Exogenous inhibition assay method

Abstract

A high-throughput, high-content assay to screen for agents that overcome the inhibition of remyelination by exogenous inhibitors.

Description

[Technical Field]

[0001] Inclusion by referencing sequence listings provided as text files The sequence listing was created on June 2, 2022, and is provided herein as a text file "2244002.txt" with a size of 693 bytes. The contents of the text file are incorporated herein by reference in their entirety.

[0002] Government funding This invention was made with government support under W81XWH-17-1-0211 granted by ARMY / MRMC and under R35 NS097976 granted by the National Institutes of Health. The government has certain rights with respect to this invention. [Background technology]

[0003] Background of the Invention Exogenous inhibitors at blood-brain barrier (BBB) ​​disruption sites and neurovascular injury sites contribute to impaired remyelination in neurological diseases. However, treatments to overcome exogenous inhibition of remyelination are not widely available, and the dynamics of glial progenitor cell niche remodeling in neurovascular dysfunction sites are largely unknown. [Overview of the project]

[0004] Since it has been shown that myelinization promoters do not rescue the exogenous inhibition of remyelination by fibrinogen, this specification provides an assay / screening (named OPC-X) for identifying compounds that promote remyelination in the presence of exogenous inhibitors.

[0005] One embodiment provides a high-throughput, high-content assay for screening agents that overcome remyelination inhibition by exogenous inhibitors, the assay comprising a) contacting oligodendrocyte progenitor cells (OPCs) with an exogenous inhibitor and a test agent, and b) detecting / quantifying the presence of 1) MBP+ myelinating oligodendrocytes (OLs) and 2) GFAP+ astrocytes by obtaining two readings in a single assay, the increase in OLs and decrease in GFAP+ astrocytes compared to control OPCs contacted with the exogenous inhibitor alone, indicates that the agent has overcome remyelination inhibition by the exogenous inhibitor.

[0006] In one embodiment, the exogenous inhibitor is an antibody, compound, small molecule, peptide, and / or nucleic acid. In another embodiment, the exogenous inhibitor is an inflammatory molecule. In one embodiment, the exogenous inhibitor is fibrinogen. In one embodiment, fibrinogen is present at physiological levels. In another embodiment, fibrinogen is added at a concentration of at least 2.5 mg / ml.

[0007] In one embodiment, OPC is primary OPC. In one embodiment, primary OPC is cultured in growth medium for 1 to 6 days prior to a). In another embodiment, the growth medium contains PDGF-AA and NT3. In one embodiment, OPC is isolated from the culture dish by proteolysis and / or collagenolysis and then seeded into a fresh culture dish. In another embodiment, OPC is cultured prior to a), 5 × 10⁶ per well of a 96-well plate. 3 In cells, or 1 × 10⁶ per well in a 384-well plate. 3 The cells are seeded. In one embodiment, the seeded OPCs are cultured for up to 24 hours before step a). In another embodiment, the OPCs are cultured for 1 to 6 days, including 1, 2, 3, 4, 5, or 6 days in step a), so that the OPCs can differentiate before step b).

[0008] In one embodiment, after a), cells are brought into contact with antibodies against MBP (oligodendrocytes) and antibodies against GFAP (astrocytes). In one embodiment, the antibodies are directly or indirectly labeled with a detectable label, and images of the labeled cells are obtained, such as automated imaging. In one embodiment, at least about 80% of the culture vessel (e.g., wells) are imaged. In another embodiment, automated quantification of MBP+ and GFAP+ is used.

[0009] One embodiment provides a high-throughput, high-content assay for screening drugs that overcome inhibition by exogenous inhibitors, the assay comprising a) contacting cells with the exogenous inhibitor and the test drug, and b) detecting and / or quantifying the cellular response to the test drug, wherein a different cellular response compared to a control in which cells were contacted with the exogenous inhibitor alone indicates that the drug was able to overcome the exogenous inhibitor. In one embodiment, the exogenous inhibitor is selected from the group consisting of chondroitin sulfate proteoglycans, hyaluronane, fibronectin aggregates, myelin debris, inflammatory cytokines (e.g., soluble TNF-α or interferon-γ), bone morphogenetic proteins, endothelin-1, semaphorin, environmental toxins, and alcohol, tobacco, illegal drugs or recreational drugs. In another embodiment, the cells are selected from the group consisting of stem cells or progenitor cells, e.g., neural stem cells and / or progenitor cells (adult and / or fetal / neonatal), radial glial cells (adult and / or fetal / neonatal), cerebellar granule neuron progenitor cells, neural crest stem / progenitor cells, vascular / endothelial stem / progenitor cells, organ stem / progenitor cells (e.g., heart, liver, lung, kidney, skeletal muscle, skin, bone, retina), mesenchymal stem / progenitor cells, placental stem / progenitor cells, embryonic stem cells, induced pluripotent stem cells (or ESC / iPSC-derived cells), and cancer / tumor-associated cells / stem cells. In one embodiment, the neural progenitor cells are oligodendrocyte progenitor cells (OPCs). [Brief explanation of the drawing]

[0010] [Figure 1-A] Figures 1'A to 1'D show the experiments and data from Example 1. [Figure 1-B]Same as above. [Figure 1-C] Same as above. [Figure 1-D] Same as above. [Figure 1A] NG2 cells cluster around blood vessels at fibrinogen deposition sites and limited remyelination sites in chronic neuroinflammation. A, NG2-CreER™:RosatdTomato / +:Cx3cr1GFP / + in vivo 2P maximum projection images of microglia (green), NG2 cells (red), and vascular system (blue, 70kDa Oregon green dextran) in age-matched healthy control mice, at peak clinical signs (peak EAE, mean score 3) and chronic EAE (mean clinical score 2.1). The images shown are from mice at 17 days (peak) and 35 days (chronic) after EAE induction. NG2tdTomato / + pericytes in control conditions are indicated by white arrows. Scale bar, 50 μm. Quantification of NG2 cell and microglia clusters in control (n=4 mice), peak (n=5 mice), and chronic (n=6 mice) EAE. Values ​​are mean ± standard error, *p<0.05, ns indicates no significant difference (two-way ANOVA with Bonferroni multiple comparison test). [Figure 1B]NG2 cells cluster around blood vessels at fibrinogen deposition sites and limited remyelination sites in chronic neuroinflammation. B. Micrographs of spinal cord sections from unimmunized healthy mice (control) and MOG35-55-EAE mice in the peak and chronic phases of the disease, immunostained for fibrinogen (green). Nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 100 μm. Quantification of dextran leakage in the spinal cord of MOG35-55-EAE mice in unimmunized healthy mice (control) (n=4 mice) and in the peak (n=5 mice) and chronic (n=6 mice) phases of the disease. Values ​​are mean ± standard error, *p<0.05 (one-way ANOVA with Tukey's multiple comparison test). Quantification of fibrinogen immunoreactivity in the spinal cord of unimmunized healthy mice (control) and MOG35-55-EAE mice (n=3 mice per group) in the peak and chronic phases of the disease. Values ​​are mean ± standard error, **p<0.01, ***p<0.001 (one-way ANOVA with Tukey's multiple comparison test). [Figure 1C] NG2 cells cluster around blood vessels at fibrinogen deposition sites and limited remyelination sites in chronic neuroinflammation. ☐C, Micrograph of ventral spinal cord sections from NG2-CreER™:RosatdTomato / +:Cx3cr1GFP / + mice with chronic EAE, immunostained for fibrinogen (green). Scale bar, 50 μm. Quantification of fibrinogen immunopositivity in NG2 clustered and non-clustered regions (n=5 mice). Values ​​are mean ± standard error, **p<0.01 (two-sided Mann-Whitney test). [Figure 1D]NG2 cells cluster around blood vessels at fibrinogen deposition sites and limited remyelination sites in chronic neuroinflammation. D. In vivo 2P maximum projection images of myelin (green) in NG2-CreER™:RosatdTomato / +:Cx3cr1GFP / + mice of chronic EAE in areas with and without NG2 clusters. The framed area is shown in the inset in the upper right to show only the myelin labeling. Scale bar, 20 μm. Quantification of the roundness of myelin in chronic EAE in areas with and without NG2 clusters (n=5 mice). Values ​​are mean ± standard error, **p<0.01 (two-sided Mann-Whitney test). A value of 1.0 indicates a perfect circle (as seen in degenerated myelin in longitudinal section). Values ​​closer to 0.0 indicate a non-circular and linear shape (longitudinal section of normal myelinated fibers). [Figure 1E] NG2 cells cluster around blood vessels at fibrinogen deposition sites and limited remyelination sites in chronic neuroinflammation. ROI tracking workflow for co-registration of E, 2P volume and SBEM volume. [Figure 1F] NG2 cells cluster around blood vessels at fibrinogen deposition sites and limited remyelination sites in chronic neuroinflammation. F-G, representative correlated SBEM images from n=3 ROIs from two different mice. Fi, CNS parenchyma in NG2 cluster regions shows inflamed spinal vessels with perivascular lesions, including activated endothelial cells (green asterisks), leukocyte adhesion to endothelium (black arrows), and predominantly demyelination (red box) and sparse remyelination (blue box). Scale bar, 20 μm. Fii, red box region shown at high magnification. Red arrows indicate demyelinate axons. Scale bar, 10 μm. Fiii, blue box region shown at high magnification. Blue arrows indicate remyelinate axons. Scale bar, 10 μm. Fiv. Correlated SBEM within CNS parenchyma in areas without NG2 clusters. The black arrow indicates a normal myelinated axon. Scale bar: 10 μm. [Figure 1G]NG2 cells cluster around blood vessels at fibrinogen deposition sites and limited remyelination sites in chronic neuroinflammation. F-G, representative correlated SBEM images from n=3 ROIs from two different mice. Gi, a representative SBEM from another ROI within the NG2 cluster region, showing veins with perivascular demyelination, gliosis (red dotted area), and some limited remyelination (blue boxed area). The gliosis region contains infiltrating macrophages (M) and astrocytes (A). The distal region has normal myelinated axons indicated by black arrows. Scale bar, 10 μm. Gii, high magnification of the blue boxed area. Blue arrows indicate remyelination axons. Black arrows indicate NG2 cells. Scale bar, 5 μm. [Figure 2A] Figures 2A-2F. RNA-seq analysis of NG2 cells in EAE revealed suppression of the anticoagulant pathway. Data are from n=3 mice per group (A-D). A. Volcano plot of DEG from RNA-seq analysis of NG2 strain cells from MOG35-55-EAE or healthy mice. Circles indicate genes that were significantly downregulated (blue, log2 ratio change <-1, FDR < 0.05) or upregulated (red, log2 ratio change > 1, FDR < 0.05) in EAE compared to healthy mice. [Figure 2B] Heatmap of data from A. Genes were clustered by HOPACH unsupervised clustering analysis (clusters 1-9). Expression values ​​were log-normalized, rows were centered, and displayed as Z-scores. Examples of important GO terms and genes are shown for each cluster. FDR < 0.05; modified by Benjamini-Hochberg. [Figure 2C] Visualization of downregulated (blue nodes) or upregulated (red nodes) co-expression GO term networks in NG2 cells from EAE compared to healthy mice. Gene set size and co-expression overlap (key) were determined by GSEA (p<0.05). [Figure 2D]Enrichment plots of the gene sets "negative regulation of coagulation" and "regulation of cell junction assembly" determined by GSEA from RNA-seq data of NG2 cells from EAE or healthy mice. The X-axis shows the rank of the gene in the dataset. NES, normalized enrichment score. [Figure 2E] Representative histograms of surface-labeled TFPI and quantification of TFPI+ cells in PDGFRα+OPC(E) or PDGFRβ+ pericytes(F) populations from E-F, healthy mice, and EAE mice. Data are from n=5 per group (mean ± standard error)**p<0.01, no significant difference in ns (two-sided Mann-Whitney U test). [Figure 2F] Same as above. [Figure 3A] Figures 3A-3G. Myelination-promoting compounds do not overcome the exogenous inhibition of OPC differentiation by fibrinogen. A. Medium-throughput workflow for OPC-X screening of myelination-promoting agents in the presence of fibrinogen. [Figure 3B] B-C, as shown, immunofluorescence of MBP (green) and GFAP (red) in primary rat OPCs treated with fibrinogen and myelin enhancer or vehicle control (dimethyl sulfoxide, DMSO). Nuclei are stained with Hoechst dye (blue). Representative images from n=3 independent experiments. Scale bar, 100 μm. [Figure 3C] Same as above. [Figure 3D] D-E. Quantification of the percentage of total cellular MBP+ or GFAP+ from automated image acquisition and quantification. Data are mean ± standard error from n=3 independent experiments. ****p<0.0001 (one-way ANOVA with Dunnett's multiple comparison test). [Figure 3E] Same as above. [Figure 3F] Phospho-SMAD1 / 5 (P-SMAD1 / 5) and ID2 protein levels in control or fibrinogen-treated primary rat OPCs in the presence of DMH1 or clemastine. Values ​​are the mean of n=3 independent experiments. [Figure 3G]Immunofluorescence of MBP (green) and GFAP (red) in primary rat OPCs treated with fibrinogen and LDN-212854 (0.18 μM) or vehicle control (DMSO) for 3 days. Nuclei are stained with Hoechst dye (blue). Representative images from n=3 independent experiments. Scale bar, 100 μm. H, quantification of the percentage of total MBP+ or GFAP+ cells from automated image acquisition and quantification. Data are mean ± standard error from n=3 independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (matched one-way ANOVA with Dunnett's multiple comparison test). [Figure 4A] 4A-4E. Therapeutic effects of type I BMP receptor inhibition in chronic neuroinflammation. A. Clinical scores of MOG35-55-EAE mice treated with LDN-212854 or saline (key) for 14 days starting from the peak of the disease. Data are mean ± standard error from n=6 mice (EAE + LDN-212854) and n=5 mice (EAE + saline). *p<0.05, (two-sided permutation test). [Figure 4B] Microscopic images visualizing myelin (green) and fibrinogen (red) by immunostaining for MBP in spinal cord sections of MOG35-55-EAE mice treated with physiological saline (left panel) or LDN-212854 (right panel). Dashed lines indicate the boundaries of demyelination in white matter. Scale bar, 50 μm. Data are mean ± standard error from n=5 mice per group. **p<0.01 (two-sided Mann-Whitney test). [Figure 4C] Clinical scores of NOD-MOG35-55EAE mice treated with LDN-212854 or physiological saline (key) for 30 days. Data are mean ± standard error for n=8 mice (EAE + LDN-212854) and n=7 mice (EAE + physiological saline). *p<0.05, (Welch two-sample t-test comparing group means of maximum score, physiological saline = 2.36, LDN-212854 = 1.75). [Figure 4D]Micrographs of spinal cord sections from NOD-MOG35-55EAE mice treated with physiological saline (left panel) or LDN-212854 (right panel). Myelin (green) was visualized and immunostained for fibrinogen (red) using a dark-field microscope. Dashed lines indicate the boundaries of demyelination in white matter. Scale bar, 100 μm. Data are mean ± standard error from n=6 mice per group. *p<0.05 **p<0.01 (two-sided Mann-Whitney test). [Figure 4E]In vivo 2P maximum projection images of NG2 cells (red) and vascular system (blue, 70kDa Oregon green dextran) in chronic EAE-treated NG2-CreERTM:RosatdTomato / + mice treated with physiological saline (left panel) and LDN-212854 (right panel). Scale bar, 50 μm. Data are mean ± standard error from n=6 (EAE + LDN-212854) and n=5 (EAE + physiological saline). *p<0.05 (unpaired two-sided t-test). F, In vivo 2P maximum projection images of NG2 cells (red) and myelin (green, MitoTracker) in chronic EAE-treated NG2-CreERTM:RosatdTomato / + mice treated with physiological saline (left panel) and LDN-212854 (right panel). Scale bar, 20 μm. Data are mean ± standard error from n=5 (EAE + LDN-212854) and n=4 (EAE + saline). *p<0.05 (two-sided Mann-Whitney test). Myelin damage was quantified by myelin roundness, where a value of 1.0 indicates a perfect circle, and values ​​closer to 0.0 indicate a non-circular, linear shape. G, Micrographs of spinal cord sections from NG2-CreER™:RosatdTomato / +MOG35-55-EAE mice after 14 days of treatment with saline (left panel) or LDN-212854 (right panel). Immunostaining of NG2 cells (red) and ID2 (green). Nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 25 μm. Data are mean ± standard error from n=6 (EAE + LDN-212854) and n=5 (EAE + saline). **p<0.01 (two-sided Mann-Whitney U test).** Fate mapping of tdTomato+OPC-derived cells using micrographs of spinal cord sections of NG2-CreER™:RosatdTomato / +MOG35-55-EAE mice treated for 14 days with H, LDN-212854, or saline. Immunostaining of NG2tdTomato+ cells (red) and mature OL marker GST-pi (green, upper panel) or astrocyte marker GFAP (green, lower panel). Scale bars, 50 μm (upper panel) and 20 μm (lower panel).Data are the mean ± standard error from n=6 (EAE + LDN - 212854) and n=5 (EAE + physiological saline). **p<0.01 (two-sided Mann-Whitney test). [Figure 5] Supplementary Figure 1. Workflow for in vivo 2P imaging and bulk RNA-seq analysis of NG2 lineage cells and microglia in MOG35-55-EAE NG2creER™:RosatdTomato / +:Cx3cr1GFP / + mice. [Figure 6] Supplementary Figures 2A-2C. In vivo 2P imaging of NG2 cells and microglia at the neurovascular interface at various stages of EAE. In vivo 2P maximum projection images of NG2 cells (red, upper panel), microglia (green, lower panel), and vascular system (blue, 70kDa Oregon green dextran) from age-matched healthy control mice. Peak EAE (mean score 3) and chronic EAE (mean clinical score 2.1). Scale bar, 100 μm. Quantification of co-localization of NG2 clusters and microglia clusters in peak (n=5 mice) and chronic (n=6 mice) EAE. Values ​​mean ± standard error, **p<0.01 (two-sided Mann-Whitney test). [Figure 7] Supplementary Figure 2B, in vivo 2P maximum projection images of NG2 cells (red) and vascular system (blue, 70kDa Oregon green dextran) from age-matched healthy control mice NG2creER™:RosatdTomato / +:Cx3cr1GFP / +, at peak clinical signs (peak EAE, mean score 3) and chronic EAE (mean clinical score 2.1). Scale bar, 50 μm. Quantification of distance of NG2 clusters from nearest blood vessels in chronic EAE (data from 45 clusters in 6 mice). NG2tdTomato+ pericytes in control conditions are indicated by white arrows. [Figure 8]Supplementary Figure 2C, in vivo 2P maximum projection of tdTomato+ (red) pericytes (left panel) and OL lineage cells associated with the vascular system (blue, 70kDa Oregon green dextran) within the spinal cord parenchyma of NG2-CreER™:RosatdTomato / +:Cx3cr1GFP / + mice. Scale bar, 20 μm. [Figure 9] Supplementary Figures 3A-3C. Endothelial activation at various stages of EAE. A. Micrographs of ventral spinal sections of NG2-CreER™:RosatdTomato / + mice with immunostaining for VCAM-1 in control, peak EAE, and chronic EAE. Red arrows indicate vascular VCAM-1 expression. Red asterisks indicate diffuse VCAM-1 positivity. Quantification of VCAM-1 immunoreactivity in the ventral spinal cord of control, peak EAE, and chronic EAE. Scale bar, 50 μm. Values ​​are mean ± standard error, **p<0.05 (one-way ANOVA with Dunnett's multiple comparison test). [Figure 10] Supplementary Figure 3B, micrographs of ventral spinal cord sections from control NG2-CreERTM:RosatdTomato / + mice, showing peak EAE and chronic EAE immunostained for PLVAP. Red arrows indicate PLVAP expression in blood vessels. Red asterisks indicate diffuse PLVAP positivity. Scale bar, 50 μm. Quantification of PLVAP+ vessels in the ventral spinal cord in control, peak EAE, and chronic EAE. Values ​​are mean ± standard error, *p<0.05 (one-way ANOVA with Tukey's multiple comparison test). [Figure 11] Supplementary Figure 3C shows inflamed spinal cord vessels with activated endothelial cells in the CNS parenchyma of the NG2 cluster region. The activated endothelium (black arrows) shown here is thicker compared to the very thin endothelium of normal blood-brain barrier (BBB) ​​vessels. These activated endotheliums form small projections (red arrows) that come into contact with leukocytes (black arrows) within the vessels. [Figure 12]Supplementary Figures 4A-4B. NG2 cell clusters associated with fibrinogen deposition and myelin disruption in chronic EAE. A. Micrograph of ventral spinal cord section of NG2-CreERTM:RosatdTomato / +:Cx3cr1GFP / + mice with chronic EAE, immunostained for fibrinogen (green). NG2tdTomato+ cells (red) cluster at fibrinogen deposition sites. Here, these are indicated by yellow ROIs (white arrows) within merge channels. Scale bar, 50 μm. [Figure 13] Supplementary Figure 4B, in vivo 2P maximum projection images of NG2tdTomato+ cells (red) and myelin (green) in chronic EAE NG2-CreER™:RosatdTomato / +:Cx3cr1GFP / + mice in the NG2 cell cluster region and the non-cluster region. Of particular note is that the myelin sheath is labeled with MitoTracker Deep Red far-red fluorescent dye (abs / em approx. 644 / 665 nm) and is pseudo-stained green here. Here, disrupted myelin or myelin vesicles are indicated by white arrows in the NG2 cell cluster region, and normal-looking myelin is indicated by white arrows in the non-cluster region. Scale bar, 20 μm. [Figure 14] Supplementary Figures 5A-5C. FACS isolation of NG2 cells. A. Representative flow cytometry plots of gating strategies of NG2tdTomato+ cells from EAE (n=3) and healthy control mouse (n=3) spinal cord against bulk RNA sequences. [Figure 15] Supplementary Figure 5B, representative flow cytometry plots of gating strategies of PDGFRα+ and PDGFRβ+ cells from the spinal cord of chronic EAE (n=5) and healthy control mice (n=5) for cell surface staining. [Figure 16] Supplementary Figure 5C, representative flow cytometry contour plots and quantification of surface MHCII in viable PDGFRα+ cells. Data are from n=5 per group (mean ± standard error)**p<0.01, (two-sided Mann-Whitney test). Percentages of cell populations are listed above gates (A-C). [Figure 17] Supplementary Figures 6A-6C. Ratio of oligodendrocytes to pericytes between NG2tdTomato+ cells in control and peak EAE. A. Micrographs of ventral spinal cord sections of NG2-CreER™:RosatdTomato / + mice in control and peak EAE. NG2tdTomato+ cells (red) were immunostained for OLIG2 (green) and PDGFRβ (stained with far-infrared channels, here pseudo-stained in blue). NG2tdTomato+OLIG2+ cells are indicated by white arrows. NG2tdTomato+PDGFRβ+ cells are indicated by white asterisks. NG2tdTomato+OLIG2-PDGFRβ- cells are indicated by white arrows. Scale bar, 20 μm. [Figure 18] Supplementary Figures 6B-C: Quantification of the percentage of NG2tdTomato+ cells (OLIG2+ and PDGFRβ+) in the control and peak EAE. [Figure 19] Same as above. [Figure 20] Supplementary Figures 7A-7B. Effect of clemastine on primary OPCs in the presence of fibrinogen. Immunofluorescence of MBP (green) in primary rat OPCs treated for 3 days with fibrinogen and clemastine (0.56 μM), DMH1 (1 μM), or vehicle control (dimethyl sulfoxide, DMSO) in differentiation medium without growth factors, A, T3, or other growth factor-free media. Nuclei are stained with Hoechst dye (blue). Representative images from n=2 independent experiments. Scale bar, 100 μm. [Figure 21] Supplementary Figure 7B, Quantification of the percentage of total cellular MBP+ from automated image acquisition and quantification. Data are mean ± standard error from n=2 independent experiments. [Modes for carrying out the invention]

[0011] Detailed description of the invention The methods and compositions described herein can be carried out using prior art in medicinal chemistry, drug formulation technology, dosage planning, molecular biology, and biochemistry, unless otherwise indicated, all of which are within the scope of the art. Specific examples of suitable techniques can be obtained by referring to the examples herein.

[0012] The following description includes many specific details to provide a more complete understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be carried out without one or more of these specific details. In other cases, features and procedures that are well known / available to those skilled in the art are not described in order to avoid complicating the invention.

[0013] Abbreviation 2P = 2 photons; BBB = blood-brain barrier; BMP = bone morphogenetic protein; CSPG = chondroitin sulfate proteoglycan; DEG = differential gene expression; EAE = experimental autoimmune encephalomyelitis; GSEA = gene set enrichment analysis; GO = gene ontology; GST-pi = glutathione s-transferase-pi; MHCII = major histocompatibility complex class II; MOG = myelin oligodendrocyte glycoprotein; NOD = non-obese diabetes; OL = oligodendrocyte; OPC = oligodendrocyte progenitor cell; RNA-seq = RNA sequencing; SBEM = serial blockface electron microscopy; TFPI = tissue factor pathway inhibitor; TGF-β = transforming growth factor beta.

[0014] explanation A remyelination assay consisting of single-reading OPCs from MBP+ oligodendrocytes is unsuitable for screening exogenous remyelination inhibitors present in the lesion environment, nor for screening cell fate switches to GFAP+ astrocytes.

[0015] By developing a screening (OPC-X) to identify compounds that promote remyelination in the presence of exogenous inhibitors, it was shown that myelin-promoting agents do not rescue exogenous inhibition of remyelination by fibrinogen. In contrast, bone morphogenetic protein (BMP) receptor blockade yielded potent therapeutic effects in a chronic model of multiple sclerosis, rescuing the inhibitory fibrinogen effect by promoting myelinating oligodendrocytes while suppressing astrocyte cell fate, and restoring the myelin-promoting precursor niche. Therefore, incomplete OPC differentiation by fibrinogen is resistant to known myelin-promoting compounds, suggesting that blocking the BMP signaling pathway may enhance remyelination by overcoming exogenous inhibition in neuroinflammatory lesions with vascular damage.

[0016] This assay can be used for a variety of purposes, including screening antibodies, compounds, small molecules, peptides, etc., to a) overcome the inhibition of remyelination by fibrin / fibrinogen, b) overcome the inhibition of remyelination by other exogenous inhibitors such as inflammatory molecules and cytokines, c) inhibit the generation of fibrous astrocytes, and d) inhibit the cell fate switching of other stem cells, such as oligodendrocyte progenitor cells (OPCs) or neural progenitor cells (NPCs), to astrocytes.

[0017] The assay provided herein offers several advantages, including: 1) screening and discovery of drugs that can rescue disease-related remyelination inhibition; 2) optimized for fibrin / fibrinogen as a disease-related exogenous inhibitor; 3) adaptable for use with or in place of other exogenous inhibitors in addition to fibrin / fibrinogen; and 4) enabling the examination of both myelinating cells and astrocytes in a single assay.

[0018] Other exogenous inhibitors, in addition to or instead of fibrin / fibrinogen, include, but are not limited to, chondroitin sulfate proteoglycans (Keough et al., 2016), hyaluronan (Srivastava et al., 2018), fibronectin aggregates (Stoffels et al., 2013), myelin debris (Kotter et al., 2006), inflammatory cytokines (e.g., soluble TNF-α (Karamita et al., 2017), interferon-gamma (Kirby et al., 2019)), bone morphogenetic proteins (Mabie et al., 1997), endothelin-1 (Hammond et al., 2014), semaphorin (Syed et al., 2011), environmental toxins, and alcohol / tobacco / illegal drugs and / or recreational drugs (Forbes and Gallo, 2017).

[0019] Cells used in the assays / screenings described herein include, but are not limited to, neural stem / progenitor cells (adult and fetal / neonatal), radial glial cells (adult and fetal / neonatal), cerebellar granule neuron progenitor cells, neural crest stem / progenitor cells, vascular / endothelial stem / progenitor cells, organ stem / progenitor cells (heart, liver, lung, kidney, skeletal muscle, skin, bone, retina), mesenchymal stem / progenitor cells, placental stem / progenitor cells, embryonic stem cells and / or induced pluripotent stem cells (and ESC / iPSC-derived cells), and cancer / tumor-associated stem cells.

[0020] Such screenings / assays can be used to identify therapeutic agents / molecules for the treatment of many conditions / diseases, including but not limited to: neurological disorders involving BBB destruction and fibrin deposition (Petersen et al., 2018), Alzheimer's disease, age-related dementia, traumatic brain and spinal cord injury, neonatal and premature brain injury, subarachnoid / intraventricular hemorrhage, stroke, infections, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, HIV encephalitis, neuropsychiatric disorders such as schizophrenia and bipolar disorder, cancer, atherosclerosis / cardiovascular disease, retinopathy / macular degeneration, chronic lung disease, peripheral autoimmune diseases (e.g., rheumatoid arthritis, colitis, lupus), epithelial-mesenchymal transition (EMT), and multiple sclerosis (MS; for example, agents that overcome the fibrinogen-rich inhibitory MS lesion environment may provide an urgently needed alternative treatment for MS). Use of test reagents The assays of the present invention are used to identify candidate therapeutic agents that inhibit exogenous inhibition. This includes assays for testing novel drugs and assays for testing the effects of known compounds (including synthetic, recombinant, or naturally occurring compounds).

[0021] In the field of pharmacy, it is known that binding affinity to a target and efficacy do not necessarily correlate, and that identifying cell-based activity changes induced by investigational drugs is a more improved functional predictor of therapeutic activity compared to drugs identified solely by affinity (e.g., drug binding to microglial receptors).

[0022] In certain embodiments, the assays of the present invention correlate with the in vivo regulation of activated fibrin-mediated signaling. Examples of cell-based assays for use in the present invention include, but are not limited to, high-throughput coupling screening; assays measuring cell activation, proliferation, differentiation, necrosis and / or apoptosis; flow cytometry assays; metabolic assays measuring labeling or turnover; phase-contrast and fluorescence microscopy; receptor phosphorylation and / or turnover; cell signaling assays; immunohistochemical studies; reporter gene assays; and intracellular fractionation and localization.

[0023] Biochemical assays can also be used to correlate binding and efficacy in the cell-based assay methods of the present invention. These include, but are not limited to, spectrophotometric assays, fluorescence assays, calorimetry assays, chemiluminescence assays, radiometric assays, chromatographic assays, colorimetric assays, and substrate specificity inhibitor kinase assays. Specific examples include luciferase assays (in which the firefly luciferase protein catalyzes the oxidation of luciferin, generating light in the reaction; this is frequently used as a reporter gene to measure promoter activity or transfection efficiency); electrophoresis; gas-liquid chromatography; and Forster resonance energy transfer (FRET).

[0024] To confirm the functional activity of the test drug, a therapeutically effective dose of the test drug of the present invention can be administered to a subject (including an animal model of a neurological disorder), and its in vivo activity can be confirmed after identification in the assay of the present invention. "Therapeutic effective dose or amount" or "effective dose" means the amount of the test drug that, when administered, elicits a positive therapeutic response. In some embodiments of the present invention, the therapeutically effective dose is within the range of approximately 0.1 mg / kg to approximately 100 mg / kg body weight, approximately 0.001 mg / kg to approximately 50 mg / kg, approximately 0.01 mg / kg to approximately 30 mg / kg, approximately 0.1 mg / kg to approximately 25 mg / kg, approximately 1 mg / kg to approximately 20 mg / kg, approximately 3 mg / kg to approximately 15 mg / kg, approximately 5 mg / kg to approximately 12 mg / kg, approximately 7 mg / kg to approximately 10 mg / kg, or any value within this range. It is recognized that the treatment method may include a single administration of the therapeutically effective dose or multiple administrations of the therapeutically effective dose.

[0025] The investigational drug is administered to deliver a desired therapeutic dose to promote a desired therapeutic response. “Desired therapeutic response” means improvement of the condition or symptoms associated with the condition. Examples of routes of administration include intravenous, intra-arterial, intra-coronary, parenteral, subcutaneous, subdermal, intraperitoneal, ventricular / intraventricular infusion, infusion catheter, balloon catheter, bolus injection, direct application to the tissue surface during surgery, or other convenient routes.

[0026] The test agent can be formulated in unit doses, such as solutions, suspensions, or emulsions, in combination with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” is intended to include any solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent, and absorption retarder suitable for drug administration. Suitable carriers are listed in the latest edition of Remington's Pharmaceutical Sciences, a standard reference in this art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, glucose solution, and 5% human serum albumin. The use of such media and agents for cell delivery is well known in the art. Their use in compositions is assumed unless conventional media or agents are incompatible with the cells or polypeptides provided herein. Auxiliary active compounds may also be incorporated into the test agent.

[0027] The solution or suspension used for such administration may contain other components such as sterile diluents like water, saline, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents like benzyl alcohol or methylparaben; antioxidants like ascorbic acid or sodium bisulfite; chelating agents like ethylenediaminetetraacetic acid; buffers like acetates, citrates, or phosphates; and tonicity adjusters like sodium chloride or glucose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The composition can be sealed in glass or plastic ampoules, disposable syringes, or vials for multiple doses.

[0028] Suitable test agents for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and as fluid as possible. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants. Inhibition of microbial activity can be achieved by various antimicrobial and antifungal agents.

[0029] Sustained absorption of injectable compositions can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of active agent into a suitable solvent containing a selected combination of components, followed by filter sterilization. Typically, dispersions are prepared by incorporating the active agent into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders used to prepare sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying, obtaining powders of the active ingredient and any additional desired components from a pre-sterilized filtered solution. In many cases, it would be preferable to include an isotonic agent.

[0030] Various delivery methods can be used to deliver the test drug, depending in part on the drug and its bioavailability. For example, bioavailable small molecules or other drugs may be administered orally, while protein-based drugs are usually (but not always) administered parenterally. Certain drugs may be administered systemically, while others may be more beneficial via local delivery. The delivery method will be apparent to those skilled in the art by reading this specification and can be determined by considering the specific characteristics of the test drug.

[0031] It is understood that the effective dose of the test drug may vary depending on the nature of the desired effect, the frequency of treatment, any concurrent treatments, the patient's health status, the recipient's weight, etc. See, for example, Berkow et al., Merck Manual, 16th edition, Merck and Co., Rahway, New Jersey (1992); Goodman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, New York (1990); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Maryland (1987); Ebadi, Pharmacology, Little, Brown and Co., Boston (1985); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, Connecticut (1992). These references and the references cited therein are incorporated herein by reference in their entirety.

[0032] definition For clarity and conciseness, features may be described herein as part of the same or different embodiments. However, it will be understood that the scope of the invention may include embodiments having all or some combinations of the described features.

[0033] The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which the invention pertains. The following definitions are intended to help the reader understand the invention and are not intended to alter or limit the meaning of such terms unless otherwise specified.

[0034] The indefinite articles “one,” “that,” and “the foregoing” used herein should be understood to include multiple references unless the context clearly indicates otherwise. For example, a reference to “one inhibitor” refers to one or more agents having the ability to inhibit a target molecule, and a reference to “the foregoing method” includes references to multiple equivalent steps and methods known to those skilled in the art.

[0035] As used herein, the phrase "and / or" should be understood to mean "either or both" of the elements joined by that phrase, for example, elements that exist jointly in one case and separately in the other.

[0036] As used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating a list of items, “and / or” or “or” shall be interpreted as inclusive, e.g., including at least one of a number of items, but also including multiple items, and optionally including additional unlisted items. Terms that explicitly indicate the opposite, such as “only one of” or “exactly one of” or, when used in claims, “consisting of” only, refer to including only one element from a number of elements or a list of elements. Generally, as used herein, the term “or” shall be interpreted as indicating an exclusive choice (i.e., “one or the other, but not both”) only when preceded by an exclusive term such as “either,” “one of,” “only one of,” or “exactly one.”

[0037] As used herein, the term "about" means plus or minus 10% of the indicated value. For example, about 100 means between 90 and 110. Where a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limits of that range, as well as other stated values ​​or intermediate values ​​within the stated range, are included in the present invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are also included within the scope of the present invention, including when subject to restrictions specifically excluded within the stated range. Where a stated range includes one or both of the upper and lower limits, the range excluding either of these included upper and lower limits is also included in the present invention.

[0038] The term "small molecule" refers to molecules that are comparable in size to organic molecules commonly used in pharmaceuticals. This term does not include biomacromolecules (e.g., proteins, nucleic acids, etc.). Organic small molecules include those with sizes up to approximately 5000 Da, up to 2000 Da, and up to approximately 1000 Da.

[0039] As used herein, “Test Agent” refers to any agent that is a candidate for treating a disease or its symptoms. Such agents include, but are not limited to, peptides; proteins (including derivatized or labeled proteins); antibodies or fragments thereof; small molecules; aptamers; carbohydrates and / or other non-protein binding sites; derivatives and fragments of naturally occurring binding partners; peptide mimes; and pharmacophores.

[0040] The term "pharmacophore" is used herein in an unconventional manner. While this term typically refers to a geometric and / or chemical description of a class or set of compounds, as used herein, it refers to a compound having a specific biochemical activity or bonding property determined by the three-dimensional physical shape of the compound and the electrochemical properties of the atoms constituting the compound. Therefore, as used herein, "pharmacophore" refers to a compound, not a set of compounds having the defined property. Specifically, a "pharmacophore" is a compound possessing these properties.

[0041] Examples The following embodiments are provided to those skilled in the art to provide a complete disclosure and explanation of the manufacturing method and use of the present invention, and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to represent or imply that the following experiments are all or only experiments in which the invention was performed. Those skilled in the art will understand that many variations and / or modifications can be made to the invention as shown in particular embodiments without departing from the spirit or scope of the invention as broadly described. Accordingly, these embodiments should be considered in all respects to be illustrative and not limiting.

[0042] Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., quantity, temperature, etc.). However, a certain degree of experimental error and deviation must be taken into consideration. Unless otherwise specified, parts are measured in parts by weight, molecular weight is the weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0043] Example 1 Assay for discovering compounds that promote remyelination Introduction A major obstacle to advances in the treatment of multiple sclerosis (MS) is the inability to overcome the inhibitory lesion environment that contributes to the failure of remyelination and axonal loss during disease progression (1). The blood protein fibrinogen is a component of the inhibitory lesion environment, deposited in the CNS after vascular injury (1). Fibrinogen promotes inflammation, demyelination, and axonal injury in the CNS and blocks the differentiation of oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes (OLs) (2-5). Subsequently, perivascular OPC clusters may contribute to persistent blood-brain barrier (BBB) ​​disruption and fibrinogen accumulation in chronic MS lesions (6).

[0044] The discovery of drugs that overcome the fibrinogen-rich, inhibitory MS lesion environment would provide a much-needed alternative treatment for MS. While therapeutic depletion of fibrinogen can promote remyelination in demyelinating animal models, its clinical use may be limited due to the potential for hemorrhagic complications (2). Recently, compounds that promote OPC differentiation have been identified through comprehensive drug screening. However, it remains unclear whether these myelin-promoting compounds can overcome the inhibitory effect of fibrinogen on myelin repair in inflammatory demyelination (7-10).

[0045] This document describes the development of a novel medium-throughput fibrinogen-OPC inhibition assay. This assay has shown that none of the recently discovered myelin-promoting compounds can rescue the effects of fibrinogen on OPCs. Only BMP type I receptor inhibitors restore the differentiation of OPCs into mature OLs in the presence of fibrinogen. The data suggest that known myelin-promoting compounds may not be able to overcome exogenous inhibitors such as fibrinogen in the MS lesion environment, and that targeting fibrinogen-induced BMP signaling could be an alternative therapeutic approach to promote functional recovery in MS. The novel assay described herein can be used to screen for compounds that overcome the inhibition of OPC maturation and remyelination by fibrinogen.

[0046] Materials and Methods / Results / Discussion A. Development of a medium-throughput fibrinogen-OPC inhibition assay To miniaturize the assay into a 96-well plate format, we first optimized the OPC density, fibrinogen concentration, staining procedure, and the protocol used for image analysis (Figure 1'A). In the optimized protocol, primary rat O4+ OPCs are isolated by immunopanning. Approximately 5 × 10⁶ OPCs are obtained per 10 cm dish. 5 The cells were seeded and incubated for 3 days to allow the OPCs to grow. The cells were then subcultured using Accutase and placed in 96-well plates in 5 × 10⁶ layers. 3Cells were reseeded in wells and allowed to recover for one day before the experiment. To achieve the optimal difference between the control and fibrinogen conditions, fibrinogen had to be added at a concentration of at least 2.5 mg / mL. OPCs were differentiated for 3 days before fixation and subsequent staining. OPCs were stained with Hoechst dye (nucleus) and antibodies against MBP (oligodendrocyte) and GFAP (astrocytocyte). Images were acquired using an Arrayscan XTI instrument. To minimize well-to-well variability, 25 images covering approximately 80% of the well surface area were taken with a 10× objective lens. Images were analyzed using HCS Studio software. Total cell count was calculated based on the number of Hoechst + nuclei. To quantify the percentage of all cells positive for either MBP or GFAP, the ring was extended from the nuclear mask (Hoechst dye) to include the cell body. For MBP, a sufficiently large ring was extended around the nucleus. The percentage of MBP+ cells relative to the total number of cells was calculated based on the number of nuclei with green fluorescent cell bodies (MBP+, 488 nm), and the percentage of GFAP+ cells was calculated based on the number of nuclei with red cell bodies (GFAP+, 549 or 647 nm).

[0047] B. Development of a high-content fibrinogen-OPC inhibition assay in a 384-well format. We developed an automated 384-well high-content screening assay using assay parameters established in a 96-well assay. OPCs were obtained and cultured as described above. Various cell densities per well were tested to adapt the assay to the 384-well format. (1 × 10⁶) 3The optimal cell count was determined for each cell / well. Cells were manually seeded using a multichannel pipette. The following steps were performed using an Agilent BRAVO liquid handler. After 24 hours of incubation, growth medium was removed from each well and thoroughly washed with DPBS containing glucose and sodium pyruvate to ensure all growth factors were removed. After removing the DPBS solution, 50 μL of medium containing 2x concentration of differentiation factors + compounds was added from a pre-diluted polypropylene plate. After 1 hour of incubation, 50 μL of normal medium containing 2x concentration of fibrinogen was manually added using a multichannel pipette, and the solution was maintained on a heating platform. After 3 days of incubation, cells were stained according to the same protocol described for the 96-well plate format, but using a BioTekEL406 liquid handler at all steps of the protocol. Image acquisition and analysis were performed using a Thermo Scientific Arrayscan XTI as described above.

[0048] Consideration The number of OPCs available per isolation was scaled up, increasing the post-growth yield from 1.5 million cells per preparation to 5 million cells per preparation. Growth can be carried out in 10 cm plates for 3-4 days in OPC medium containing PDGF-AA and NT3. ACCUTASE® can be used to aid cell subculturing with incubation at 37°C for approximately 5 minutes. Re-seeding can be done in 96-well or 384-well plates. For example, 96-well plate = 5000 cells per well; 384-well plate = 1000 cells per well. For reproducible OPC differentiation, a period of time (e.g., approximately 3 days) in differentiation medium is required. To inhibit OPC differentiation to MBP+ cells and maximize OPC differentiation to GFAP+ cells, fibrinogen can be used at a concentration of at least approximately 2.5 mg / ml.

[0049] We developed workflows to test multiple compounds simultaneously on the same plate. For example, using multichannel administration of 2× compound followed by 2× fibrinogen reduces the time required to administer compounds and fibrinogen outside the incubator. Using Agilent BRAVO liquid handlers for programming and 384-well format minimizes cell loss during medium transfer. The BioTekEL406 liquid handler can be used for immunohistochemical staining procedures in 384-well format.

[0050] The target cells / cellular components / proteins can be labeled / stained with, for example, MBP (1:250) and GFAP (1:500) primary antibodies, secondary fluorescent antibody (1:500), and Hoechst nuclear dye (2 μg / mL), and used for automated detection. Automated image acquisition can be performed using Thermo Scientific Arrayscan XTI. For example, acquiring 25 images at 10x magnification maximizes the quantifiable area of ​​each well (covering approximately 80% of the well surface area of ​​a 96-well plate). A wide area is required because cells tend to be dense in some areas. Therefore, imaging the entire well more accurately captures the therapeutic effect and reduces inter-well variability in quantification. Staining and labeling of MBP+ cells, GFAP+ cells, etc., can be automated for quantification (quantification methods can be designed using HCS Studio software (Thermo Scientific)). As an example, to quantify the percentage of GFAP-positive total cells, the ring was expanded from the nuclear mask (Hoechst dye) to include the cell bodies. For MBP+ cells, the ring was expanded to include cellular processes beyond the cell body to ensure that only mature OL cells were included in the analysis. Cells were identified as positive by the software if the fluorescence intensity measured within the ring exceeded a set threshold relative to the fluorescence intensity generated by the secondary antibody-only control. Overall, this technique eliminates bias and reduces well-to-well and plate-to-plate variability in quantification.

[0051] Furthermore, all the steps described here improve the speed at which results are obtained. Manual image acquisition and quantification of a 96-well plate takes 1-2 weeks, but automated image acquisition and quantification can be done in one day.

[0052] References

[0053] [Table 1-1]

[0054] [Table 1-2]

[0055] Example 2 Blocking BMP receptors overcomes exogenous inhibition of remyelination and restores neurovascular homeostasis. Introduction CNS myelin regeneration fails in several neurological disorders, including multiple sclerosis, neonatal brain injury, and stroke (Franklin and Ffrench-Constant, 2017). Under these conditions, extracellular cues in the microenvironment inhibit remyelination by blocking the differentiation of pluripotent OPCs into mature myelin-producing oligodendrocytes (OLs) (Forbes and Gallo, 2017). A major barrier to advancement in the treatment of chronic demyelinating diseases such as multiple sclerosis is the inability to overcome this inhibitory lesion environment and halt disease progression (Reich et al., 2018). Small molecules that enhance the intrinsic pathways of OPC differentiation and remyelination have been identified through drug screening (Fancy et al., 2011; Deshmukh et al., 2013; Mei et al., 2014; Najm et al., 2015; Mei et al., 2016). However, these drugs failed to overcome disease-related exogenous inhibitors of OPC differentiation, such as chondroitin sulfate proteoglycans (CSPGs) and inflammatory cytokines, and were unable to promote OL differentiation in aged OPCs or OPCs in multiple sclerosis patients with an inflammatory environment (Keough et al., 2016; Neumann et al., 2019; Starost et al., 2020). It remains unclear whether myelination-promoting compounds can overcome the inhibitory microenvironment in areas with increased vascular permeability.

[0056] In multiple sclerosis, disruption of the blood-brain barrier (BBB) ​​allows the blood clotting factor fibrinogen to enter the central nervous system (CNS) (Petersen et al., 2018). Fibrinogen deposition is one of the earliest events in the development of multiple sclerosis, persisting in chronic demyelinating lesions but being minimal in remyelination lesions and absent in normal white matter (Vos et al., 2005; Petersen et al., 2017; Lee et al., 2018). In progressive multiple sclerosis, fibrinogen is detected in the cortex and cerebrospinal fluid and correlates with neuronal and cortical loss (Yates et al., 2017; Magliozzi et al., 2019). In demyelinating injury models, genetic or pharmacological depletion of fibrinogen promotes remyelination in the CNS and peripheral nervous system (Akassoglou et al., 2002; Petersen et al., 2017). Fibrinogen inhibits remyelination and neurogenesis, respectively, by activating BMP receptor signaling in OPCs and neural progenitor cells (Petersen et al., 2017; Pous et al., 2020). Fibrinogen induces a fate switch of NG2+ (encoded by CSPG-4) OPCs to astrocytes via BMP receptor activation (Petersen et al., 2017). This suggests a role for fibrinogen in the exogenous inhibition of remyelination by inducing OPC-induced astrocyte formation in the neurovascular niche. Furthermore, the conversion of fibrinogen to fibrin induces oxidative stress and pro-inflammatory polarization in microglia and macrophages (Ryu et al., 2015; Mendiola et al., 2020), which is toxic to OPCs and contributes to impaired remyelination (Back et al., 1998; Miron et al., 2013). This suggests a role for increased vascular permeability and fibrinogen deposition in maintaining the inhibitory microenvironment in chronic neurological diseases. However, the remodeling of the neurovascular niche at BBB disruption sites and its relationship to impaired remyelination remain not fully understood.

[0057] Here, it has been shown that the signaling pathway of OPCs that could not be overcome by known myelinization-promoting compounds such as clemastine is activated by exogenous inhibition of remyelination by fibrinogen. In contrast, inhibition of BMP signaling rescued the inhibitory effect of fibrinogen on remyelination by restoring the cell fate of OPCs to mature OLs with a therapeutic effect in the chronic EAE model. By integrating transcriptomics and in vivo two-photon (2P) imaging synchronized with electron microscopy in chronic neuroinflammatory lesions, it has been shown that OPCs accumulate at fibrinogen deposition sites with active BMP signaling and limited remyelination. Therefore, known myelinization-promoting compounds cannot overcome BMP receptor activation by fibrinogen and incomplete OPC differentiation, suggesting that BMP pathway inhibition may enhance the regenerative capacity of the myelinization-promoting progenitor niche at the cerebrovascular injury site.

[0058] Materials and Methods Animals C57BL / 6, NOD, B6.Cg-Tg(Cspg4-cre / Esr1 * )BAkik / J(NG2-CreER TM ) 1 , B6.Cg-Gt(ROSA)26 Sortm14(CAG-tdTomato)Hze / J(Rosa tdTomato ) 2 , and B6.129P-Cx3cr1 tm1Litt / J(CX3CR1 GFP ) 3 Mice were purchased from the Jackson Laboratory. Mice were housed in groups of five per cage under standard housing conditions and a 12-hour light-dark cycle. Sprague-Dawley female rats with littermates were purchased from Charles River, and P1 - P7 male and female rats were used for OPC isolation. All animal protocols were approved by the University of California, San Francisco Institutional Animal Care and Use Committee and followed the National Institutes of Health and ARRIVE guidelines.

[0059] EAE induction and clinical scoring Activated EAE was administered to NG2-CreER 9-10 week old fish. TM :Rosa tdTomato / + :Cx3cr1 GFP / + In female mice, 35-40 days after the last tamoxifen injection, 75 μg of MOG was administered in an incomplete Freund's adjuvant (Sigma-Aldrich) supplemented with 400 μg of thermo-inactivated Mycobacterium tuberculosis H37Ra (Difco Laboratories). 35-55 Emissions were induced by subcutaneous immunization with the peptide (MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 1); Auspep). On days 0 and 2 after immunization, mice were intraperitoneally injected with 200 ng of pertussis toxin (Sigma-Aldrich). In the chronic NOD EAE model, 10-12 week old NOD mice were immunized with 150 μg of MOG. 35-55 The patients were immunized with peptides, followed by the administration of 200 ng of pertussis toxin on days 0 and 2, as described. 4 .

[0060] For therapeutic treatment, mice were administered 6 mg / kg of LDN-212854 (Axon Medchem #2201) or saline twice daily (10-14 hour intervals) for 14 days at peak +2 days. Mice were randomly assigned to the treatment group, scored, and drug treatment was performed in a blinded manner. To avoid experimenter bias, the treatment assignment was left open at the end of the experiment. Mice that did not develop symptoms of EAE were excluded from treatment and analysis. Mouse body weight was measured and scored daily. Neurological impairment was assessed on a 5-point scale by observers who were not informed of the treatment: 0, no symptoms; 1, loss of tail tone; 2, ataxia; 3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; 5, mortality. A score > 2.5 was defined as the EAE peak.

[0061] Fluorescence-activated cell sorting of NG2 cells To select NG2 cells, spinal cord tissue was collected from perfused female mice as previously described. 5A single-cell suspension was prepared from the entire spinal cord using a modified method from the instructions provided by the manufacturer of the adult brain dissection (ABD) kit (Miltenyi Biotec). In short, finely chopped tissue was treated with 15 μM actinomycin D (ActD; Sigma). 6 The tissue was incubated individually with ABD Mix1 containing the specified substance at 34°C for 15 minutes, then ABD Mix2 was added to the solution at 34°C for 10 minutes. The tissue was gently pulverized and incubated at 34°C for 10 minutes. The homogenized tissue solution was passed through a 70 μm smart strainer (Miltenyi Biotec), washed with cold Dulbecco phosphate-buffered saline, and centrifuged at 4°C and 450 × g for 7 minutes. Tissue debris was removed according to the debris removal step of the ABD kit, passed through a 30 μm smart strainer (Miltenyi Biotec), and centrifuged at 450 × g and 4°C for 7 minutes. All of the above steps were performed in the presence of 3 μM ActD. The single-cell suspension was incubated with 1 μM Sytox blue live / dead stain (Thermo Fisher Scientific) at 4°C for 5 minutes, and then cell sorting was performed on FACSARIA II (BD Biosciences) with BD FACSDiva® v8 software. All cells were gated based on SSC-A and FSC-A size, and doublet identification was performed using FSC-H and FSC-W parameters. (Sytox blue) - NG2 tdtomato+Cells were directly sorted into tubes containing RLT Plus lysis buffer (Qiagen) supplemented with 1% 2-mercaptoethanol (Sigma) and 0.25% reagent DX (Qiagen). Cell lysates were frozen on dry ice and stored immediately at -80°C until use. To measure TFPI and MHC class II expression, single-cell suspensions of C57BL / 6 spinal cord tissue were prepared as described above without the addition of ActD. Cells were incubated on ice with Fc Block (BioLegend) for 15 minutes, followed by incubation on ice for 30 minutes with fluorescently conjugated Abs and anti-TFPI in FACS staining buffer (BD). Cells were then stained on ice with a fluorescently conjugated secondary antibody in PBS using the Aqua Live / Dead Staining Kit (Thermo Fisher Scientific) for 30 minutes. Samples were immediately run on LSRFortessa (BD Biosciences) using BD FACSDiva® v8 software. All FACS plots were generated in Flowjo. The following antibodies were used: APC / Cy7 anti-mouse CD11b (BioLegend, #101225, 1:200), PE anti-mouse CD3 (BioLegend, #100206, 1:200), PE / Cy7 anti-mouse PDGFRA (Invitrogen, #25-1401-82, 1:50), Alexa Fluor488 anti-mouse PDGFRB (Invitrogen, 53-1402-82, 1:50), BV650 anti-mouse MHCII (BD, #743873, 1:200), rabbit anti-mouse TFPI (Invitrogen, PA5-34578, 1:100), BV421 donkey anti-rabbit IgG (Biolegend, 406410, 1:200), and LIVE / DEAD™ fixable aqua dead cell staining kit (Invitrogen, L34957, 1:500).

[0062] Bulk RNA sequencing Frozen NG2 cell lysates in RLT buffer were thawed at 24°C and lysed using a QIA shredder (Qiagen) according to the manufacturer's instructions. Total RNA was isolated from the cell lysates using the RNAeasy microkit (Qiagen) as is. RNA quality and quantity were determined by Bioanalyzer pico-chip analysis (Agilent), and all samples with an RNA integrity number > 8 were used for RNA-seq library preparation. cDNA libraries were generated from total RNA using the Ovation RNA-seq System V2 (NuGEN). The libraries were quantified and quality-checked by KAPA qPCR (Roche) and Bioanalyzer DNA chip analysis (Agilent), respectively. The libraries were pooled, and paired-end 75-base pair reads were sequenced across 8 lanes using Nextseq500 (Illumnia). The sequencing depth per library exceeded 40 million reads. FASTQ files were generated in Biospace according to the manufacturer's guidelines (Illumina).

[0063] Analysis of bulk RNA sequencing For each sample, the FASTQ files of read 1 and read 2 were ligated separately, the Illumnia adapter was trimmed, and the quality of the FASTQ files was checked using FASTQC. Next, the sequencing reads were aligned to the mouse reference genome mm10 using STAR, and the count per gene was quantified by featureCounts as previously described. 5 The DEG was identified by EdgeR (version 3.24.3). 7 A cutoff for log2 scaling change greater than 1 or less than -1 was used, and the false positive rate (FDR) p-value was set to less than 0.05. Clustering analysis of DEGs was performed using R (version 3.5.0) and K-meansHOPACH (version 2.42.0), visualized using the pheatmap package (version 1.0.12), and volcano plots were generated using the ggplot2 package (version 3.2.1).

[0064] Functional enrichment and gene network analysis Functional enrichment analysis of HOPACH-clustered DEGs was performed in Metascape using default parameters. 8 Important gene ontology (GO) terms were identified by FDRp values ​​< 0.05. Gene network analysis was performed in GSEA using the GO molecular signature database biological process (C5.bp.v7.1symbols.gmt) with default settings, using RNA-seq normalized counts per million datasets. 9、10 The GO term with a p-value < 0.10 is Cytoscape (version 3.7.2). 11 It was used to visualize the enrichment map and fairly clustered using the default settings of the AutoAnnotate plugin (version 1.3.2).

[0065] In vivo multiphoton microscopy An Ultima IV 2P microscope (Prairie Technologies / Bruker) equipped with Mai Tai eHP DeepSee and InsightX3 Ti sapphire femtosecond lasers (pulse width <120 fs, tuning range 690–1040 nm (Mai Tai) and 680–1300 nm (InsightX3), repetition rate 80 MHz, Spectra-Physics / Newport) was used. The laser was tuned to excitation wavelengths of 910–1150 nm depending on the fluorescent dye. Imaging was performed approximately 80–120 μm below the dura mater using either an Olympus 25×1.05NA with 1.6 zoom or a Nikon 10×0.4NA immersion lens with a z-step of either 1.0–1.5 μm or 3–4 μm, at 40× or 10× magnification, respectively. Throughout all imaging experiments, the maximum laser output emitted from the objective lens was less than 40 mW. An IR blocking filter and 560 nm dichroic light were placed in the primary emission beam path before the undiscanned detector. 660 nm dichroic light and 692 / 24 nm + 607 / 45 nm bandpass filters were used to separate the fluorescence emissions of Mito Tracker Red / far-infrared and tdTomato / rhodamine, respectively. 520 nm dichroic light and 542 / 27 nm + 494 / 41 nm bandpass filters were used to separate the fluorescence emissions of YFP and GFP, respectively.

[0066] In vivo spinal cord imaging In vivo spinal cord imaging was performed as previously described. 12In short, the spinal cord was exposed to the desired level (T11) by a single laminectomy, and the mice were placed on a spinal stabilization device. Using Flow-It® ALC (Pentron), a well was created around the exposed spinal cord, and a drop of pre-warmed (37°C) artificial cerebrospinal fluid (ACSF, HEPES-based, in mM units: 125 NaCl, 10 glucose, 10 HEPES, 3.1 CaCl2, 2.7 KCl, and 1.3 MgCl2; pH 7.4) was applied. The dura mater was then gently washed with pre-warmed ACSF to wash away and remove any potential dural hemorrhage. Mice that sustained accidental injuries during laminectomy, or showed signs of subdural hemorrhage, were excluded from the study because these events could trigger inflammatory or other neurodegenerative responses unrelated to the experimental design. The vascular system was labeled by injecting 100 μl of a 3% 70 kDa Oregon green-conjugated dextran (Thermo Fisher Scientific) solution in ACSF posteriorly into the orbit, and then the mice were placed under a 2P imaging microscope. For in vivo myelin imaging, the meninges (dura and arachnoid membranes) were carefully removed using a subcutaneous needle, and the exposed spinal cord beneath was immersed for 30 minutes in MitoTracker Deep Red (Thermo Fisher Scientific) dissolved at a concentration of 8 μM in ACSF. 13 Next, before the imaging session, the spinal cord was carefully washed 4-5 times with pre-warmed ACSF.

[0067] Processing of in vivo image data To generate images for the figures, the Z-stack was intensity-projected along the Z-axis using the ImageJ (NIH) summation projection algorithm to recreate a 2D representation of the imaged volume. Image brightness / contrast, background noise, and sharpness were adjusted using background subtraction, outlier removal, and unsharp mask algorithms in ImageJ. The GFP and YFP signals were separated using the ImageJ spectral separation algorithm and then pseudocolorized.

[0068] Quantification of cell clusters NG2-CreER TM :Rosa tdTomato / + :Cx3cr1 GFP / + Z-stacks of images from healthy control mice or EAE challenge mice were Z-projected and automatically thresholded to account for differences in signal intensity between experiments (ImageJ's default algorithm). NG2 and microglia clusters were defined as regions where four or more cell bodies were in contact with each other and the cell density was at least twice as high as that of a healthy spinal cord. The number of clusters and the distance to the nearest blood vessel were measured using ImageJ.

[0069] Myelin's circularity Myelin damage was quantified by the circularity of the myelin. A value of 1.0 indicates a perfect circle (as seen in the longitudinal section of degenerated myelin), while a value closer to 0.0 indicates a non-circular, linear shape (as seen in the longitudinal section of normal myelinated fibers).

[0070] Electron microscopy Tissue preparation for SBEM. To elucidate the vascular system visualized with tdTomato+NG2 lineage cells, microglia, and dextran, NG2-CreER TM :Rosa tdTomato / + :Cx3cr1 GFP / + In vivo 2P imaging of mice was performed under chronic EAE. After the imaging session, the animals were perfused with Ringer's solution, followed by perfusion with 0.5% glutaraldehyde / 2% PFA in cacodylic acid. The spinal cord region was excised from the perfused spinal cord under the imaging window and fixed with 0.5% glutaraldehyde / 2% PFA in cold cacodylic acid for 2 hours. The specimen was then fixed overnight with 4% PFA in cold cacodylic acid. The dorsal side of the spinal cord was cut into 150 μm thick horizontal vibratome sections. The sections were fixed overnight with 2% glutaraldehyde in cold cacodylic acid. The sections were stained as previously described. 14In short, the tissue was stained with 2% osmium tetroxide (Ted Pella), 0.5% thiocarbohydrazide (Electron Microscopy Sciences) aqueous solution, 2% osmium tetroxide aqueous solution, 2% uranyl acetate (Ted Pella) aqueous solution, and lead aspartate in 0.15 M cacodylic acid. 15 The sections were thoroughly washed with water between each staining solution. The sections were then dehydrated through ethanol and acetone, and subsequently impregnated with Durcupan ACM (Millipore Sigma). The sections were embedded flat between glass slides coated with a release agent (Electron Microscopy Sciences, Hatfield, Pennsylvania) and cured at 60°C for 72 hours.

[0071] ROI targeting using X-ray microscopy and SBEM. The specimen was imaged with XRM to find and orient ROIs for SBEM imaging. 16The specimen was scanned with a Zeiss Versa 510. The first scan of the entire vibratome section was acquired with a 0.4× objective lens, 80kV, and a pixel size of approximately 5μm. After comparing the vascular system observed by XRM with the two-photon volume, the ROI was identified and cut out using a razor blade. The specimen was bonded to ACLAR (Ted Pella), which itself was bonded to a dummy block using cyanoacrylate adhesive. The ventral side of the vibratome section was positioned upwards. Using the XRM volume as guidance, the specimen was approached using a glass blade on a Leica EM UC6 ultramicrotome, ensuring that the cutting plane was parallel to the desired final cutting plane in the SBEM. After removing excess epoxy and exposing the tissue, the specimen was removed from the dummy block and mounted on an A3 SBEM specimen pin (RMC Boeckler) using conductive silver epoxy (Ted Pella), this time dorsal side upwards. The A3 pin was placed in an A3 specimen holder and scanned with XRM using a 4× objective lens at 80kV and a pixel size of approximately 1.5μm. This XRM volume was used to precisely adjust the tilt of the specimen block, remove excess resin from the back of the block, and identify the ROI location for SBEM imaging.

[0072] SBEM imaging. Specimens were imaged using a Zeiss Gemini 300 VP SEM equipped with a focal charge compensation system and a Gatan 2XP 3View system. Volumes were acquired at 2.5kV with a residence time of 1 μs, 10 nm pixels, a 50 nm step size, and focus gas injection with nitrogen gas enabled. The scope was run in analytical mode and high current mode. The resulting image stacks were symmetrical using a custom Python script with the IMOD program. 17 .

[0073] OPC-X Screening First generation Rat O4 +OPCs were isolated by sequential immunopanning of papain-dissociated cortical cell suspension in three dishes: RAN-2 (negative selection), O1 (negative selection), and O4 (positive selection), as previously described. 18 O4+OPC, 5 x 10 per plate. 5 Cells were seeded at an initial density onto 10 cm culture plates coated with polyethyleneimine (PEI, Sigma-Aldrich) and grown in growth medium for 3 days in a 5% CO2 incubator at 37°C. The cells were then subcultured using Accutase and transferred to PEI-coated μClear® 96-well plates (Greiner Bio-One) at a rate of 5 × 10⁶ cells per well. 3 Cells were re-seedled. Cells were incubated in growth medium for 1 day before experimental treatment in differentiation medium. Chemically defined base media included DMEM (4.5 g / L glucose, + pyruvate, + glutamine; Thermo Fisher Scientific), 1 × B27 (Thermo Fisher Scientific), 1 × N2 (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific), and 50 ng ml. -1 The culture medium was NT3 (Peprotech). The growth medium was 20 ng ml. -1 The differentiation medium consisted of a basic medium supplemented with PDGF-AA (Peprotech). -1 CNTF (Peprotech) and 40 ng ml -1 The medium consisted of a basal medium without PDGF-AA, supplemented with triiodothyronine (T3, Sigma-Aldrich). A "slow" differentiation medium (basal medium without NT3, additional growth factors, or T3) was used in a clemastine dose-response study to replicate previously reported conditions. 19 .

[0074] To mimic an inhibitory lesion environment, fibrinogen (Millipore Sigma) is used, and 1.5 mg ml is used for screening myelin-promoting compounds. -1For all other in vitro studies, use 2.5 mg ml at this concentration. -1 These were added to the differentiation medium at the concentrations listed above. These are physiological plasma concentrations known to inhibit OPC differentiation into mature OL. 18 The myelin-promoting compound was dissolved in DMSO and added to four wells at concentrations previously shown to promote OPC differentiation to OL, one hour before fibrinogen treatment. The final compound concentration was 1 μM benztropin. 19 , clemastine 1 μM 19 Quetiapine 1 μM 19 Miconazole 1 μM 20 Clobetasol 5 μM 20 (±)U-50488 1μM 21 , and XAV-939 0.1μM 22 It was DMH1 (1 μM). 18 LDN-212854 served as a positive control in all assay plates. Cells were exposed to DMSO concentrations up to 0.1%, and the control contained the same concentration of DMSO. All conditions were tested in four wells and repeated in three independent experiments for N=3 biological replication. For dose-response curves, LDN-212854 and clemastine were added to four wells in 3-fold serial dilutions (5 μM to 2 nM) one hour before fibrinogen treatment. Dose-response experiments were repeated in two or three independent experiments. After differentiating cells for three days, fixation, staining, and quantification were performed. To test combinations of BMP receptor inhibitors and other myelination-promoting compounds, LDN-212854 (0.1 μM) and clemastine (0.5 μM) were added individually or together to four wells one hour before fibrinogen treatment in three independent experiments. After differentiating cells for two days, fixation, staining, and quantification were performed.

[0075] OPCs were fixed with 4% paraformaldehyde, blocked and permeabilized with 5% normal goat serum / 0.1% Triton®-X100, and stained with 2 μg / mL Hoechst nuclear dye (Thermo Fisher Scientific), anti-MBP antibody (Abeam ab92406 or Abeam ab7349), and anti-GFAP antibody (Cell Signaling #12389), followed by goat secondary antibody (Thermo Scientific). Images were acquired using an ArrayscanXTI instrument (Thermo Scientific) with a 10× objective lens, 386 / 23 filter for Hoechst dye detection, 485 / 20 filter for MBP / Alexafluor-488 detection, and 549 / 18 filter for GFAP / Alexafluor-546 fluorescence detection. To reduce inter-well variability, 25 images covering approximately 80% of the well surface area were taken. Images were analyzed using HCS Studio software (Thermo Scientific). The total number of cells was measured using Hoechst. + Calculated based on the number of nuclei. To quantify the percentage of all cells positive for either MBP or GFAP, the cell body (GFAP) was used. + The ring was expanded from the nuclear mask (Hoechst dye) to include the cells. For MBP+ cells, the ring was expanded to include the OL processes beyond the cell body to ensure that only mature OLs were included in the analysis. Using HCS Studio software, MBP + and GFAP + The percentage of cells, MBP per total number of cells + and GFAP + The calculation was based on the number of cells. If the fluorescence intensity measured within the ring exceeded a set threshold relative to the fluorescence intensity generated by a control with only the secondary antibody, the cells were identified as positive by the software.

[0076] immunohistochemistry Mice were perfused transcardially with 4% PFA under deep abeltin or ketamine / xylazine anesthesia. Tissue was removed, fixed overnight with 4% PFA, cryoprotected with 30% sucrose / PBS, frozen in Neg-50 medium (Thermo Scientific), and frozen-sectioned into 10–12 μm sections, which were placed on tissue tack microscope slides (Polysciences, Inc.). Sections were permeabilized in 0.1–0.3% Triton® X-100, blocked with 5% BSA or 5% normal donkey serum, incubated overnight with primary antibody at 4°C, and then incubated with fluorescent secondary antibody at room temperature for 1–2 hours. Slides were covered with coverslips containing DAPI (Thermo Fisher Scientific) and Prolong Gold or SlowFade Gold anti-fading agents.

[0077] The following primary antibodies were used: fibrinogen (mouse IHC: 1:1000, rabbit polyclonal, J. Degen, donated from Cincinnati); GFAP (1:200, rat monoclonal, #13-0300, Thermo Fisher Scientific); GST-pi (1:200, rabbit polyclonal, #312, MBL International), ID2 (1:2000, rabbit monoclonal, #M213, CalBioreagents); MBP (1:500, #ab7349, Abeam), OLIG-2 (1:200, rabbit polyclonal, #ab9610, EMD Millipore), PDGFRβ (1:100, goat polyclonal, #AF1042, R&D Systems), PLVAP (1:100, rat monoclonal, #553849, BD) Pharmingen), VCAM-1 (1:50, rat monoclonal, #550547, BD Pharmingen).

[0078] Images were acquired using an Axioplan II epifluorescence microscope (Carl Zeiss) equipped with a dry Plan-Neofluar objective lens (10×0.3NA, 20×0.5NA, or 40×0.75NA), an Axiocam HRc CCD camera, and Axiovision image analysis software; a BIOREVO BZ-9000 inverted fluorescence microscope (Keyence) with a Nikon CFI 60 series infinite optical system and Keyence imaging software; or an Olympus Fluoview confocal microscope with a 20×NA1.0 objective lens. All images were processed and analyzed in ImageJ. Depending on the staining, quantification was performed on thresholded binary images or cell counts by researchers who were not informed of the mouse treatment group.

[0079] Imbubrot Cells or tissues were dissolved in RIPA lysis buffer (Thermo Fisher Scientific) supplemented with a protease / phosphatase inhibitor cocktail (Calbiochem), and the lysate was removed by centrifugation at 13,000 × g for 15 minutes at 4°C. Equal volumes of protein were loaded onto 4%–12% Bistrice gels (Thermo Fisher Scientific) and analyzed by Western blotting. Bands were visualized with HRP-conjugated secondary antibody (Cell Signaling Technology). Concentration measurements were performed using ImageJ software (NIH), and the values ​​of each band were normalized against a GAPDH loading control from the same membrane. The primary antibodies were as follows: Id2 (1:1000, rabbit monoclonal, #M213, CalBioreagents); phospho-Smadl / 5 (1:1000, rabbit monoclonal, #9516, Cell Signaling Technology); GAPDH (1:1000, rabbit monoclonal, #2118, Cell Signaling Technology).

[0080] statistical analysis Statistical analysis was performed using GraphPad Prism (version 8). Data are presented as mean ± standard error. No statistical methods were used to pre-determine sample size, but the sample size is similar to that previously reported. Statistical significance was determined using Student's t-test (two-tailed, unpaired), Mann-Whitney test (two-tailed), or one-way or two-way analysis of variance (ANOVA) followed by Dunnett or Tukey's post-hoc test for multiple comparisons, as indicated in the figure captions. A p-value ≤ 0.05 was considered significant. Mice with similar EAE scores (score difference ≤ 0.5) were randomly assigned to experimental groups, and animals from each treatment group were placed in separate cages to minimize confounding factors. EAE clinical scoring, histopathological analysis, and quantification were performed blinded. To compare EAE clinical scores, the statistical significance of the daily mean clinical score change in the EAE experiments was estimated using permutation tests. 23 The corresponding p-values ​​were estimated using 1000 permutations. In each permutation, the mice were randomly rearranged. In the NOD-EAE model, the mean of the maximum score from the last 20 days of treatment was compared between each group using Welch's two-sample t-test.

[0081] References

[0082] [Table 2-1]

[0083] [Table 2-2]

[0084] result NG2 cells undergo limited remyelination in chronic neuroinflammation and cluster around blood vessels at fibrinogen deposition sites.

[0085] NG2 cells, also known as OPCs, are progenitor cells of the adult CNS closely associated with the vascular system and possess a unique ability to promote remyelination (Dimou and Gallo, 2015). To study NG2 cells and neurovascular dysfunction in neuroinflammation, NG2-CreER TM :Rosa tdTomato / + :Cx3cr1 GFP / + A mouse was created. The epitopes of amino acids 35-55 of myelin oligodendrocyte glycoprotein (MOG) ("MOG 35-55 In vivo 2P imaging and transcriptome profiling of NG2 cells and microglia in chronic experimental autoimmune encephalomyelitis (EAE) induced by EAE were performed (Supplementary Figure 1). Extravascular leakage of 70 kilodaltons of Oregon green dextran was used as a marker for acute BBB leakage, and fibrinogen immunohistochemistry was used as a marker for chronic BBB leakage and local coagulation. At the peak of EAE, perivascular clusters consisted mainly of microglia, and NG2 cells were uniformly distributed within the spinal cord parenchyma (Figure 1A, Supplementary Figure 2A). However, in chronic EAE, perivascular clusters also consisted of NG2 cells, and more than 80% of NG2 cell clusters were located in or within 30 μm of blood vessels (Figure 1A, Supplementary Figure 2B). NG2 cells within clusters tdTomato+ The cells have a glial-like morphology characterized by multiple branching processes within the spinal cord parenchyma, and NG2 has elongated processes along the blood vessel walls. tdTomato+They were distinguishable from pericytes (Supplementary Figure 2C). VCAM1 (Lengfeld et al., 2017), a marker of endothelial activation, and PLVAP (Niu et al., 2019), a marker of fenestrated endothelial cells in leaking CNS vessels, were increased in the peak and chronic EAE white matter (Supplementary Figures 3A, B). This suggests disruption of neurovascular homeostasis. Fibrinogen deposition is a prominent feature of neurovascular pathology in EAE and is necessary for disease development (Adams et al., 2007; Davalos et al., 2012; Ryu et al., 2018). Acute dextran leakage was highest at the peak of EAE, but fibrinogen deposition increased over time and was highest during chronic EAE (Figure 1B). This suggests persistent fibrinogen deposition even when active BBB disruption decreases. In chronic EAE, NG2 clusters aggregated around blood vessels only at fibrinogen deposition sites (Figure 1C, Supplementary Figure 4A), and in many cases co-localized with microglia clusters (Figure 1A, Supplementary Figure 2A). These results suggest dynamic glial modeling of the neurovascular interface at fibrinogen deposition sites during neuroinflammation.

[0086] To evaluate myelin within perivascular NG2 clusters using in vivo 2P imaging, we applied Mito Tracker Deep Red (Romanelli et al., 2013), a mitochondrial dye that also labels myelin when used at high concentrations. Significant myelin disruption, characterized by blister formation in the myelin sheath, was present near NG2 clusters, while normal-looking myelin sheaths appeared in areas without clusters (Figure 1D, Supplementary Figure 4B). To study the ultrastructure of myelin, a co-registration technique was developed using microcomputed tomography to correlate 2P-imaged volumes with 3D serial block-face electron microscopy (SBEM) (Figure 1E). Using this technique, SBEM images were collected from the exact same regions of perivascular NG2 clusters in EAE mice imaged with in vivo 2P microscopy. Endothelial activation, leukocyte adhesion on the endothelial surface, perivascular astrogliosis, and inflammation were observed in inflamed veins with macrophages partially containing debris (Figure 1Fi, Gi, Supplementary Figure 3C). Two distinct patterns were found in parenchymal lesions. The first was characterized by cellular infiltration of elongated cells with low cell density, some of which contained osmium affinity degradation products. In these regions, most axons were demyelinate, and remyelination was sparse (Figure 1Fii, Gi). In other regions, there were denser clusters of small cells with small perinuclear cytoplasmic margins that contained some mitochondria but few other organelles reminiscent of NG2 cells (Figure 1Gii). Remyelinated axons were closely adjacent to these cell clusters, but in regions away from the clusters, the axons were demyelinate (Figure 1Fiii, Gii). Away from perivascular NG2 cells, seemingly normal perivascular CNS tissue, astrocyte glial boundary membranes, and axons with normal myelin thickness were observed (Figure 1Fiv). These results suggest that perivascular NG2 clusters are associated with inflammation, gliosis, apparent demyelination, and limited remyelination. Transcriptome profiling of NG2 cells in EAEs reveals vascular homeostasis and suppression of anticoagulant pathways.

[0087] To study transcriptome changes in NG2 cells in chronic EAE, MOG 35-55 NG2 collected from the spinal cord of EAE mice or healthy controls. tdTomato+ RNA-seq was performed on cells (Supplementary Figure 3A). Compared to the control, a total of 1,241 differentially expressed genes (DEGs) (FDR < 0.05; ±1 log2 change) were identified in the chronic EAE setting, of which 738 (60%) were downregulated and 503 (40%) were upregulated (Figure 2A). Unsupervised gene clustering analysis identified nine distinct gene clusters (Figure 2B). Gene ontology (GO) analysis revealed that chronic EAE activates inflammatory and antigen-presenting genes in clusters 1-4, which include GO pathway terms such as "positive regulation of acute inflammatory response," "positive regulation of T cell-mediated cytotoxicity," "antigen processing and presentation," and "cellular response to interferon beta" (Figure 2B). Standard antigen-presenting genes such as Cd74, H2-dma, and B2m were significantly upregulated in EAE (Figure 2B). This is consistent with reports suggesting immune-like functions of OL lineage cells in disease (Falcao et al., 2018; Kirby et al., 2019). Interestingly, GO analysis of downregulated gene clusters 5-9 revealed pathways related to vascular and BBB homeostasis, including angiogenesis, regulation of Wnt signaling pathways, vascular formation, vascular development, and cell junction organization (Figure 2B). Accordingly, gene networks involved in vascular maintenance, wound healing and coagulation, and tight junctions were generally suppressed in EAE (Figure 2C). Gene set enrichment analysis (GSEA) of DEG identified the top two downregulated gene sets as "regulation of cell junction assembly" (normalized enrichment score (NES) 1.7, p<0.01) and "negative regulation of coagulation" (NES 1.7, p<0.01) (Figure 2D). The expression of tissue factor pathway inhibitors (Tfpi), major inhibitors of blood coagulation and fibrin formation (Wood et al., 2014), was significantly reduced in NG2 cells of EAE.tdTomato+ Since the population includes OPC and pericyte lineages, we are MOG 35-55 -PDGFRα from the spinal cord of EAE mice or healthy controls + OPC and PDGFRβ + Pericytes were isolated (Supplementary Figure 3B), and major histocompatibility complex class II (MHCII) and TFPI were labeled on the cell surface to evaluate antigen presentation and anticoagulation pathways, respectively. Consistent with our bulk RNA-seq and previous studies (Kirby et al., 2019), PDGFRα of EAEs was identified. + MHCII levels were increased in OPCs (Supplementary Figure 3C). TFPI was expressed by OPCs but not in pericytes in healthy controls, and was significantly suppressed in EAEs (Figures 2E, F). Overall, these results highlight dysregulation of antigen presentation, coagulation, and vascular homeostasis pathways in OPCs in chronic neuroinflammation.

[0088] Myelinization-promoting compounds do not overcome the exogenous inhibition of fibrinogen in OPC differentiation. OPCs can differentiate into myelinating OLs or astrocyte-like cells in response to exogenous signals such as fibrinogen and BMPs found in multiple sclerosis lesions (Mabie et al., 1997; Petersen et al., 2017; Hackett et al., 2018). We investigated the maturation of OPCs in the presence of exogenous inhibitors. + Promote differentiation into OL, GFAP + To identify compounds that reduce the OPC fate switch to astrocytes, we developed the OPC-X screening, a medium-throughput, high-content imaging assay (Figure 3A). In the OPC-X assay, fibrinogen compared to the control showed a decrease in MBP + Reduce mature OL, GFAP +The number of astrocyte-like cells was increased by approximately 60% (Figures 3B-D). Seven compounds, namely benztropine, clemastine, quetiapine, miconazole, clobetasol, (±)U-50488, and XAV-939, have been previously shown to promote the endogenous pathway of OPC differentiation (Fancy et al., 2011; Mei et al., 2014; Najm et al., 2015; Mei et al., 2016). However, these myelination-promoting compounds did not overcome the exogenous inhibition of OPC differentiation by fibrinogen (Figures 3B-D). In contrast, the BMP receptor inhibitor DMH1 (Hao et al., 2010) rescued the inhibitory effect of fibrinogen and restored OPC differentiation to mature OL to control levels (Figures 3B-D). Fibrinogen-mediated OPC to GFAP + Cell fate switching in cells was also deactivated by DMH1 (Figure 3D). Clemastine, a muscarinic receptor antagonist, promotes the remyelination ability of OPCs and is currently in clinical trials for multiple sclerosis (Mei et al., 2014; Green et al., 2017). As expected, clemastine was effective in controlling MBP + While it increased the number of cells, it did not promote the differentiation of OPCs into mature OLs in the presence of fibrinogen (Supplementary Figure 4). Clemastin did not block the phosphorylation of the BMP signaling transducer SMAD1 / 5 or the expression of the BMP target protein ID2, which are induced by fibrinogen (Figure 3E). In contrast, DMH1 blocked the phosphorylation of SMAD1 / 5 and the expression of ID2, which are induced by fibrinogen (Figure 3E). Therefore, compounds identified to date that promote OPC differentiation may not be able to overcome exogenous inhibitory signaling pathways at the site of vascular injury.

[0089] Therapeutic effects of type I BMP receptor inhibition in neuroinflammation BMP expression and downstream receptor signaling are increased in human multiple sclerosis lesions (Costa et al., 2019; Harnisch et al., 2019). The BMP target protein ID2 is also increased in lesions with extensive fibrinogen deposition (Petersen et al., 2017). The finding that DMH1 effectively blocked fibrinogen-induced BMP receptor activation and restored OPC differentiation in vitro (Figure 3) suggested that targeting BMP signaling may promote the repair of neuroinflammation. However, because DMH1 is not water-soluble, its use in vivo is limited. Therefore, we tested LDN-212854, which has a molecular structure similar to DMH1 and is a type I BMP receptor inhibitor biased towards the water-soluble activin A receptor type I (ACVR1) (Mohedas et al., 2013), in the OPC-X screening. LDN-212854 restored mature OL differentiation and dose-dependently blocked the formation of GFAP+ astrocytes from fibrinogen-treated OPCs (Figure 3F, G).

[0090] To determine the therapeutic potential of LDN-212854, two models of EAE: NG2-CreER TM :Rosa tdTomato / + mice-induced chronic MOG 35-55 EAE and progressive EAE induced in non-obese diabetic (NOD) mice by the epitope of amino acids 35-55 of MOG ( "NOD-MOG 35-55 EAE") (Mayo et al., 2014) were selected. Therapeutic administration of LDN-212854 significantly improved the clinical score (Figure 4A-D), and fibrinogen deposition and demyelination were reduced in both models (Figure 4A-D). In addition, LDN-212854 significantly reduced perivascular NG2 clusters and myelin damage in MOG 35-55 EAE, as revealed by in vivo 2P imaging (Figure 4E, F). Furthermore, LDN-212854 reduced ID2 expression in NG2 cells in the EAE white matter (Figure 4G). This is consistent with the inhibition of BMP signaling in the NG2 cell lineage.

[0091] Since the important mechanisms of fibrinogen and BMP receptor signaling are the cell fate switch from OPCs to astrocytes (Mabie et al., 1997; Petersen et al., 2017), we tested whether LDN-212854 promotes OPC differentiation into myelinated cells in MOG35-55 EAE. To track the cell fate of OPCs in vivo, NG2-CreER TM :Rosa tdTomato / + mice were induced with EAE, enabling tamoxifen-induced expression of tdTomato in NG2 + OPCs and their progeny (Petersen et al., 2017; Hackett et al., 2018). Glutathione s-transferase-pi (GST-pi) labels mature OLs, and GFAP labels gene-labeled tdTomato + NG2 + astrocytes derived from OPCs. Therapeutic administration of LDN-212854 increased the proportion of NG2 + OPCs that differentiated into mature OLs compared to controls, and the formation of GFAP tdTomato+ astrocytes derived from NG2-CreER TM :Rosa tdTomato / + MOG 35-55 in EAE mice disappeared (Figure 4H). Taken together, these results suggest that inhibition of type I BMP receptors restores the cell fate of OPCs to mature OLs and has therapeutic potential for neuroinflammatory diseases involving fibrinogen deposition and active BMP signaling. + <00,00536>

[0092] Discussion The data provided herein reveal the dynamic cellular remodeling of the neurovascular niche at BBB dysfunction sites in neuroinflammation and identify pathways that could serve as drug discovery targets to promote myelin repair. Perhaps in neuroinflammation, perivascular NG2 + ​OPC clusters contribute to a procoagulation environment, leading to excessive fibrinogen deposition, activation of BMP receptor signaling in OPCs, and exogenous inhibition of remyelination at the site of vascular injury. This model is consistent with chronic demyelinating multiple sclerosis lesions with fibrinogen deposition, impaired fibrinolysis, BMP pathway activation, and gliosis, where perivascular OPC clusters are localized at the boundary of active lesions (Petersen et al., 2017; Yates et al., 2017; Lee et al., 2018; Niu et al., 2019). Through OPC-X screening, we found that the therapeutic capacity of many myelination-promoting agents may be limited to the sites of vascular injury and fibrinogen deposition, highlighting an unmet clinical need for therapeutic strategies to overcome exogenous inhibition in diseases with chronic demyelination. This specification provides the concept that inhibiting the activation of the BMP pathway can promote myelin repair by overcoming the failure of OPC differentiation in neurovascular dysfunction sites. Therefore, BMP inhibitors can expand the toolbox of myelinization-promoting agents and provide additional treatment options for patients suffering from BBB disruption and white matter pathology.

[0093] Using in vivo 2P imaging, we discovered significant changes in microglia and perivascular glial cell composition associated with demyelination at the peak of disease, followed by the formation of perivascular NG2 clusters with restricted remyelination in chronic neuroinflammation. The clustering of NG2 cells at fibrinogen deposition sites suggests that OPC migration or adhesion may be altered at vascular injury sites, or that OPCs themselves may contribute to BBB disruption or local coagulation. This study suggests a previously unknown function of OPCs in the expression of genes regulating coagulation. TFPI (Wood et al., 2014), a potent inhibitor of coagulation factor X and tissue factor-mediated coagulation, was expressed in OPCs and suppressed by chronic neuroinflammation. Interestingly, hemostatic biomarkers, including TFPI, are altered in multiple sclerosis patients (Ziliotto et al., 2019), suggesting an imbalance between anticoagulant and procoagulant pathways in neuroinflammatory diseases. Pro-oxidative microglia may also contribute to a pro-coagulation environment in the lesion microenvironment through the expression of coagulation proteins such as coagulation factor X (Mendiola et al., 2020). Therefore, transcriptional changes at the neurovascular interface may establish a local pro-coagulation environment that contributes to the excess or persistent deposition of fibrin observed in many neurological diseases (Petersen et al., 2018). Therapeutic strategies targeting the NG2 cell-vascular-fibrinogen axis or downstream fibrinogen signaling may provide therapeutic means to overcome exogenous inhibition in the neuroinflammatory lesion environment.

[0094] This study suggests that myelination-promoting agents suppress signaling pathways activated by exogenous inhibitors in the pathological environment in different ways. Indeed, clemastine neither inhibited SMAD1 / 5 phosphorylation, a key downstream pathway of BMP receptor activation, nor rescued the OPC cell fate switch to astrocytes. Fibrinogen, in addition to activating BMP receptor signaling in OPCs, stimulates CSPG production from astrocytes and is a carrier of transforming growth factor beta (TGF-β) (Schachtrup et al., 2010). CSPG partially inhibits remyelination in OPCs through activation of the protein tyrosine phosphatase sigma receptor (Pendleton et al., 2013). Age-related loss of OPC function may occur in response to TGF-β signaling or increased rigidity of the OPC niche, followed by signaling via the mechanoresponsive ion channel Piezol (Baror et al., 2019; Segel et al., 2019). Therefore, assays that better replicate the inhibitory lesion environment and downstream signaling are needed to improve the selection of agents that increase remyelination in inflammatory lesions involving gliosis, vascular injury, and BBB disruption. Furthermore, the selection of myelination-promoting agents in clinical practice may need to consider their efficacy in the exogenous inhibitory environment of patients with demyelinating neurological diseases. Targeting multiple inhibitory pathways with drug combinations may result in additive or synergistic effects on remyelination and may provide a means to maximize the therapeutic effect of myelination-promoting compounds in the inhibitory lesion environment.

[0095] Therapeutic fibrinogen depletion with anticoagulants can suppress neuroinflammation and promote myelin regeneration (Akassoglou et al., 2002; Petersen et al., 2017), but the clinical utility of this approach may be limited by hemorrhagic complications. This study identified LDN-212854, an ACVR1-biased BMP receptor inhibitor, as a potential therapeutic agent for chronic neuroinflammation. Activation of fibrinogen and BMP signaling in damaged perivascular niches leads OPC cell fate to astrocytes rather than OL remyelination (Petersen et al., 2017; Baror et al., 2019), which may contribute to pathological gliosis at vascular injury sites. LDN-212854 increased myelinating OLs and eliminated OPC differentiation to astrocytes. LDN-212854 was well-tolerated at the dose used in the study, but human toxicity data are limited. The clinical use of ACVR1-selective BMP inhibitors has recently attracted attention in the treatment of fibrodysplasia ossificans progressive (CBD), a rare disease characterized by excessive BMP signaling leading to ectopic ossification and myelin abnormalities (Kan et al., 2012). LDN-212854 and other safe ACVR1-selective inhibitors may be treatment options for neurological disorders involving BBB disruption and myelin abnormalities, including multiple sclerosis, Alzheimer's disease, neonatal brain injury, and traumatic brain injury.

[0096] References

[0097] [Table 3-1]

[0098] [Table 3-2]

[0099] [Table 3-3]

[0100] [Table 3-4]

[0101] [Table 3-5]

[0102] All publications, nucleotide and amino acid sequences identified by accession numbers, patents and patent applications are incorporated herein by reference. While the above details describe the present invention in relation to its specific embodiments and include many details for illustrative purposes, it will be apparent to those skilled in the art that additional embodiments are possible and some of the details described herein can be substantially modified without departing from the basic principles of the invention.

[0103] The specific methods and compositions described herein are representative and illustrative embodiments and do not limit the scope of the invention. Other purposes, aspects, and embodiments will be conceivable to those skilled in the art by examining this specification and are included within the spirit of the invention as defined by the claims. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described exemplary herein can be adequately implemented without any elements or limitations not specifically disclosed herein as essential. The methods and processes described exemplary herein can be adequately implemented in different order of steps, and the methods and processes are not necessarily limited to the order of steps shown herein or in the claims.

[0104] Under no circumstances shall the patent be construed as being limited to any specific example, embodiment, or method specifically disclosed herein. Under no circumstances shall the patent be construed as being limited by any statement made by an examiner, other officer or employee of the Patent and Trademark Office, except when such a statement is specifically, unqualified, or expressly adopted without reservation in the applicant's written response.

[0105] The terms and expressions used are for illustrative purposes only and are not limiting. The use of such terms and expressions is not intended to exclude any features or equivalents thereof that are shown or described. It should be understood that various modifications are possible within the scope of the invention as described in the claims. Therefore, although the invention is specifically disclosed by embodiments and optional features, those skilled in the art will understand that modifications and changes to the concepts disclosed herein can be relied upon, and such modifications and changes will be considered within the scope of the invention as defined by the appended claims and description of the invention.

Claims

1. A high-throughput, high-content assay method for screening drugs that overcome remyelination inhibition by exogenous inhibitors, a) Contacting oligodendrocyte progenitor cells (OPCs) with an exogenous inhibitor, which is at least one of an antibody, compound, small molecule, peptide, and nucleic acid, and a test drug, and b) Detect / quantify the presence of 1) myelin basic protein (MBP) + myelin-forming oligodendrocytes (OL) and 2) glial fibrillary acidic protein (GFAP) + astrocytes by obtaining two readings in a single assay. Includes, The OPC is cultured in a) for 3 days before b) so that the OPC can differentiate. a) After this, the OPC is brought into contact with Hoechst dye (nucleus), an antibody against MBP, and an antibody against GFAP, and an automated image is acquired so that 80% of the culture vessel is imaged. The automated image is analyzed to quantify the percentage of all cells that are positive for either MBP or GFAP. In the analysis, for GFAP+ cells, the ring is expanded from the nuclear mask (Hoechst dye) to include the cell body, and for MBP+ cells, the ring is expanded beyond the cell body to include the OL protrusion so that only mature OL is included in the analysis. If the fluorescence intensity measured within the ring exceeds a threshold set relative to the fluorescence intensity produced by a control with only the secondary antibody, the cell is determined to be positive. An assay method in which an increase in OL and a decrease in GFAP+ astrocytes compared to a control OPC exposed only to the exogenous inhibitor indicates that this drug overcame the inhibition of remyelination by the exogenous inhibitor.

2. The assay method according to claim 1, wherein the exogenous inhibitor is an inflammatory molecule.

3. The assay method according to claim 1, wherein the exogenous inhibitor is fibrinogen.

4. The assay method according to claim 3, wherein fibrinogen is present at physiological levels.

5. The assay method according to claim 3, wherein fibrinogen is added at a concentration of at least 2.5 mg / ml.

6. The assay method according to claim 1, wherein the OPC is the first generation OPC.

7. The assay method according to claim 6, wherein the primary OPC is cultured in growth medium for 1 to 6 days prior to a).

8. The assay method according to claim 7, wherein the growth medium comprises platelet-derived growth factor recombinant protein (PDGF-AA) and neurotrophin-3 recombinant protein (NT3).

9. The assay method according to claim 7, wherein the OPC is separated from the culture dish by proteolysis and / or collagenolysis, and then seeded into a fresh culture dish.

10. The aforementioned OPC is used before a), with 5 × 10 per well of the 96-well plate. ３ In cells, or 1 x 10⁶ per well in a 384-well plate. ３ The assay method according to claim 9, wherein the cells are seeded.

11. The assay method according to claim 10, wherein the seeded OPC is cultured for up to 24 hours prior to a).

12. The assay method according to claim 1, wherein the differentiation medium comprises ciliary neurotrophic factor recombinant protein (CNTF), triiodothyronine (T3), and PDGF-AA.

13. The assay method according to claim 1, wherein the antibody is directly or indirectly labeled with a detectable label, and an image of the labeled cells is obtained.

14. The assay method according to any one of claims 1 to 13, wherein automated quantification of MBP+ and GFAP+ is used.

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