0 T magnetic resonance system (Philips Medical System Ingenia scanner) with dStream head coil. Structural images of the whole brain were scanned using 3D fast spoiled gradient-echo sequence. FLAIR data were scanned using TR = 7000 ms, Flip Angle 90, TE = 125 ms, acquisition matrix = 272 × 176, and slice thickness 6 mm. First, the whole hippocampus was automatically segmented and calculated using FreeSurfer version 7.1.1 and the hippocampal subfields were automatically segmented and measured using a package available. Using this algorithm, the hippocampus was accurately segmented into the following subfields: parasubiculum, presubiculum, subiculum, CA1, CA2/3, CA4, GC-DG, hippocampus-amygdala transition area (HATA) , fimbria, molecular layer, fissure, hippocampal tail.
Clinical screening
We recorded the demographic and clinical information of all patients, which included age, sex, education and disease duration. The Mini-Mental State Examination Scale (MMSE) , Symbol Digit Modalities Test (SDMT) and Brief Visuospatial Memory Test Revised (BVMT-R) can comprehensively, accurately and quickly reflect the subjects’ mental state and the degree of cognitive impairment. The CVLT assessed the subjects’ learning and memory abilities, including immediate recall (Trials 1-5, T1-5) , short delayed free recall (SDFR) , short delayed cue recall (SDCR) , long delayed cue recall (LDCR) and long delayed free recall (LDFR) . General anxiety and depression-relevant behaviors were assessed using the Hamilton Anxiety Rating Scale (HARS) and Hamilton Depression Rating Scale (HDRS) , respectively. Behavioral symptoms were assessed via interview with the informant and quantified using the Frontal Behavioral Inventory.
Results
CHI3L1 is induced in astrocytes activated by pro-inflammatory stimuli of cytokines and antibody-mediated immune response
We first confirmed the induction of CHI3L1 in activated astrocytes by analyzing two independent transcriptome datasets of mouse astrocytes undergoing neurotoxic activation after inflammatory stimulation in vivo and in vitro. In astrocytes acutely purified from young adult mice subject to intraperitoneal injection of lipopolysaccharide (LPS) , CHI3L1 level was dramatically increased (fig. S1A in vivo; Hasel, P., et al. Neuroinflammatory astrocyte subtypes in the mouse brain. Nature neuroscience24, 1475-1587 (2021) ) ; the increase was even more obvious in the cultures of primary astrocytes treated with pro-inflammatory cytokines of IL-1, TNF and C1q (ITC; Fig. S1A in vitro; Guttenplan, K.A, et al. Knockout of reactive astrocyte activating factors slows disease progression in an ALS mouse model. Nature communications 11, 3753 (2020) ) . To further investigate the role of CHI3L1 in immune-mediated neuroinflammation, we prepared primary astrocyte cultures from neonatal wildtype C57BL/6, and treated them with IL-1b, monoclonal mouse antibodies against AQP4 (msAQP4-IgG) and human anti-AQP4 autoantibodies purified from NMO patients’ plasma (hsAQP4-IgG) , with proper control conditions of vehicle (PBS) , mouse control IgG (msCtrl-IgG) , and human control IgG (hsCtrl-IgG, pooled from healthy subjects’ plasma) , respectively (Fig. 1A) . We observed marked induction of CHI3L1 mRNA level in all three treatment conditions by qPCR assays, with the hsAQP4-IgG being the most robust one (Fig. 1B) . All three treatments also caused a surge in protein levels of CHI3L1 and GFAP, signifying the activation of astrocytes (Fig. 1C and D) . Only the treatments with anti-AQP4 antibodies (Fig. 1D) , but not IL-1b (Fig. 1C) , led to the abolishment of AQP4 expression. To systemically probe the cellular consequences of immune-mediated astroglial activation, we referred to prior studies based on the use of human IgG derived from NMO patients and RNA-sequenced mouse astrocytes non-treated or treated with three independent replicate batches of our hsAQP4-IgG or hsCtrl-IgG (by using the Illumina HiSeq 2500 system) . Overall, we noticed a  considerable change in gene profile that distinctly separated the hsAQP4 treatment cluster from the control IgG group (fig. S1B, by the functional principal component analysis) . We identified a total of 152 differentially expressed genes (DEGs) that met our statistical criteria, with 110 upregulated and 42 downregulated ones (fig. S1C, Fig. 1E and F) . The gene ontology (GO) enrichment analysis showed a clear picture of inflammatory and immunological responses (fig. S1D) . The induction of a reactive program was further corroborated after we contrasted our data with a similar astroglial transcriptome dataset (from Walker-Caulfield, M. E et al. NFkappaB signaling drives pro-granulocytic astroglial responses to neuromyelitis optica patient IgG. J Neuroinflammation 12, 185 (2015) ) , based on array-based gene expression analysis (Illumina mouse WG-6 v2.0 Beadchip) . The overlap of 91 genes out of our 152 DEGs was considerable (Fig. 1E and F) , and the hierarchical clustering of the heatmap based on these 91 overlapped DEGs clearly discerned the hsCtrl-IgG and hsAQP4-IgG samples and depicted the transcriptional signature of immune-mediated astroglial activation (Fig. 1E) . The most significantly enriched GO pathways for these overlapped genes (Fig. 1G) were akin to the analysis of our own DEGs (fig. S1D) , and included cellular responses to pro-inflammatory cytokines and viral infections. The 21 secreted proteins that overlapped between the two studies outlined the core response of immune-activated astrocytes, with CHI3L1 as a key responder (Fig. 1H) . We integrated the data from our untreated samples to discriminate the effect from hsCtrl-IgG treatment and pinpointed the GO pathway of neuroinflammation. We recognized a group of C-C and C-X-C motif chemokine genes, including CCL2 5, 6, 7, 17, and CXCL1 and 10, together with IL-1a and b that denoted the hsAQP4-IgG-induced neuroinflammatory feature (Fig. 1I) . The analysis of the overlap by a published gene change pattern of astrocyte activation revealed CHI3L1 as a top gene that distinguished the hsCtrl-IgG and hsAQP4-IgG-treated astrocytes (Fig. 1J) . Additionally, we inquired into the cellular pathways by employing the computational tool of Pathview to integrate our 91 overlapped DEGs with the pathway-based data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) . The NFκB canonical pathway –which has been documented to be critical for neurotoxic inflammation of astrocytes and can be activated by CHI3L149 –was the most prominent one in hsAQP4-IgG-induced astroglial activation (fig. S1E) . These findings jointly supported the notion that CHI3L1 is induced in astrocytes activated by immune-mediated inflammatory stimuli and is an important secreted protein in neuroinflammation associated with the astroglial autoimmune response of hsAQP4-IgG expression.
CHI3L1 secretion correlates with neurocognitive impairment and hippocampal dysfunction in human brains affected by autoimmune-mediated astrocyte activation
To investigate the clinical relevance of an elevated CHI3L1 level in neuroinflammation, we examined our multi-center, longitudinal NMO patient cohort with complete disease records, which is larger than all prior studies to date and comprises 44 patients and 20 age-matched healthy controls  (Table 1) . The autoantibodies against AQP4 were detected in all patients, and, in agreement with prior studies, the demographic data well reflected the dominance of young women (mean age: 43.2 ± 13.4 years; male/female = 4/40) and the high prevalence of brain demyelinating lesions, myelitis, and optic neuritis (Table 2 and Fig. S2A) . Importantly, the CHI3L1 levels were significantly higher in serum and CSF samples from NMO patients as compared to controls (Fig. 2A) . Additionally, we performed immunohistochemical examinations on biopsied live brain tissues from one NMO patient and one control of head trauma patient free of existing health condition (table S1) . In addition to the characteristic reduction of AQP4 expression within the end-feet of astrocytes (along the vasculature labeled by endothelial marker CD31) , we found nearly all astrocytes (labeled with GFAP) were positive for CHI3L1 (Fig. 2B) . Unique to our cohort is the comprehensive neuropsychological assessments, including the batteries of Hamilton Anxiety Rating Scale (HARS) and Hamilton Depression Rating Scale (HDRS) for general anxiety and depression-like symptoms, as well as the standard tests of Mini-Mental State Examination (MMSE) , Symbol Digit Modalities Test (SDMT) , Brief Visuospatial Memory Test Revised (BVMT-R) and five formats of California Verbal Learning Test (CVLT-LDFR, -SDCR, -SDFR, -LDCR and -T1.5) for learning and memory-related performance. The difference between healthy controls and NMO patients was significant in all the assessments, substantiating the cognitive impairment that could be overshadowed by the neurological focal signs of motor and visual function losses (fig. S2B) . Strikingly, the neuropsychological disabilities documented here greatly correlated with the CHI3L1 CSF levels in individual NMO patients. HARS and HDRS assessments corresponded positively with CHI3L1 levels and indicated the heightened mental stress reactions in response to the disease burden (Fig. 2C) . There was a negative correlation between CHI3L1 level and the severity of learning and memory defects measured by MMSE, SDMT, BVMT-R and all five sub-forms of CVLT (Fig. 2C) . To emphasize the brain structural basis of the cognitive impairment associated with CHI3L1 expression in NMO, we carried out detailed analyses of the well-defined hippocampal regions based on the MRI scans. Consistent with case reports in the literature, our NMO patients had apparent volume atrophy of the hippocampus (Fig. 2D) , which was assigned as a biomarker for poor cognitive performance. Our additional analyses of hippocampal sub-regions (e.g., subiculum, CA1/3, dentate gyrus and fimbria) (fig. S2C and D) further highlight the hippocampal dysfunction in cognitive impairment potentially modulated by CHI3L1 in neuroinflammation.
Table 2. The clinical characteristics and demography of health controls and NMO patients enrolled in this study.
Astrocyte-secreted CHI3L1 reduces proliferation and differentiation of neural stem cells in immune-mediated neuroinflammation
To determine whether CHI3L1 secreted by activated astrocytes contributes to the hippocampal dysfunction underlying the neuroinflammation-related cognitive impairment, we decided to focus on a specialized hippocampal cell type, the neural stem cell (NSC) , which is important in maintaining proper size and function of the mammalian hippocampi and highly sensitive to inflammatory microenvironment. We thus purified and cultured primary mouse NSCs from the dissected dentate gyri of 8-week-old young adult wildtype mice. We then treated the cultured NSCs with recombinant CHI3L1 proteins (Fig. 2E) , a method that our collaborative group and others have shown to well mimic the secretion of CHI3L1 by immune cells. The CHI3L1 treatment (100 ng/ml for 3 days) lowered the proliferation of NSCs by ～50%, quantified by EdU labeling (Fig. 2F) . We evaluated the effect of CHI3L1 on neuronal differentiation, the process of neurogenesis in vitro, by differentiating NSCs grown in a differentiation medium with or without concomitant incubation of CHI3L1 (Fig. 2E; for additional 3 days) . Our assays of multiple fluorescence immunostaining for neural markers showed that CHI3L1 treatment significantly shrank the population of the newly differentiated neurons (Fig. 2G; labeled by Tuj1) , implying a hampered neurogenesis process. To directly examine the effect of CHI3L1 secreted by immune-activated astrocytes on NSCs, we treated primary mouse astrocytes with hsCtrl-IgG or NMO patient derived hsAQP4-IgG as in Fig. 1 (100 ng/ml IgG for 24h) and then harvested the astrocyte conditioned media (ACM; Fig. 2H) . The ACM from hsAQP4-IgG treated astrocytes contained a noticeably higher level of secreted CHI3L1 (Fig. 2I) . Given we showed earlier that multiple secretary proteins could be induced by hsAQP4-IgG (Fig. 1F, I and J) , we sought to isolate CHI3L1 and determine its necessity by removing CHI3L1 with a monoclonal neutralizing antibody targeting against CHI3L1 –an approach which efficiently blocks the  function of the secreted CHI3L1. As expected, ACM from hsAQP4-IgG-treated astrocytes diminished the NSC proliferation (Fig. 2J and K) and differentiation into neurons (Fig. 2L and M) . Remarkably, the administration of the CHI3L1 neutralizing antibody (anti-CHI3L1) abolished the inhibitory effect of the hsAQP4-IgG ACM on NSC proliferation and the in vitro neurogenesis. These findings collectively suggested that CHI3L1 secreted by activated astrocytes impairs NSC proliferation and neurogenesis and precipitates hippocampal dysfunction and cognitive impairment.
Immune-mediated astrocytopathy impairs hippocampal neurogenesis and hippocampus dependent learning behaviors in young adult mouse brains
Having shown that CHI3L1 secreted by immune-activated astrocytes reduced NSC proliferation and differentiation in vitro, we next sought to determine whether the same effect could be observed in vivo. We utilized the passive immunization method to construct the mouse model of immune-mediated astrocytopathy as in NMO, via passive transfer of the msAQP4-IgG or msCtrl-IgG (Fig. 1B and D) . We first stereotaxically injected the control or anti-AQP4 antibodies (1 μg/μL in 1 μl) into the dentate gyri of 8-week-old wildtype C57BL/6 mice on both sides (Fig. 3A, left) . To assess the adult hippocampal neurogenesis, these injected mice then received pulse labeling of bromodeoxyuridine (BrdU) or 5-ethynyl-2’-deoxyuridine (EdU) by intraperitoneal injections, and were sacrificed at two different time points: one week for the EdU groups (two hours after the labeling) , and 4-5 weeks for the BrdU groups after behavioral tests (Fig. 3A) . One week after the stereotaxic IgG delivery, we found that, while brain vasculature remained unperturbed (labeled by the endothelial marker CD31) , the msAQP4-IgG group evidently exhibited the NMO diagnostic feature of reduced coverage of AQP4+ astrocyte end-feet (along the micro-vessels) , as compared to controls of msCtrl-IgG group (Fig. 3B) . Consistent with our findings on primary astrocytes (Fig. 1D) and biopsied human brain tissues (Fig. 2B) , the msAQP4-IgG instigated a marked induction of GFAP (Fig. 3C) and CHI3L1 (Fig. 3D) in astrocytes. We also dissected out the injected hippocampal tissues without fixation and lysed them for further analyses. The CHI3L1 level was noticeably higher in the lysates from msAQP4-IgG group after the ELISA measurements (fig. S3A) . We further confirmed the pro-inflammatory features we characterized in vitro (fig. S3B) with qPCR assays showing an induction in almost all tested neuroinflammation and astrocyte activation genes identified from our transcriptomic meta analyses (Fig. 1H-J) .
We continued to characterize the cognitive effects from immune-mediated astrocyte activation by performing behavioral assays that corresponds to the cognitive impairment and hippocampal dysfunction observed in NMO patients (Fig. 2C and fig. S2B) . Four to five weeks after the stereotaxic transfer of IgGs (Fig. 3A, red arrows) , the injected mice underwent standardized behavioral assays of the open field test and the Morris water maze. Compared with the control msCtrl-IgG group (fig. S3C) , the msAQP4-IgG  group of mice overall traveled shorter total distances (fig. S3D) and spent less time in the center area (fig. S3E) in the open field test. Such defects indicated a decrease in locomotor activity and an increase in anxiety-like behavior owing to the brain inflammation. The Morris water maze task, a widely used assay to assess the hippocampus-dependent spatial learning and memory (Fig. 3E) , revealed in msAQP4-IgG group the faulty behaviors with decreased numbers of the platform crossing (Fig. 3F) and extended escape latencies (Fig. 3G) . These behavioral findings, together with the histopathological data above (Fig. 3B-D) , demonstrated our AQP4 antibody-induced astrocytopathy mouse model recapitulated key aspects of NMO disease manifestations (Fig. 2A-D) .
To investigate whether the cognitive dysfunction stemming from immune-mediated neuroinflammation would be associated with a change in neurogenesis, we then checked the proliferation and differentiation of NSCs within the hippocampus subgranular zone (SGZ) by standardized methodology for quantification of neurogenesis. One week after the injections of antibodies, the EdU-labeled mice from msCtrl-IgG and msAQP4-IgG groups had comparable sizes of dentate gyrus after the acute experiment period (Fig. 3A) , but the msAQP4-IgG group showed much fewer EdU+ cells (Fig. 3H) . Further analyses showed that the populations of the radial glia-like cells (triple-labeled by EdU+GFAP+Sox2+) and the transiently amplifying progenitor cells (EdU+GFAP-Sox2+) were both reduced (Fig. 3I) , suggesting an inhibition of NSC proliferation. Moreover, to determine the effect on neuronal differentiation of NSC after our single-dose AQP4-IgG administration, we analyzed the BrdU-labeled mice, labeling four to five weeks after the intra-hippocampal delivery of msCtrl-IgG or msAQP4-IgG (Fig. 3A) . The numbers of newly-differentiated immature neurons (double-labeled by BrdU+DCX+) and mature neurons (BrdU+NeuN+) were appreciably lower (Fig. 3J and K) . Consistent with our in vitro results (Fig. 2E-M) , our findings here demonstrated a defective process of neurogenesis in the neuroinflammation triggered by the immune response.
In parallel to the mouse model based on stereotaxic injections of antibodies, we also leveraged mini-osmotic pump implantation to develop the passive transfer mouse model. The msCtrl-IgG or msAQP4-IgG were chronically infused (1 μg/100 μl PBS at the rate of 0.25 μl per hour for 14 days) into the hippocampal regions of young adult wildtype C57BL/6 mice on both sides at the age of eight weeks (Fig. 3M) . The BrdU labeling was administered soon after the start of IgG perfusion for us to evaluate the neuronal differentiation of NSCs (Fig. 3L) ; EdU was given at the end of the 2-week perfusion and followed by sacrifice and fixation of the brain to examine NSC proliferation (Fig. 3L) . In the EdU-labeled hippocampal slices, the perfusion of msAQP4-IgG, as compared to the msCtrl-IgG control condition, resulted in a decline in the numbers of total proliferating cells (fig. S3F and Fig. 3M, EdU+) , radial glia-like cells (Fig. 3N, EdU+GFAP+Sox2+) and transiently amplifying progenitor cells (Fig. 3O, EdU+GFAP-Sox2+) in the SGZ (fig. S3F, Fig. 3M) . Furthermore, at the end of the fourth week of  experimentation, our quantification of BrdU labeling and neuronal marker expression showed that the msAQP4-IgG perfusion contracted the cell populations of newly-differentiated immature neurons (fig. S3G and Fig. 3P, double-labeled by BrdU+DCX+) and mature neurons (fig. S3H and Fig. 3Q, BrdU+NeuN+) . Together, two different approaches –stereotaxic injection and chronic perfusion –of msAQP4-IgG delivery generated results that mirrored each other and jointly suggested the inhibition of adult hippocampal neurogenesis in immune-mediated astrocyte activation, potentially mediated by the secreted CHI3L1 in a paracrine manner.
CHI3L1 is sufficient to reduce neurogenesis and impair hippocampal-dependent learning behaviors in young adult mice
We showed that, among the plethora of pro-inflammatory secretory proteins released by activated astrocytes (Fig. 1H) , CHI3L1 is important for the inhibition of NSC proliferation and differentiation following inflammatory responses in vitro (Fig. 2E-G, treatment of recombinant CHI3L1; Fig. 2J-M, the use of neutralizing anti-CHI3L1 antibody) . To directly test the effect of CHI3L1 on cognitive performance and neureogenesis, we overexpressed CHI3L1 in the mouse hippocampus and assayed the cognitive performance and NSC proliferation and differentiation by an experiment design similar to that described above (Fig. 3A and B) . We constructed lentiviral vectors expressing CHI3L1 plus eGFP (Lenti-CHI3L1, eGFP-2A-CHI3L1 with CMV promoter) or eGFP alone as a control (Lenti-Ctrl) , and stereotaxically injected the prepared lentiviruses into the dentate gyrus in 8-week-old wildtype C57BL/6 mice (Fig. 4A) . Following the lentiviral injections, the labeling of BrdU or EdU was carried out at the specific time points to allow our measurements of NSC proliferation and neuronal differentiation (Fig. 4B) . Two weeks after the injections, we sacrificed a subset of mice, dissected out the injected hippocampal tissues without fixation, and confirmed the overexpression of CHI3L1 by immunoblotting on the hippocampal lysates (Fig. 4C) . We then probed the effect of CHI3L1 overexpression on cognitive function and performed the behavioral assays four to six weeks after the lentiviral injections (Fig. 4B) . In the open field test (fig. S4A) , mice overexpressing CHI3L1 traveled for a total distance significantly shorter than the controls (expressing eGFP alone) , indicating a reduced level of locomotor activity (fig. S4B) . Tracing the movements of crossing the central area showed that CHI3L1 overexpression led to a strong preference for the edge and corner locations, which suggested an elevated level of anxiety-like behavior (fig. S4C) . Meanwhile, the Morris water maze (Fig. 4D) revealed that CHI3L1-overexpressing mice made less platform crossovers (Fig. 4E) and took significantly more time to escape (Fig. 4F) , as compared to the control group. These behavioral changes induced by CHI3L1 resemble our findings on the msAQP4-IgG-treated mice (Fig. 3F-H and fig. S3C-E) . We next sought to investigate the effect of CHI3L1 overexpression on neurogenesis, and analyzed the fixed and immune-stained hippocampal slices  by confocal imaging. In the EdU-labeled mice (Fig. 4B) , we detected abundant eGFP expression in the dentate gyri from both control (eGFP alone) and CHI3L1 overexpression (CHI3L1 plus eGFP) groups, and validated the successful lentiviral transduction two weeks after the lentiviral injections (Fig. 4G, GFP) . To assess the proliferation, we quantified the EdU signals and noted a decrease in the number of EdU+ cells in the SGZ of the hippocampus (Fig. 4G and H) . We classified these proliferating cells by co-staining of GFAP and by the typical NSC morphology, and we found CHI3L1 overexpression lessened the radial glia-like cells (EdU+GFAP+) as well as the transiently amplifying progenitor-like cells (EdU+GFAP-) in the examined hippocampal tissues (Fig. 4I and J) . We again used the fluorescence signals from BrdU and markers for immature (DCX) and mature neurons (NeuN) to evaluate the neuronal differentiation from NSCs (Fig. 4B) . Overexpression of CHI3L1 reduced both the pools of newly-differentiated immature neurons (double-labeled by BrdU+DCX+) and mature neurons (BrdU+NeuN+) , yielding one more line of evidence for the inhibitory role of CHI3L1 for adult hippocampal neurogenesis. Importantly, the virtual reproduction of the msAQP4-IgG effects (Fig. 3) by CHI3L1 overexpression here (Fig. 4) shows that CHI3L1 overexpression is sufficient to convey the detrimental effects of neuroinflammation on hippocampal function and cognitive performance.
CHI3L1 cooperated with CRTH2 receptor to inhibit adult hippocampal neurogenesis by reducing β-catenin signaling
To delineate the CHI3L1 signaling mechanism leading to impaired neurogenesis in neuroinflammation, we pursued identifying the responsible receptor and downstream pathway in NSCs. CHI3L1 functions through a set of defined transmembrane receptors, such as interleukin 13 receptor α2 (IL-13Rα2) , transmembrane protein 219 (TMEM219) and chemoattractant receptor homologous with Th2 cell (CRTH2) , by activating several signaling cascades (Fig. 5A) . To uncover potential CHI3L1 receptors on NSCs, we first performed immunostaining to analyze the expression patterns of the individual candidate receptors in the SGZ of dentate gyrus in young adult mouse brains (8-week-old wildtype C57BL/6 mice) . CHI3L1 receptor IL-13Rα2 was not expressed in NSCs (fig. S5A, EdU+GFAP+Sox2+radial glia-like cells) , and neither was TMEM219 (fig. S5B) . CRTH2 was expressed in NSCs (GFAP+Sox2+) and to a lesser extent in the immature neurons (DCX+) , but not in mature granular neurons (NeuN+) (Fig. 5B) . After the recognition of CRTH2 as a potential CHI3L1 receptor on NSCs, we focused on the specific downstream pathway that is mediated by GSK3β and β-catenin (Fig. 5A) . β-catenin is a transcription factor in Wnt signaling and governs the process of adult hippocampal neurogenesis. GSK3β, a serine/threonine protein kinase stimulated by CRTH2 activation, phosphorylates and targets β-catenin for proteosomal degradation unless the kinase function is inactivated by phosphorylation at Ser962. We analyzed the perturbation of GSK3β and β-catenin expression in purified  mouse NSCs treated with recombinant CHI3L1. Strikingly, CHI3L1 treatment rapidly accumulated active GSK3β with a concomitant reduction in the inhibitory Ser9 phosphorylation within 30 minutes (Fig. 5C, protein levels of total and inactive/phosphorylated GSK3β) . CHI3L1 simultaneously caused a prolonged decrease in the β-catenin protein level that persisted more than two hours (Fig. 5C) , indicating destabilization and degradation triggered by the increased GSK3β kinase activity, which would be directly downstream to CRTH2 activation by CHI3L1 binding.
To confirm the participation of CRTH2 receptor in the identified CHI3L1 signaling pathway, we designed shRNAs targeting all three tested receptors, CRTH2, IL-13Rα2 and TMEM219, and lentivirally transduced them (shCRTH2, shIL-13Rα2 and shTMEM219) and a scrambled non targeting control shRNA (shCtrl) individually in cultured mouse NSCs (Fig. 5D, with co expression of eGFP to ensure adequate lentiviral transduction efficiency >99%) . The knockdown efficiency was satisfactory at a level of 75-90%across all designed shRNAs, as compared to the receptor protein levels in control condition (Fig. 5E, fig. S5C and D) . Upon CHI3L1 treatment for two hours, the shRNA-mediated knockdown of CRTH2 in NSCs maintained the Ser9 inhibitory phosphorylation of GSK-3β and stabilized β-catenin from degradation (Fig. 5F) , suggesting CRTH2 depletion blocked the CHI3L1 effects on GSK-3βactivation and β-catenin inhibition. We continued to investigate whether the CRTH2-dependent signaling pathway is involved in CHI3L1-induced inhibition of NSC proliferation and neuronal differentiation. In cultures of primary mouse NSCs expressing control shRNA (plus eGFP) , CHI3L1 treatment reduced the numbers of proliferating cells (Fig. 5G, GFP+EdU+ cells in conditions of shCtrl+PBS vs. shCtrl+CHI3L1) as well as the newly differentiated neurons (Fig. 5H, GFP+Tuj1+) as we expected. The cultured NSCs made deficient of CRTH2 by shRNA, however, displayed unchanged levels of proliferation (Fig. 5G, GFP+EdU+ cells in conditions of shCRTH2+PBS vs. shCRTH2+CHI3L1) and neuronal differentiation (Fig. 5H, GFP+Tuj1+) . Conversely, the knockdown of the receptors IL-13Rα2 and TMEM219 did not render NSCs insensitive to CHI3L1 treatment and still showed the suppressed proliferation (fig. S5E) and neuronal differentiation (fig. S5F) . Taken together, we demonstrated that CHI3L1 engages CRTH2 receptor and activates GSK-3β, which in turn destabilizes β-catenin and thus perturbs the transcriptional network for neurogenesis driven by β-catenin. Our findings also suggested that blocking the CHI3L1 receptor and signaling pathway would reinstate neurogenesis under the circumstance of astrocyte activation in neuroinflammation.
Depletion of CHI3L1 secreted by astrocytes improves adult hippocampal neurogenesis and function in immune-mediated neuroinflammation
We continued to test if this CHI3L1-CRTH2-GSK-3β/β-catenin signaling cascade could be modulated to modify the inhibitory effect of CHI3L1 on hippocampal function and neurogenesis in vivo.  As CHI3L1 is primarily secreted by activated astrocytes, we first targeted the astroglial expression of CHI3L1 in the immune-mediated neuroinflammation. We used our validated Chi3l1-floxed (Chil1f/f) mouse strain and achieved astrocyte-specific conditional knockout by AAV injections transducing Cre recombinase under the control of GFAP promoter (Fig. 6A, with co-expression of eGFP for our evaluation of lentiviral transduction efficiency) . Specifically, these AAVs were stereotaxically injected into the hippocampal regions of the homozygous mutant mice (Chil1f/f, exon 5 flanked by loxP sites) or heterozygous littermates (Chil1f/+) at the age of 8 weeks (Fig. 6A) . We first checked the specificity and efficiency of the intended conditional knockout in astrocytes. Two weeks after the AAV injections (Fig. 6A, first sacrifice) , we made the observation that the majority of GFP+ cells were co-labeled with GFAP signals, indicating that nearly all AAV-transduced cells were astrocytes (Fig. 6B) . The brains of a subset of the AAV-injected Chil1f/+ and Chil1f/f mice were harvested without fixation and then their hippocampi were dissected and lysed to measure the CHI3L1 mRNA levels by qPCR. The knockout efficiency was over 75%in comparing the Chil1f/f samples to the Chil1f/+ ones (Fig. 6C) . Having validated the astrocyte-specific depletion of CHI3L1 in the hippocampus, we carried out the experiments to assess the hippocampal function by using the model of stereotaxic IgG injection as we described above (Fig. 3A-K) . We stereotaxically delivered msCtrl-IgG or msAQP4-IgG into the hippocampal region after a four-week recovery from the AAV-mediated CHI3L1 depletion, and performed the behavioral tests at week eight (Fig. 6A) . In the CHI3L1-expressing Chil1f/+ mice, we noticed that msAQP4-IgG provoked the abnormalities of lower locomotor activity, anxiety-like behaviors and impaired memory operation, as in the wildtype mice receiving intra-hippocampal injection of msAQP4-IgG (Fig. 3E-G and sig. S3C-E) : the open field test (OFT) (fig. S6A) showed a shorter total travel distance (fig. S6B) and the reluctance to explore and cross the central area (fig. S6C) ; while the MWM (Fig. 6D) revealed a decreased number of the platform crossing (Fig. 6E) and extended escape latencies (Fig. 6F) . In the Chil1f/f mice undergoing the diminution of CHI3L1 by AAV-Cre, the aforementioned behavioral abnormalities induced by msAQP4-IgG were alleviated, according to our analyses of OFT (fig. S6A-C) and MWM (Fig. 6E-G) . These results of CHI3L1 deletion, in conjunction with our findings on CHI3L1 overexpression (Fig. 4A-F) , firmly argued for the inhibitory role of astrocyte-secreted CHI3L1 on the hippocampal function in an inflammatory condition with astroglial activation.
We next sought to decipher the effect of astrocyte-secreted CHI3L1 on hippocampal neurogenesis in immune-mediated neuroinflammation by labeling the Chil1f/+ and Chil1f/f mice with BrdU and EdU after the astroglial activation by mini-pump perfusion of msAQP4-IgG (Fig. 6G) . The BrdU was administrated right after the AAV injections in one group of Chil1f/+ and Chil1f/f mice and the EdU was given 4 weeks later in the other group in order to facilitate our measurements of NSC proliferation (by EdU labeling) and differentiation in neurons (by BrdU) . The msCtrl-IgG or msAQP4-IgG was chronically  perfused into the dentate gyrus for a period of two weeks by a minipump to elicit astrocyte activation and immune-mediated neuroinflammation. The treated mice were then sacrificed at the indicated times to prepare the hippocampal slices for our confocal microscopy (Fig. 6G) . At the completion of IgG perfusion, we found that msAQP4-IgG significantly decreased the number of total proliferating cells in the SGZ from the CHI3L1f/+ groups but did not alter that from the Chil1f/f groups (Fig. 6H) . The similar msAQP4-IgG effect was observed on the number of proliferating NSC-like cells (Fig. 6I, EdU+GFAP+with a proper NSC morphology) . We next analyzed the BrdU-labeled NSC lineage cells for their expression of neuronal markers DCX (immature) and NeuN (mature neurons) . As expected, msAQP4-IgG perfusion diminished both the newly differentiated immature and mature neurons in the CHI3L1-expressing hippocampal tissues from the CHI3L1f/+ groups and CHI3L1f/+ groups (Fig. 6K and L) . Of note, the CHI3L1 depletion in astrocytes counteracted the inhibitory effect from msAQP4-IgG and made no obvious alternation in the pools of immature (BrdU+DCX+) and mature (BrdU+NeuN+) neurons as compared to the control msCtrl-IgG group (Fig. 6J and K) . These data showed that the astrocyte-secreted CHI3L1 negatively affects hippocampal neurogenesis and function in immune-mediated neuroinflammation and that the deletion of CHI3L1 in astrocytes could reverse such inflammatory sequelae.
Inhibition of CHI3L1 receptor pathway rescues the neurogenic and cognitive deficits in immune-mediated neuroinflammation
We further investigated whether blocking the CHI3L1 signaling pathway would be able to rescue the impaired neurogenesis resulting from astroglial activation in neuroinflammation. To disrupt activation of CRTH2 receptor by CHI3L1 binding, we injected the lentiviruses expressing the control shRNA or the validated shRNA against CRTH2 (Fig. 5D-H, shCtrl and shCRTH2, with eGFP co-expression) into the dentate gyrus of the hippocampus to knock CRTH2 down in 8-week-old young adult wildtype mice (Fig. 7A) . We then implanted the mini-pump for a 2-week perfusion of msCtrl-IgG or msAQP4-IgG for 2 weeks, and administered EdU and BrdU at the indicated times to evaluate NSC proliferation and neuronal differentiation, respectively (Fig. 6M) . In the control groups (shCtrl) , msAPQ4-IgG markedly attenuated NSC proliferation, as the numbers of total proliferating cells (GFP+EdU+) and proliferating NSC-like cells (GFP+EdU+GFAP+, with a typical NSC morphology) were both lowered (Fig. 7B) . In contrast, CRTH2 knockdown abolished such repressive influence from msAQP4-IgG and led to little or no apparent change in NSC proliferation (Fig. 7B) . In neuronal differentiation assays, CRTH2 knockdown also preserved the numbers of immature (GFP+BrdU+DCX+) and mature (GFP+BrdU+NeuN+) newborn neurons, which were both reduced in the presence of CRTH2 to convey CHI3L1 signaling in neuroinflammation (Fig. 7C and 7D) . Together, these findings corroborated the role of CRTH2 as a  receptor for CHI3L1 signaling in NSCs and suggested that the CHI3L1-CRTH2-GSK-3β/β-catenin pathway can be targeted to rescue the impaired neurogenesis in the context of neuroinflammation.
Finally, we examined the translational potential in targeting this identified CHI3L1 signaling mechanism by exploring the growing list of CRTH2 antagonists, a promising class of anti inflammatory drugs in multiple clinical trials already. To test the feasibility, we chose the compound AZD1981 (AstraZeneca, Gothenburg, Sweden) , a potent, selective and non-competitive CRTH2 antagonist that went through a Phase IIb study as an adjuvant for asthma therapy. For the in vitro neurogenesis assay, in cultured mouse NSCs labeled with EdU and allowed to differentiate into neurons as we described earlier (Fig. 2E-G) , we stimulated with PBS (control) or CHI3L1 (100 ng/ml) , with or without co-incubation of AZD1981 (10 nM) (fig. S7A) . The AZD1981 treatment alone did not alter the basal level of NSC proliferation (fig. S7B, EdU+ cells in PBS vs. PBS+AZD1981) ; it nevertheless offset the suppression of proliferation by CHI3L1 and sustained the number of proliferating cells as in the control PBS conditions (EdU+ cells in CHI3L1 vs. CHI3L1+AZD1981) . Along the in vitro neuronal differentiation process, cultured NSCs treated with AZD1981 were insensitive to the CHI3L1 stimulation and still able to differentiate into neurons to the extent of their control counterparts (fig. 7C) . Next, we turned to our established settings of chronic msAQP4-IgG perfusion and labeling with BrdU and EdU to evaluate the functional output of AZD1981 in vivo. Given the great tolerance, the mice were dosed with AZD1981 at the higher range of 1mg/kg or with just vehicle DMSO as the control by daily intraperitoneal (i.p. ) injection for four weeks (Fig. 7E) . We found that AZD1981 relieved the repression of neurogenesis in the neuroinflammatory condition initiated by msAQP4-IgG perfusion: in msAQP4-IgG groups (msAQP4-IgG alone and msAQP4-IgG+AZD1981) , AZD1981-treated mice had significantly more proliferating cells (Fig. 7F, EdU+) that were radial glia-like NSCs (Fig. 7G, EdU+GFAP+Sox2+) and had also more newborn neurons that were immature (Fig. 7H, BrdU+DCX+) or mature (Fig. 7I, BrdU+NeuN+) at the time of examination. The rescue of neuronal differentiation was indeed partial (Fig. 7H and I, msCtrl-IgG+AZD1981 vs. msAQP4-IgG+AZD1981) , which was anticipated considering that the pharmacological blockade of CRTH2 would not be complete due to the suboptimal efficiency of AZD1981 in crossing the blood-brain barrier. In summary, these results of genetic knockdown or pharmacological blockade demonstrated that the CRTH2 receptor could be targeted to shut down the CHI3L1 signaling that adversely impact hippocampal neurogenesis and function after astroglial activation in neuroinflammation.
OTHER EMBODIMENTS
Specific compositions and methods for the treatment of NMOSD have been described. The scope of the invention should be defined by the claims. The detailed description in this specification is  illustrative and not restrictive or exhaustive. This invention is not limited to the particular methodology, protocols, and reagents described in this specification and can vary in practice. When the specification or claims recite ordered steps or functions, alternative embodiments might perform their functions in a different order or substantially concurrently. Other equivalents and modifications besides those already described are possible without departing from the concepts described in this specification, as persons having ordinary skill in the biomedical art recognize.
All patents and publications cited throughout this specification are incorporated by reference to disclose and describe the materials and methods used with the technologies described in this specification. The patents and publications are provided solely for their disclosure before the filing date of this specification. All statements about the patents and publications' disclosures and publication dates are from the Applicant’s information and belief. The Applicant makes no admission about the correctness of the contents or dates of these documents. Should there be a discrepancy between a date provided in this specification and the actual publication date, then the actual publication date shall control. Should there be a discrepancy between the scientific or technical teaching of a previous patent or publication and this specification, then the teaching of this specification and these claims shall control.
The foregoing written specification is considered sufficient to enable one skilled in the biomedical art to practice the present aspects and embodiments. The present aspects and embodiments are not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect and other functionally equivalent embodiments are within the scope of the disclosure. Various modifications besides those shown and described herein will become apparent to those skilled in the biomedical art from the foregoing description and fall within the scope of the appended claims. The advantages and objects described herein are not necessarily encompassed by each embodiment. Those skilled in the biomedical art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by these claims.

Claims (24)

  1. A method of treating neuroinflammation in a subject, the method comprising:
    administering to the subject an effective amount of a CRTH2 inhibitor,
    thereby treating the neuroinflammation in the subject.
  2. The method of claim 1, wherein the subject has a Neuromyelitis Optica Spectrum Disorder (NMOSD) .
  3. The method of claim 1, wherein the subject has Alzheimer’s disease, malignancy of glioblastoma, or an autoimmune disorder
  4. The method of claim 3, wherein the autoimmune disorder is multiple sclerosis
  5. A method of treating a Neuromyelitis Optica Spectrum Disorder (NMOSD) in a subject, the method comprising:
    administering to the subject an effective amount of a CRTH2 inhibitor,
    thereby treating the NMOSD in the subject.
  6. The method of any of the preceding claims, wherein the CRTH2 inhibitor comprises a small molecule, an antibody molecule, a nucleic acid, or a polypeptide.
  7. The method of any of the preceding claims, wherein the CRTH2 inhibitor comprises AZD1981, fevipiprant, OC00459, BI671800, AMG853, TQC3564, setipiprant, ASP5642, ARRY-502, AZD6430, ADC-3680, MK-1029, MK-7246, or BI1021958.
  8. The method of any of the preceding claims wherein the CRTH2 inhibitor is administered at a dose of 25 to 1200mg.
  9. The method of any of the preceding claims wherein the CRTH2 inhibitor is administered daily or twice daily.
  10. The method of any of the preceding claims, wherein the CRTH2 inhibitor is administered intravenously, orally, parenterally, or topically.
  11. The method of any of the preceding claims, wherein the NMO is monophasic NMO, relapsing NMO, acute NMOSD, chronic NMOSD, AQP4 antibody negative NMOSD, AQP4 antibody positive NMOSD, MOG antibody negative NMOSD, and/or MOG antibody positive NMOSD.
  12. The method of any of the preceding claims, wherein the subject is suffering from a neurological symptom, neuroinflammation, cognitive impairment, loss of spinal cord functions, loss of optic nerve functions, vision loss, and/or motor deficits.
  13. The method of any of the preceding claims, wherein treatment results in lessening of cognitive impairment in the subject.
  14. The method of any of the preceding claims, wherein treatment results in an increase in neurogenesis in the subject.
  15. The method of any of the preceding claims, wherein treatment results in an increase in neural stem cell proliferation in the subject.
  16. The method of any of the preceding claims, wherein treatment results in an increase in neural stem cell differentiation in the subject.
  17. The method of any of the preceding claims, wherein the subject has detectable levels of CHI3L1 prior to the administration.
  18. The method of any of the preceding claims, wherein the subject has an elevated level of CHI3L1 compared to a healthy subject prior to the administration.
  19. The method of any of the preceding claims, wherein the subject has an elevated level of astrocyte activation prior to the administration.
  20. The method of any of the preceding claims, wherein the subject has detectable levels of CRTH2 prior to the administration.
  21. The method of any of the preceding claims, wherein the subject is a human.
  22. A method of treating neuroinflammation in a subject, the method comprising:
    administering to the subject an effective amount of a CHI3L1 inhibitor,
    thereby treating the neuroinflammation in the subject.
  23. The method of claim 22, wherein the CHI3L1 inhibitor comprises a small molecule, an antibody molecule, a nucleic acid, or a polypeptide.
  24. The method of claim 22 or 23, wherein the subject has a Neuromyelitis Optica Spectrum Disorder (NMOSD) .
PCT/CN2023/083374 2023-03-23 2023-03-23 First in class use of the crth2 antagonist in treating neuromyelitis optica spectrum disorder (nmosd) and relevant autoimmune cns disorders Pending WO2024192765A1 (en)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
WO2006095183A1 (en) * 2005-03-11 2006-09-14 Oxagen Limited 1-acetic acid-indole derivatives with pgd2 antagonist activity
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WO2022189531A1 (en) * 2021-03-09 2022-09-15 Fondazione Centro San Raffaele Compounds for use in progressive multiple sclerosis
CN115701997A (en) * 2020-08-10 2023-02-14 江苏恒瑞医药股份有限公司 Use of BTK inhibitors for treating diseases

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006095183A1 (en) * 2005-03-11 2006-09-14 Oxagen Limited 1-acetic acid-indole derivatives with pgd2 antagonist activity
KR20210035425A (en) * 2019-09-24 2021-04-01 충북대학교 산학협력단 N-Allyl-2-[(6-butyl-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidin-5-yl)sulfanyl]acetamide for treating and preventing anxiety or Neuroinflammatory diseases
CN115701997A (en) * 2020-08-10 2023-02-14 江苏恒瑞医药股份有限公司 Use of BTK inhibitors for treating diseases
WO2022189531A1 (en) * 2021-03-09 2022-09-15 Fondazione Centro San Raffaele Compounds for use in progressive multiple sclerosis

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MOHRI IKUKO, TANIIKE MASAKO, TANIGUCHI HIDETOSHI, KANEKIYO TAKAHISA, ARITAKE KOSUKE, INUI TAKASHI, FUKUMOTO NORIKO, EGUCHI NAOMI, : "Prostaglandin D 2 -Mediated Microglia/Astrocyte Interaction Enhances Astrogliosis and Demyelination in twitcher", THE JOURNAL OF NEUROSCIENCE, SOCIETY FOR NEUROSCIENCE, US, vol. 26, no. 16, 19 April 2006 (2006-04-19), US , pages 4383 - 4393, XP093211884, ISSN: 0270-6474, DOI: 10.1523/JNEUROSCI.4531-05.2006 *

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