Compositions and methods for promoting myelin formation

Abstract

The present invention provides methods and compositions useful for treating diseases, disorders, conditions, or injuries characterized by insufficient myelin formation. [Solution] A method for increasing axon myelination is provided, comprising the step of contacting oligodendrocyte progenitor cells (OPCs) in the presence of axons with a drug that inhibits GPR17 and / or a drug that removes and / or inhibits activated microglia, thereby increasing axon myelination.

Description

Technical Field

[0001] Statement of Rights to Inventions Made Under Federally Sponsored Research This invention was made with government support under grant number 5R01EY026939 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0002] Cross - Reference to Related Applications This application is an international PCT application claiming the priority and benefits of U.S. Provisional Application No. 63 / 018,939, filed on May 1, 2020, the entire contents of which are hereby incorporated herein by reference.

Background Art

[0003] Background of the Invention Functional impairments associated with CNS injuries, such as traumatic brain injury and spinal cord injury, are mainly caused by the severance of long - projection axons and subsequent disruption of related circuits. However, despite remarkable progress in developing strategies to promote axon regeneration, the behavioral improvements achieved by these methods are still limited, even in experimental injury models. For example, regenerating axons induced by operations that enhance the intrinsic regenerative ability of retinal ganglion cells (RGCs) can form functional synapses at their appropriate targets, such as the superior colliculus, but are in an unmyelinated (not myelinated) state. Given the well - established role of myelin (myelin sheath) in promoting axon conduction, these observations suggest hypomyelination of regenerating axons as a barrier to functional recovery that has not been properly evaluated, and point to the need to understand the regulatory mechanisms of myelin formation of regenerating axons in adults.

[0004] Myelination is well known to continue even after the completion of neurogenesis in adult central nervous system (CNS). This is achieved by oligodendrocyte progenitor cells (OPCs), which are universally distributed throughout the CNS. In successful myelination, resident OPCs often proliferate, followed by a multi-step, but not fully understood, differentiation process to ultimately become myelinate oligodendrocytes. The initiation and timing of CNS differentiation and myelination are tightly controlled by both endogenous and exogenous factors. On the other hand, myelinogenesis failure underlies many neurological diseases, including multiple sclerosis (MS), leukodystrophy, and neurodegenerative Alzheimer's disease. For example, in advanced MS, known as progressive MS, some proliferating OPCs remain in the center of the lesion but are unable to differentiate into mature oligodendrocytes. Therefore, considerable effort has been spent developing strategies to promote OPC proliferation and differentiation. However, in the most commonly used demyelination models, such as experimental allergic encephalomyelitis (EAE), some degree of spontaneous remyelination may occur, making it difficult to determine whether pro-myelination therapy works by accelerating the spontaneous process or / or by initiating de novo myelogenesis. Furthermore, the multi-step differentiation required for OPCs to mature into oligodendrocytes may necessitate interventions that address multiple stages.

[0005] Currently, there are no effective treatments for diseases associated with myelin dysplasia, such as multiple sclerosis (MS), leukodystrophy, and neurodegenerative Alzheimer's disease, or for central nervous system injuries associated with myelin dysplasia (e.g., traumatic brain injury, spinal cord injury). [Overview of the project]

[0006] As described below, the present invention features methods and compositions useful for treating diseases, disorders, conditions, and injuries characterized by insufficient myelin formation.

[0007] In one aspect, the present invention provides a method for increasing axon myelination, the method comprising contacting oligodendrocyte progenitor cells (OPCs) in the presence of axons with a GPR17 inhibitor and / or an agent that removes and / or inhibits activated microglia, thereby increasing axon myelination. In some embodiments, the axons are damaged and / or demyelinate.

[0008] In another aspect, the present invention provides a method for increasing axon myelination, the method comprising contacting oligodendrocyte progenitor cells (OPCs) in the presence of axons with a GPR17 inhibitor and / or a TNFα receptor 2 or TNFα inhibitor, thereby increasing axon myelination. In some embodiments, the TNFα receptor 2 or TNFα inhibitor is thalidomide.

[0009] In yet another aspect, the present invention provides a method for increasing the number and / or differentiation of OPCs, the method comprising contacting oligodendrocyte progenitor cells (OPCs) with a GPR17 inhibitor and / or an agent that removes or inhibits activated microglia, thereby increasing the number and / or differentiation of OPCs. In any one aspect of the above, the GPR17 inhibitor is montelukast or pranlukast.

[0010] In yet another aspect, the present invention provides a method for increasing the number and / or differentiation of OPCs, the method comprising contacting oligodendrocyte progenitor cells (OPCs) with a drug that inhibits GPR17 and / or a drug that inhibits TNFα receptor 2 or TNFα, thereby increasing the number and / or differentiation of OPCs. In some aspects of the above aspects, the drug that removes or inhibits activated microglia is PLX3397. In some aspects, the drug that inhibits TNFα receptor 2 or TNFα is thalidomide.

[0011] In another aspect, the present invention provides a method for increasing axon myelination, the method comprising contacting oligodendrocyte progenitor cells (OPCs) in the presence of axons with one or more agents, which are benztropine mesylate, clemastine, montelukast, pranlukast, and thalidomide, thereby increasing axon myelination. In some embodiments, the agent inhibiting GPR17 is montelukast.

[0012] In another aspect, the present invention provides a method for increasing the number and / or differentiation of OPCs, the method comprising contacting oligodendrocyte progenitor cells (OPCs) with one or more agents, which are benztropine mesylate, clemastine, montelukast, pranlukast, and thalidomide, thereby increasing the number and / or differentiation of OPCs. In some embodiments, the agent that inhibits GPR17 is montelukast.

[0013] In some aspects of the above aspects, the method increases the number of CC1 and / or Oligo1-positive OPCs. In some aspects, the drugs are administered simultaneously or sequentially. In some aspects, the GPR17 inhibitor is administered simultaneously with the drug that removes or inhibits activated microglia. In some aspects, the GPR17 inhibitor is administered at least one week before the drug that removes or inhibits activated microglia. In some aspects, these drugs are administered before, simultaneously with, or after injury. In some aspects, these drugs are administered several days or weeks after injury. In some aspects, these drugs are administered one to two weeks after injury. In some aspects, these drugs are administered for at least 14 to 28 days. In some aspects, the method is performed in vivo or in vitro.

[0014] In another aspect, the present invention provides a method for increasing axonal myelination in a subject, the method comprising administering to the subject an agent that inhibits GPR17 and / or an agent that removes or inhibits activated microglia, thereby increasing axonal myelination.

[0015] In another aspect, the present invention provides a method for increasing the number and / or differentiation of OPCs in a subject, the method comprising administering to the subject an agent that inhibits GPR17 and / or an agent that removes or inhibits activated microglia, thereby increasing the number and / or differentiation of OPCs. In some aspects of the two aspects, the agent that inhibits GPR17 is montelukast or pranlukast. In some aspects, the agent that removes or inhibits activated microglia is PLX3397.

[0016] In another aspect, the present invention provides a method for increasing axon myelination in a subject in need thereof, the method comprising administering to the subject one or more agents, which are benztropine mesylate, clemastine, montelukast, pranlukast, and thalidomide, thereby increasing axon myelination.

[0017] In another aspect, the present invention provides a method for increasing the number and / or differentiation of OPCs in a subject in need, the method comprising administering to the subject one or more of the following agents: benztropine mesylate, clemastine, montelukast, pranlukast, and thalidomide, thereby increasing the number and / or differentiation of OPCs.

[0018] In another aspect, the present invention provides a method for treating a subject having a disease or injury associated with myelin dysplasia, the method comprising administering to the subject an agent that inhibits GPR17 and / or an agent that removes or inhibits activated microglia. In some aspects of any of the above aspects, or in some aspects of any other aspects of the present invention as herein, the method increases the number of CC1 and / or Oligo1-positive OPCs. In some aspects of any of the above aspects, or in some aspects of any other aspects of the present invention as herein, the subject has a disease associated with myelin dysplasia, such as multiple sclerosis (MS), leukodystrophy, neurodegenerative Alzheimer's disease, traumatic brain injury, spinal cord injury, or optic nerve injury. In some forms, leukodystrophy is adrenoleukodystrophy (ALD), Aicardi-Goutieres syndrome, Alexander disease, Canavan disease, cerebrotendinous xanthomatosis (CTX), globoid cell leukodystrophy (Krabbe disease), metachromatic leukodystrophy (MLD), Pelizaeus-Merzbacher disease (X-linked spastic paraplegia), or child ataxia with central nervous system hypomyelination (CACH).

[0019] In some aspects of any of the above aspects, or in some aspects of any other aspects of the present invention as herein, the agents are administered simultaneously or sequentially. In some aspects, the GPR17 inhibitor is administered simultaneously with the agent that removes or inhibits activated microglia. In some aspects, the GPR17 inhibitor is administered at least one week before the agent that removes or inhibits activated microglia. In some aspects, the agent is administered before, simultaneously with, or after the injury. In some aspects, the agent is administered several days or weeks after the injury. In some aspects, the agent is administered one to two weeks after the injury. In some aspects, the traumatic brain injury is a concussion. In some aspects, the oligodendrocyte progenitor cells are CC1- and have Oligo1 localized in the nucleus. In some aspects, the OPC is an early differentiated oligodendrocyte that is CC1+ and has Oligo1 localized in the nucleus.

[0020] In any one aspect of the above, the OPC is a differentiated oligodendrocyte that is CC1+ and has Oligo1 localized in the cytoplasm.

[0021] In another aspect, the present invention provides a composition comprising a GPR17 antagonist and a microglia inhibitor or scavenger, a TNFα receptor 2 inhibitor, or a TNFα inhibitor. In some embodiments, the GPR17 antagonist is montelukast. In some embodiments, the microglia inhibitor or scavenger is PLX3397. In some embodiments, the TNFα inhibitor is thalidomide.

[0022] In another aspect, the present invention provides a method for identifying compounds that induce the differentiation of oligodendrocytes or oligodendrocyte progenitor cells, the method comprising the steps of: damaging the optic nerve of a mouse; contacting the optic nerve with an axon-regenerating agent; administering a candidate compound to the mouse to induce the differentiation of oligodendrocyte progenitor cells; administering a known microglia inhibitor or scavenger; and determining the differentiation state of the oligodendrocytes or oligodendrocyte progenitor cells, wherein an increase in CCl+ oligodendrocytes compared to an untreated control indicates that the candidate compound induced the differentiation of oligodendrocyte progenitor cells.

[0023] In another aspect, the present invention provides a method for identifying compounds that induce differentiation of oligodendrocytes or oligodendrocyte progenitor cells, the method comprising the steps of: damaging the optic nerve of a mouse; contacting the optic nerve with an axon-regenerating agent; administering to the mouse a compound known to induce differentiation of oligodendrocyte progenitor cells; administering a predicted microglia inhibitor or scavenger; and determining the differentiation state of the oligodendrocytes or oligodendrocyte progenitor cells, wherein an increase in cytoplasmic Oligo1-containing CCl+ oligodendrocytes compared to an untreated control indicates that the predicted microglia inhibitor or scavenger has effectively inhibited or removed microglial cells.

[0024] The compositions and articles defined by the present invention were separated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the present invention will become apparent from the detailed description and claims.

[0025] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in the field to which this invention pertains. The following references provide general definitions of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Unless otherwise specified, the following terms have the meanings set forth below.

[0026] "Drug" means a small molecule compound, antibody, nucleic acid molecule, polypeptide, or fragment thereof. In one embodiment, the drug is a GPR17 antagonist, or a microglia inhibitor or scavenger. In another embodiment, the drug is a compound that increases the number of differentiated OPCs in damaged optic nerve (e.g., 5, 10, 20, 30, 40, 50, 75, 85, 90, 95, or 100%). In another embodiment, the drugs include the M1 / M3 muscarinic receptor antagonist benztropine mesylate (Bzp) (Deshmukh et al., Nature 502, 327-332 (2013)), antihistamines, and anticholinergics, such as the M1 / M3 muscarinic receptor antagonist clemastine (Clem) (Mei et al., Nat.Med. 20, 954-960 (2014)), the M3 muscarinic receptor antagonist solifenacin (Sli) (Abiraman et al., J. Neurosci. 35, 3676-3688 (2015)), and the retinoid X receptor agonist bexarotene (Bex) (Natrajan et al., Brain 138, 3581-3597). (2015)), imidazole (Imi), an anti-cholesterol synthesis compound (Hubler et al., Nature 560, 372-376 (2018)), ibudilast (Ibud), a clinically approved phosphodiesterase (PDE) inhibitor (Fox et al., N. Engl. J. Med. 379, 846-855 (2018)), and two different GPR17 antagonists, montelukast (Mon) and pranlukast (Pra) (Fumagalli et al., J. Biol. Chem. 286, 10593-10604 (2011); Marschallinger et al., Nat. Commun. 6. (2015); Ou et al., J. Neurosci. 36, 10560-10573) (2016)) These are mTOR inhibitors such as rapamycin (Rap) or thalidomide (a TNFα inhibitor).

[0027] "Change" means a change in myelin formation or a change (increase or decrease) in markers associated with myelin formation (e.g., polynucleotides, polypeptides) as detected by standard known methods as described herein. As used herein, a change includes a 10% change in expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression level.

[0028] "To improve" means to reduce, suppress, alleviate, decrease, halt, or stabilize the onset or progression of a disease (for example, a disease associated with myelin dysplasia).

[0029] An "analog" refers to a molecule that is not identical but possesses similar functional or structural characteristics. For example, a polypeptide analog may have certain biochemical modifications that enhance the function of the analog compared to the naturally occurring polypeptide, while retaining the biological activity of the corresponding naturally occurring polypeptide. Such biochemical modifications can increase the protease resistance, membrane permeability, or half-life of the analog, for example, without altering ligand binding. The analog may contain non-natural amino acids.

[0030] In this disclosure, the terms "comprise," "comprising," and "containing" are used. Words such as "have" and "include" can have the meanings defined for them in U.S. patent law and may mean "includes" and "includes"; similarly, "consisting essentially of" or "consists essentially" also have the meanings defined for them in U.S. patent law, and this term is open-ended and allows for the existence of more than what is listed, provided that the basic or novel characteristics of what is listed are not altered by the existence of more than what is listed, but with the exception of embodiments of the prior art.

[0031] "Detecting" refers to identifying the presence, absence, or quantity of an analyte that needs to be detected.

[0032] A “detectable label” means a composition that, when bound to a target molecule, makes that molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Examples of useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens.

[0033] "Disease" means any condition, injury, or impairment that impairs or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include, but are not limited to, diseases involving insufficient, absent, or undesirable reduction of myelin formation, such as multiple sclerosis (MS), leukodystrophy, neurodegenerative diseases, Alzheimer's disease, ALS, traumatic brain injury, and spinal cord injury. Examples of leukodystrophy include, but are not limited to, 18q syndrome with myelin basic protein deficiency, adrenoleukodystrophy (ALD), adrenal spinal neuropathy (AMN), adult-onset autosomal dominant leukodystrophy (ADLD), and adult polyglucosan body disease. Diseases, Ecardi-Goutier syndrome, Alexander disease, Autosomal dominant diffuse leukoencephalopathy with nerve axonal spheroids (HDLS), AARS, AARS2, Canavan disease, Cathepsin A-associated arterial disease with stroke and leukoencephalopathy (CARASAL), Cerebral autosomal dominant arterial disease with subcortical infarction and leukoencephalopathy (CADASIL), Cerebral autosomal recessive arterial disease with subcortical infarction and leukoencephalopathy (CARASIL), Cerebral retinal microangiography with calcification and cysts, Cerebral tendon xanthomatous neuropathy (CTX), Childhood ataxia with central nervous system hypomyelination (CACH), ClC2-associated leukoencephalopathy, Coates plus, Cockayne syndrome, Elongation of Very Long-Chain Fatty Acids Acids-4:ELOVL4; Pseudo-Sjogren-Larsson; Fatty Acid 2-Hydroxylase Deficiency; Fucosidosis; Congenital Muscular Dystrophy; Globoid Cell Leukodystrophy (Krabbe Disease); GM1 Gangliosidosis; GM2 Gangliosidosis (Tay-Sachs Disease); Hypomyelination with Basal Ganglia and Cerebellar Atrophy (H-ABC); Hypomyelination; Hypogonadotropin Secretion; Hypogonadism and Tooth Dysfunction (Hypomyelination, Hypogonadotropic)Hypogonadism and Hypodontia (4H syndrome), Hypomyelination with Brainstem and Spinal Cord Involvement and Leg Spasticity (HBSL), Hypomyelination with Congenital Cataract (HCC), Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Elevated Lactate (LBSL), Leukoencephalopathy with Calcification and Cysts (LCC), Leukoencephalopathy with Thalamic and Brainstem Involvement and Hyperlactate (LTBL), Lipometabolic Dysplasia with Leukodystrophy (Nasu disease), Metachromatic Leukodystrophy (MLD), Macrocephalic Leukodystrophy with Subcortical Cysts (MLC), Mitochondrial Leukodystrophy, Multiple Sulfatase Deficiency, Axonal Sclerosis Neuroaxonal leukoencephalopathy with spheroids (hereditary diffuse leukoencephalopathy with spheroids (HDLS)), neonatal adrenoleukodystrophy (NALD), occulodetatoldigital dysplasia with cerebral white matter abnormalities, orthochromatic leukodystrophy with pigmented glia, ovarian leukodystrophy syndrome, Pelizaeus-Merzbach disease (X-linked spastic paraplegia), Pelizaeus-Merzbach-like disease (PMLD), RARS2-associated hypomyelination, Refsum disease, RNAse T2-deficient leukoencephalopathy, sialic acid storage disorders (Sala disease, childhood sialic acid storage disease, and intermediate forms), Sjögren-Larsson syndrome, SOX10-associated PCWH: peripheral demyelinating neuropathy, central myelinogenesis disorder leukodystrophy, Waardenburg syndrome, and Hirschsprung's disease, vanishing white matter disease (VWM) or childhood ataxia with diffuse central nervous system hypomyelination (CACH), X-linked adrenoleukodystrophy (X-ALD), and Zellweger spectrum (Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease).

[0034] "Effective dose" means the amount of therapeutic composition required to improve the symptoms of a disease, disorder, condition, or injury compared to an untreated patient. The effective dose of the active compound used in the implementation of the present invention for the therapeutic treatment of a disease, disorder, condition, or injury varies depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate dose and administration plan. Such a dose is called the "effective" dose. In one embodiment, the effective dose is the amount that increases neuronal myelin formation, increases OPC proliferation, increases the number of OPCs after injury, or promotes the differentiation of OPCs into CC1 and Oligo1-positive cells.

[0035] The method of the present invention provides a simple means for identifying therapeutic methods that are safe for use on a subject. Furthermore, the method of the present invention provides a means for analyzing substantially a number of compounds for their effects on the diseases described herein with high throughput, high sensitivity, and low complexity.

[0036] "Insufficient myelin formation" means that the level of myelin formation in the target neuron is reduced compared to the level of myelin formation observed in the corresponding control neuron.

[0037] A "marker" refers to any protein or polynucleotide whose expression level or activity is altered in relation to a disease or disorder.

[0038] As used herein, "obtain a drug" includes synthesizing, purchasing, or otherwise acquiring a drug.

[0039] "Decrease" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0040] "Standard" refers to a standard or comparison condition.

[0041] "Subjects" means mammals, and includes, but is not limited to, humans or non-human mammals such as cows, horses, dogs, sheep, and cats.

[0042] The ranges provided herein are understood to be abbreviated representations of all values ​​within that range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or partial range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0043] As used herein, terms such as “to treat,” “to cure,” and “to treat” refer to reducing or improving a disease, disorder, condition, injury, and / or symptoms associated therewith. It will be understood that treating a disease, disorder, condition, or injury does not require the complete elimination of the disease, disorder, condition, injury, or symptoms associated therewith, although it does not mean eliminating it.

[0044] Unless otherwise specified or evident from the context, the term "or" as used herein is understood to be inclusive. Unless otherwise specified or evident from the context, the terms "a," "an," and "the" as used herein are understood to be singular or plural.

[0045] Unless otherwise specified or the context makes clear, the term “about” as used herein is understood to mean within the normal range of acceptance in the art, for example, within two standard deviations of the mean. “About” may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context makes clear, all numerical values ​​stated herein are modified by the term “about.”

[0046] In this specification, the listing of chemical groups in the definition of a variable includes defining the variable as any single group or as a combination of the listed groups. In this specification, descriptions of any aspect of a variable include describing it as any single aspect or in combination with any other aspect or part thereof.

[0047] Any composition or method provided herein may be combined with any one or more of the other compositions and methods provided herein. [Brief explanation of the drawing]

[0048] [Figure 1A] Figures 1A–1P show increased proliferation and impaired differentiation of OPCs in injured optic nerves. Figure 1A includes representative electron microscopy images of intact optic nerves or injured optic nerves with regenerated axons, and quantification of axonal myelination rates. In intact optic nerves, most axons are myelinate, and after optic nerve crush injury in adult mice, very few regenerated axons exhibit spontaneous myelination. n=6 mice / group. Scale bar: 600nm. [Figure 1B] Figures 1A–1P show increased OPC proliferation and impaired differentiation in damaged optic nerves. Figures 1B–1D show the experimental scheme for evaluating OPC proliferation in both damaged (ipsilateral) and intact (contralateral) optic nerves of PDGFRα-H2B-GFP reporter mice. Figure 1B is an explanatory diagram of the damaged ipsilateral and intact contralateral optic nerves. Arrows indicate the site of crush injury, and the gray area indicates the region of interest analyzed. [Figure 1C] Figures 1A–1P show increased proliferation and impaired differentiation of OPCs in injured optic nerves. Figures 1B–1D show the experimental scheme for evaluating OPC proliferation in both injured (ipsilateral) and intact (contralateral) optic nerves of PDGFRα-H2B-GFP reporter mice. Figure 1C includes immunofluorescence staining images of injured optic nerves at different time points after injury. n=3–8 mice / group. Scale bar: 100 μm. [Figure 1D] Figures 1A–1P show increased proliferation and impaired differentiation of OPCs in injured optic nerves. Figures 1B–1D show the experimental scheme for evaluating OPC proliferation in both injured (ipsilateral) and intact (contralateral) optic nerves of PDGFRα-H2B-GFP reporter mice. Figure 1D is a graph showing the quantification of OPC counts in injured optic nerves at different time points after injury. n=3–8 mice / group. [Figure 1E] Figures 1A–1P show increased proliferation and impaired differentiation of OPCs in injured optic nerves. Figure 1E includes representative images of optic nerves (injured or control) stained with Oligo2 and / or GFP from PDGFRα-H2B-GPP reporter mice, showing the dynamic changes in total OPC count at different time points after injury. Scale bar: 100 μm. [Figure 1F] Figures 1A–1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1F includes representative images of BrdU / Oligo2 double-positive cells in damaged optic nerves. [Figure 1G] Figures 1A-1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1G is a graph showing the quantification of BrdU / Oligo2 double-positive cells in damaged optic nerves. [Figure 1H] Figures 1A-1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1H is an explanatory diagram of the different differentiation stages of OPCs and their respective markers. Scale bar: 50 μm. [Figure 1I] Figures 1A-1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1I is a schematic diagram of the experimental design for tracking OPC offspring using PDGFRα-CreER / iRTM mice, as shown in Figures 1J-1O. [Figure 1J] Figures 1A–1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1J includes representative images of CC1+ and RTM+ cells in damaged and intact optic nerves. [Figure 1K]Figures 1A–1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1K is a graph of CC1+ and RTM+ cell counts in damaged and intact optic nerves. [Figure 1L] Figures 1A-1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1L is a graph showing the proportion of CC1+ and RTM+ cells in damaged and intact optic nerves. In contrast to the intact (contralateral) nerve, the proportion of RTM+CC1+ cells was significantly lower in the damaged nerve. n=6 individuals / group. [Figure 1M] Figures 1A–1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1M includes representative images of three different populations (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes) in damaged and intact optic nerves. Contralateral arrows indicate RTM+ / CC1+ / Oligo1-C, and ipsilateral arrows indicate RTM+ / CC1- / Oligo1-N (undifferentiated cells). n=6 cells / group. Scale bars: 100 μm, 10 μm. [Figure 1N] Figures 1A-1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1N is a graph showing the cell numbers of three different populations (CC1- / Oligo1-N (nuclear Oligo1) in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes) in damaged and intact optic nerves. *,**,*** represent p<0.05, 0.01, and 0.001, respectively. [Figure 10] Figures 1A-1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1O is a graph showing the proportions of three different populations (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes) in damaged and intact optic nerves. *,**,*** represent p<0.05, 0.01, and 0.001, respectively. [Figure 1P] Figures 1A–1P show increased proliferation and impaired differentiation of OPCs in damaged optic nerves. Figure 1P includes representative images of damaged optic nerves from PDGFRα-CreER / RTM mice, showing no overlap between RTM and GFAP. Scale bar: 50 μm. *,**,*** p<0.05, 0.01, 0.001, respectively. [Figure 2A] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2A is a schematic diagram of in vivo compound screening in wild-type C57 mice. BrdU was injected daily into dpi4–10 to label proliferating OPCs. Damaged optic nerves from mice treated with each compound for 4 weeks were analyzed with the indicated antibodies. [Figure 2B] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2B includes representative images of damaged optic nerves stained with anti-CC1 and anti-BrdU. n=4–13 individuals / group. [Figure 2C] Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2C is a graph quantifying damaged optic nerves stained with anti-CC1 and anti-BrdU. n=4-13 individuals / group. [Figure 2D] Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2D is a schematic diagram of the experimental design for the montelukast study shown in Figures 2E-2G using PDGFRα-CreER:RTM mice. n=6 mice / group. [Figure 2E]Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2E includes representative images of damaged or intact optic nerves of different populations of RTM+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes) stained with antibodies against Oligo1, CC1, and RTM, as well as DAPI anti-CC1 and BrdU. [Figure 2F] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2F is a graph showing the density of different populations of RTM+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes) in damaged or intact optic nerves, stained with antibodies against Oligo1, CC1, and RTM, as well as DAPI anti-CC1 and BrdU. [Figure 2G] Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2G is a graph showing the proportions of different populations of RTM+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes). [Figure 2H] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2H includes representative insight hybridization images of damaged optic nerves showing injury-induced Gpr17 expression. Scale bar: 100 μm. [Figure 2I]Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2I is a graph quantifying optic nerves showing injury-induced Gpr17 expression in Figure 2H. n=6 individuals / group. [Figure 2J] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figures 2J–2L show that Gpr17 knockout did not affect OPC proliferation. Figure 2J includes representative images of damaged optic nerves of GPR17 knockout mice and their controls, stained with GFP (GPR17), BrdU, and / or Oligo2, and injected with BrdU daily between 4 dpi and 10 dpi. Scale bar: 200 μm. [Figure 2K] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figures 2J–2L show that Gpr17 knockout did not affect OPC proliferation. Figure 2K is a graph quantifying the damaged optic nerves from Figure 2J stained with GFP (GPR17), BrdU, and Oligo2. [Figure 2L] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figures 2J–2L show that Gpr17 knockout did not affect OPC proliferation. Figure 2L is a graph quantifying the damaged optic nerves from Figure 2J stained with BrdU and Oligo2. [Figure 2M] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in injured optic nerves. Figures 2M–2O show OPC differentiation analysis using antibodies against GFP(GPR17) or CC1 in injured optic nerves of GPR17 knockout mice. Figure 2M includes representative images of injured optic nerves taken from GPR17 knockout or control mice 28 days after injury. [Figure 2N]Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figures 2M-2O show OPC differentiation analysis using antibodies against GFP(GPR17) or CC1 in damaged optic nerves of GPR17 knockout mice. Figure 2N is a graph quantifying the density of GFP+CC1+ cells from GPR17 knockout mice with damaged optic nerves and their controls. [Figure 2O] Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figures 2M-2O show OPC differentiation analysis in damaged optic nerves of GPR17 knockout mice using antibodies against GFP (GPR17) or CC1. Figure 2O is a graph quantifying the proportion of CC1+ cells among GFP+ cells from GPR17 knockout mice with damaged optic nerves and their control. [Figure 2P] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2P includes representative images of damaged optic nerves from GPR17 knockouts and controls, stained with GFP (GPR17) and CC1. n=6 individuals / group. Scale bar: 200 μm. [Figure 2Q] Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2Q is a graph quantifying the proportion of CC1+ cells among GFP+ cells in GPR17 knockout mice with damaged optic nerves and their controls. [Figure 2R] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2R is a graph quantifying the density of GFP+CC1+ cells from GPR17 knockout mice with damaged optic nerves and their controls. Figures 2Q and 2R show that GPR17 knockout significantly increased CC1+ cells in damaged optic nerves, rather than in intact optic nerves. [Figure 2S]Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in injured optic nerves. Figures 2S–2U show differentiation analysis of OPCs in injured optic nerves (28 days post-injury) from GPR17 knockout mice using antibodies against GFP(GPR17), CC1, Oligo1, and DAPI. Figure 2S includes representative images of injured optic nerves from GPR17 knockout mice and controls stained with antibodies against Oligo1, CC1, and RTM, as well as DAPI anti-CC1 and BrdU. Scale bar: 50 μm. [Figure 2T] Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figures 2S-2U show differentiation analysis of OPCs in damaged optic nerves (28 days post-injury) of GPR17 knockout mice using antibodies against GFP (GPR17), CC1, Oligo1, and DAPI. Figure 2T is a graph quantifying the density of different populations of GFP+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes) in GPR17 knockout mice with damaged optic nerves and their control. n=6 individuals / group. *,**,*** p<0.05, 0.01, 0.001, respectively. [Figure 2U] Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figures 2S-2U show differentiation analysis of OPCs in damaged optic nerves (28 days post-injury) of GPR17 knockout mice using antibodies against GFP (GPR17), CC1, Oligo1, and DAPI. Figure 2U is a graph quantifying the proportion of different populations of GFP+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes) in GPR17 knockout mice with damaged optic nerves and their controls. n=6 mice / group. *,**,*** p<0.05, 0.01, 0.001, respectively. [Figure 2V] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in injured optic nerves. Figures 2V–2X show OPC differentiation analysis in injured optic nerves (7 days post-injury) from GPR17 knockout mice using antibodies against GFP (GPR17), CC1, Oligo1, and DAPI. Figure 2V includes representative images of injured optic nerves from GPR17 knockout mice and controls stained with antibodies against Oligo1, CC1, and RTM, as well as DAPI anti-CC1 and BrdU, and quantitative results of density (J) or proportion (K) of different populations of GFP+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes). Scale bar: 100 μm. [Figure 2W] Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figures 2V-2X show OPC differentiation analysis using antibodies against GFP (GPR17), CC1, Oligo1, and DAPI in damaged optic nerves (7 days post-injury) of GPR17 knockout mice. Figure 2W is a graph quantifying the density of different populations of GFP+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes) from GPR17 knockout mice with damaged optic nerves and their control. n=6 individuals / group. [Figure 2X]Figures 2A-2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figures 2V-2X show OPC differentiation analysis in damaged optic nerves (7 days post-injury) of GPR17 knockout mice using antibodies against GFP (GPR17), CC1, Oligo1, and DAPI. Figure 2X is a graph quantifying the proportion of different populations of GFP+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes) from GPR17 knockout mice with damaged optic nerves and their control. n=6 mice / group. [Figure 2Y] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2Y includes representative images of damaged optic nerves from GPR17 knockout mice and controls that received daily BrdU injections from 4 dpi to 10 dpi, stained with GFP (GPR17) and CC1 (L). [Figure 2Z] Figures 2A–2Z show that GPR17 is an endogenous blocker of early oligodendrocyte differentiation of OPCs in damaged optic nerves. Figure 2Z is ​​a graph quantifying the proportion of CC1+ cells among BrdU+ cells from GPR17 knockout mice with damaged optic nerves and their controls. n=6 mice / group. *,**,*** p<0.05, 0.01, 0.001, respectively. [Figure 3A] Figures 3A–3G show that activated microglia in the injured optic nerve were depleted by PLX3397 treatment. Figure 3A includes representative images of the optic nerve from adult mice 4 weeks after unilateral optic nerve crush injury, stained with antibodies against GFAP, CD68, or P2Y12. Scale bar: 50 μm. [Figure 3B] Figures 3A-3G show that activated microglia in damaged optic nerves were depleted by PLX3397 treatment. Figure 3B is a graph showing the quantification of GFAP immune response signals. n=10 individuals / group. [Figure 3C]Figures 3A-3G show that activated microglia in damaged optic nerves were depleted by PLX3397 treatment. Figure 3C is a graph showing the quantification of the P2Y12 immune response signal. n=10 individuals / group. [Figure 3D] Figures 3A-3G show that activated microglia in damaged optic nerves were depleted by PLX3397 treatment. Figure 3D is a graph showing the quantification of the CD68 immune response signal. n=10 individuals / group. [Figure 3E] Figures 3A–3G show that activated microglia in damaged optic nerves were depleted by PLX3397 treatment. Figure 3E includes images showing persistent activated microglia throughout the optic nerve distal to the lesion, collected from adult mice 6 weeks after injury. Scale bar: 500 μm. [Figure 3F] Figures 3A-3G show that activated microglia in damaged optic nerves were depleted by PLX3397 treatment. Figure 3F is a graph quantifying the CD68 immune response signal. n=10 individuals / group. [Figure 3G] Figures 3A–3G show that activated microglia in the injured optic nerve were depleted by PLX3397 treatment. Figure 3G includes representative images of the optic nerve from adult mice treated with and without PLX3397, 2 weeks after unilateral optic nerve crush injury, stained with an antibody against CD68. Scale bar: 40 μm. [Figure 3H] Figures 3H-3O show that microglia are necessary for OPC proliferation but detrimental to their maturation. Figure 3H is a graph quantifying the CD68 immune response signal. 6 = 10 individuals / group. Scale bar: 40 μm. *, **, *** p < 0.05, 0.01, 0.001, respectively. [Figure 3I] Figures 3H-3O show that microglia are necessary for OPC proliferation but detrimental to their maturation. Figure 3I is a schematic diagram of an experiment to evaluate OPC proliferation in PDGFRα-H2B-GFP mice treated with PLX3397 (PLX) or its vehicle control (Vec). PLX treatment was performed for 14 days (7 days before and after injury), and BrdU was injected 48 hours before the end of treatment. [Figure 3J] Figures 3H-3O show that microglia are necessary for OPC proliferation but detrimental to their maturation. Figure 3J includes representative images of damaged optic nerves stained with GFP, Oligo2, or BrdU. Scale bar: 100 μm. [Figure 3K] Figures 3H-3O show that microglia are necessary for OPC proliferation but detrimental to their maturation. Figure 3K is a graph quantifying the density of GFP+Oligo2+BrdU+ cells. n=6 cells / group. [Figure 3L] Figures 3H-3O show that microglia are necessary for OPC proliferation but detrimental to their maturation. Figure 3L is a schematic diagram of an experimental design to analyze the effect of delayed PLX3397 treatment on OPC differentiation in damaged optic nerves in PDGFRα-CreER:RTM mice, based on the results shown in Figures 3M-O. [Figure 3M] Figures 3H-3O show that microglia are necessary for OPC proliferation but detrimental to their maturation. Figure 3M includes representative images of damaged or intact optic nerves stained with antibodies against Oligo1, CC1, and RTM, as well as DAPI anti-CC1 and BrdU. Scale bar: 100 μm. [Figure 3N] Figures 3H-3O show that microglia are necessary for OPC proliferation but detrimental to their maturation. Figure 3N is a graph quantifying the density of different populations of RTM+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes). n=6 cells / group. *,**,*** p<0.05, 0.01, 0.001, respectively. [Figure 3O]Figures 3H-3O show that microglia are necessary for OPC proliferation but detrimental to their maturation. Figure 3O is a graph quantifying the proportion of different populations of RTM+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes). n=6 cells / group. *,**,*** p<0.05, 0.01, 0.001, respectively. [Figure 4A] Figures 4A–4I demonstrate that combined treatment with montelukast and PLX3397 results in robust myelin formation of regenerating axons in injured optic nerves of adult mice. Figure 4A includes representative images of injured optic nerves from adult PDGFRα-CreER:RTM mice treated with montelukast and PLX3397, stained with antibodies against Oligo1, CC1, and RTM, as well as DAPI anti-CC1 and BrdU. Samples were collected at the end of week 4 post-injury. Scale bar: 20 μm (A). [Figure 4B] Figures 4A-4I show that combination treatment with montelukast and PLX3397 results in robust myelin formation of regenerated axons in damaged optic nerves of adult mice. Figure 4B is a graph quantifying the density of different populations of RTM+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes). n=6 individuals / group. [Figure 4C] Figures 4A-4I show that combination treatment with montelukast and PLX3397 results in robust myelin formation of regenerated axons in damaged optic nerves of adult mice. Figure 4C is a graph quantifying the proportion of different populations of RTM+ cells (CC1- / Oligo1-N in undifferentiated OPCs, CC1+ / Oligo1-N in premyelinating oligodendrocytes, and CC1+ / Oligo1-C in mature myelinating oligodendrocytes). n=6 mice / group. [Figure 4D]Figures 4A–4I show that combination treatment with montelukast and PLX3397 results in robust myelin formation of regenerating axons in injured optic nerves of adult mice. Figures 4D–H show transmission electron microscopy imaging results of myelin formation of regenerating axons (4 weeks post-injury) of injured optic nerves from mice treated with montelukast and / or PLX3397. Figure 4D is a low-magnification image of the coronal section of the crushed optic nerve from each treatment group. Scale bar: 2 μm. [Figure 4E] Figures 4A–4I show that combination treatment with montelukast and PLX3397 results in robust myelin formation of regenerated axons in injured optic nerves of adult mice. Figures 4D–H show transmission electron microscopy imaging results of myelin formation in regenerated axons (4 weeks post-injury) of injured optic nerves from mice treated with montelukast and / or PLX3397. Figure 4E is a magnified image of ongoing myelin formation in regenerated axons from the combination treatment group. The thin layer of myelin and large inner tongue suggested ongoing new myelin formation. Scale bar: 500 nm. [Figure 4F] Figures 4A–4I show that combination treatment with montelukast and PLX3397 results in robust myelin formation of regenerating axons in injured optic nerves of adult mice. Figures 4D–H show transmission electron microscopy imaging results of myelin formation of regenerating axons (4 weeks post-injury) of injured optic nerves from mice treated with montelukast and / or PLX3397. Figure 4F is a montage of images from longitudinal sections of crushed optic nerves treated with the combination. A complete internode is indicated by "X". Arrows indicate the location of adjacent nodes of Ranvier. Scale bar: 1400 nm. [Figure 4G]Figures 4A–4I show that combination treatment with montelukast and PLX3397 results in robust myelin formation of regenerated axons in injured optic nerves of adult mice. Figures 4D–H show transmission electron microscopy imaging results of myelin formation of regenerated axons (4 weeks post-injury) of injured optic nerves from mice treated with montelukast and / or PLX3397. Figure 4G is a magnified image of half of the nodes of Ranvier on the regenerated axon shown in G. (H) Quantification of panel D. n=4 mice / group. Scale bar: 200 nm. [Figure 4H] Figures 4A–4I show that combination treatment with montelukast and PLX3397 results in robust myelin formation of regenerated axons in injured optic nerves of adult mice. Figures 4D–H show transmission electron microscopy imaging results of myelin formation in regenerated axons (4 weeks post-injury) of injured optic nerves from mice treated with montelukast and / or PLX3397. Figure 4H is a graph quantifying the percentage of myelinate axons observed in Figure 4D. [Figure 4I] Figures 4A–4I demonstrate that combination treatment with montelukast and PLX3397 results in robust myelin formation of regenerating axons in injured optic nerves of adult mice. Figure 4I includes representative images of combined-treated injured optic nerves stained with Caspr, AnkG, and sodium channel NaV1.6, markers of the nodes of Ranvier. Scale bar: 3.5 μm. [Figure 4J] Figures 4J–4L show better preservation of regenerated axons in mice treated with the combination of montelukast and PLX3397. Figure 4J includes representative images of CTB-labeled regenerating axons in damaged optic nerves (42 dpi) from wild-type mice treated with or without montelukast and PLX3397, which underwent intravitreal injection of AAV2 / 2-CNTF / IGF / OPN followed by optic nerve contusion. Scale bar: 750 μm. [Figure 4K] Figures 4J-4L show that regenerated axons are better preserved in mice treated with a combination of montelukast and PLX3397. Figure 4K is a graph quantifying the area of ​​regenerated axons as a function of time after injury. n=4 mice / group. [Figure 4L] Figures 4J-4L show that regenerated axons are better preserved in mice treated with a combination of montelukast and PLX3397. Figure 4L is a graph quantifying the intensity of signals associated with regenerating axons as a function of distance from the crush site. n=4 mice / group. [Figure 5A-1] Figures 5A–5E show the inhibition of TNFα in cell populations after optic nerve contusion. Figure 5A includes a Venn diagram and heatmap showing that TNFα is upregulated after optic nerve contusion. [Figure 5A-2] Refer to the explanation in Figure 5A-1. [Figure 5B] Figures 5A–5E show the inhibition of TNFα in cell populations after optic nerve crushing. Figure 5B includes fluorescence images showing adult optic nerves after crushing that have been probed for TNFα. [Figure 5C] Figures 5A–5E show the inhibition of TNFα in cell populations after optic nerve crush. Figure 5C includes fluorescence images and graphs showing a strong increase in CC1+ oligodendrocytes in the distal region near the crush site in TNFR1 KO mice. [Figure 5D] Figures 5A–5E show the inhibition of TNFα in cell populations after optic nerve contusion. Figure 5D includes a fluorescence image showing a potent increase in BrdU+ / CC1+ / Olig2+ cells in the distal region of axons in mice treated with the TNFα inhibitor thalidomide. TNFi represents thalidomide. [Figure 5E] Figures 5A–5E show the inhibition of TNFα in cell populations after optic nerve contusion. Figure 5E includes a schematic diagram and images comparing mice treated with vehicle alone and mice treated with thalidomide. [Modes for carrying out the invention]

[0049] Detailed description of the invention The present invention features compositions and methods useful for promoting or increasing myelin formation, particularly in cases of demyelination resulting from nerve damage or disease.

[0050] This invention is at least in part based on the finding that inhibition of GPR17 and removal of activated microglia resulted in robust myelin formation of regenerating axons.

[0051] Myelin formation promotes axonal conduction, enabling efficient communication between different parts of the nervous system. While manipulations that enhance the endogenous regenerative capacity of neurons result in robust axonal regeneration after optic nerve injury, these regenerating axons do not undergo spontaneous myelin formation. The underlying mechanisms of this myelin formation deficiency remain unclear.

[0052] As described herein, in an adult mouse model of optic nerve injury reminiscent of progressive multiple sclerosis, optic nerve oligodendrocyte progenitor cells (OPCs) undergo transient proliferation but fail to differentiate into myelin-forming mature oligodendrocytes. Mechanistically, both endogenous GPR17 in OPCs and chronically activated microglia inhibit different stages of OPC differentiation. Importantly, inhibition of both GPR17 and microglia led to robust myelin formation in regenerating axons. In addition to elucidating the regulatory mechanisms of stage-dependent OPC differentiation, the presented results demonstrate that the agents described herein provide robust denovomyelin formation even in the presence of chronic inflammatory conditions in the adult CNS.

[0053] Neuronal myelin formation For nerves to function properly, myelination of regenerating or demyelinating axons by mature myelin-forming oligodendrocytes is necessary. Because oligodendrocytes are involved in myelination, promoting the expansion and maturation of oligodendrocyte progenitor cells (OPCs) offers significant improvements in treating demyelination caused by nerve injury or disease. Undifferentiated, early-differentiated, and mature myelin-forming oligodendrocytes can be distinguished based on the expression profiles of CC1 and Oligo1 in the cells. For example, undifferentiated oligodendrocytes are characterized as CC1-negative cells with Oligo1 localized in the nucleus. Early-differentiated oligodendrocytes are characterized as CC1-positive cells with Oligo1 localized in the nucleus. Mature myelin-forming oligodendrocytes are characterized as CC1-positive cells with cytoplasmic Oligo1. In response to nerve injury, OPCs proliferate and expand. In some embodiments, the maturation of undifferentiated OPCs to early differentiated OPCs can be facilitated by contacting the cells with a GPR17 antagonist or inhibitor. In some embodiments, the GPR17 antagonist or inhibitor may be benztropine mesylate, an M1 / M3 muscarinic receptor antagonist; clemastine, an antihistamine and anticholinergic agent and M1 / M3 muscarinic receptor antagonist; solifenacin, an M3 muscarinic receptor antagonist; bexarotene, a retinoid X receptor agonist; imidazole, an anticholesterol synthesis compound; ibudilast, a clinically approved phosphodiesterase (PDE) inhibitor; montelukast or pranlukast; or thalidomide.

[0054] Early-differentiated oligodendrocytes (OPCs) can be further differentiated into mature myelin-forming oligodendrocytes by removing microglia present in the surrounding environment. Removal agents that effectively remove microglia are known in the art. In some aspects of the present invention, contact between microglia and an inhibitor or removal agent provides an environment that promotes the differentiation of early-differentiated oligodendrocytes into mature myelin-forming oligodendrocytes.

[0055] The present invention's agent The agents described herein, such as small molecule compounds, are useful for increasing myelin formation, enhancing OPC proliferation or differentiation, or increasing the number of OPCs. In one embodiment, the agent of the present invention is pexidartinib (also known as PLX3397), a small molecule having multikinase inhibitory activity. Pexidartinib (CAS Registry No. 1029044-16-3; C 20 -H 15 -Cl-F3-N5) has the following structure: TIFF2026082934000001.tif25128

[0056] In another embodiment, the agent of the present invention is bexarotene (e.g., 100 mg / kg, oral (po)), a low molecule having retinoid X receptor binding and activating activity. Bexarotene (CAS Registry No. 153559-49-0; C 24 H 28 O2) has the following structure: TIFF2026082934000002.tif36128

[0057] In another embodiment, the agent of the present invention is benztropine mesylate (e.g., 10 mg / kg, intraperitoneal (ip)), a small molecule central muscarinic antagonist having dopamine reuptake inhibitory activity. Benztropine mesylate (CAS Registry No. 132-17-2; C 22 H 29 NO4S) has the following structure: TIFF2026082934000003.tif35128

[0058] In another embodiment, the agent of the present invention is clemastine fumarate (e.g., 10 mg / kg, po), a low molecular weight substance having anticholinergic, sedative, and histamine H1 antagonistic effects. Clemastine fumarate (CAS Registry No. 14976-57-9; C 25 H 30 ClNO5) has the following structure: TIFF2026082934000004.tif33128

[0059] In another aspect, the agent of the present invention is ibudilast (e.g., 10 mg / kg, i.p.), which is a small molecule having cyclic nucleotide phosphodiesterase inhibitory activity. Ibudilast (CAS registration number 50847-11-5; C 14 H 18 N2O) has the following structure: TIFF2026082934000005.tif23128

[0060] In another aspect, the agent of the present invention is imidazole (e.g., 10 mg / kg, i.p.), which is a base and an excellent nucleophile. Imidazole (CAS registration number 288-32-4; C3H4N2) has the following structure: TIFF2026082934000006.tif16128

[0061] In another aspect, the agent of the present invention is montelukast (e.g., 25 mg / kg, p.o.), which is a leukotriene receptor (e.g., GRP17) antagonist. Montelukast (CAS registration number 158966-92-8; C 35 H 36 ClNO3S) has the following structure: TIFF2026082934000007.tif47128

[0062] In another aspect, the agent of the present invention is pranlukast (e.g., 0.5 mg / kg, i.p.), which is a leukotriene receptor (e.g., GRP17) antagonist. Pranlukast (CAS registration number 103177-37-3; C 27 H 23 N5O4) has the following structure: TIFF2026082934000008.tif27128

[0063] In another embodiment, the agent of the present invention is rapamycin (e.g., 6 mg / kg, ip) having mTOR inhibitory activity. Rapamycin (CAS Registry No. 53123-88-9; C 51 H 79 NO 13 ) has the following structure: TIFF2026082934000009.tif48128

[0064] In another embodiment, the agent of the present invention is solifenacin succinate (e.g., 20 mg / kg, ip), a low molecular weight substance having anticholinergic and anticonvulsant effects. (Solifenacin succinate (CAS Registry No. 242478-38-2; C) 27 H 32 N2O6 has the following structure: TIFF2026082934000010.tif46128

[0065] In another embodiment, the agent of the present invention is thalidomide (e.g., 50 mg / kg, ip), a small molecule that inhibits the production of tumor necrosis factor α (TNFα). Thalidomide (CAS Registry No. 50-35-1; C 13 H 10 N2O4 has the following structure: TIFF2026082934000011.tif25128

[0066] Treatment method The present invention provides a method for treating a disease, disorder, or injury, or a symptom thereof, characterized by unmyelinated or demyelinated neurons, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an agent described herein (e.g., a G protein-coupled receptor 17 (GPR17) antagonist, a microglia inhibitor, or a scavenger). In some embodiments, the disease is characterized by demyelination of nerves. In some embodiments, the disease is a neurodegenerative disease such as multiple sclerosis or Alzheimer's disease. In some embodiments, the injury being treated is traumatic brain injury.

[0067] In some embodiments, the agent is benztropine mesylate, clemastine, solifenacin, bexarotene, imidazole, ibudilast, montelukast, pranlukast, or thalidomide. In some embodiments, the microglia inhibitor or scavenger is PLX3397. Thus, one embodiment is a method for treating a subject that is suffering from or susceptible to a disease or disorder or its symptoms. The method includes the step of administering to a mammal a therapeutic amount of the agent described herein sufficient to treat the disease or disorder or its symptoms, under conditions such that the disease or disorder is treated.

[0068] Identifying subjects requiring such treatment can be left to the judgment of the subject or healthcare professional and may be subjective (e.g., opinion) or objective (e.g., measurable by tests or diagnostic methods). Such treatment would be appropriately administered to subjects, particularly humans, who have, are susceptible to, or are at risk of having, a disease, disorder, or its symptoms. The determination of subjects at "risk" can be made by objective or subjective judgment, such as diagnostic tests or the opinion of the subject or healthcare professional (e.g., genetic testing, enzyme or protein markers, family history, etc.). The compounds described herein may also be used to treat other diseases in which myelin dysplasia or deficiency may be involved.

[0069] composition GPR17 antagonists and microglia inhibitors or scavengers, when administered in combination, are useful in treating diseases, disorders, or injuries characterized by insufficient myelin formation in neurons. In some embodiments, the GPR17 antagonist is montelukast or pranlukast. In some embodiments, the microglia inhibitor or scavenger is PLX3397. In certain embodiments, combination therapy of a GPR17 antagonist and a microglia inhibitor or scavenger can increase myelin formation in target neurons by at least 10%, 25%, 50%, 75%, or even 100%.

[0070] pharmaceutically acceptable salts of GPR17 antagonists or microglia inhibitors or scavengers, or both, are intended herein to increase myelin formation of target neurons. The term “pharmaceutically acceptable salt” refers to a salt prepared from a GPR17 antagonist or microglia inhibitor or scavenger having an acidic functional group such as a carboxylic acid functional group, and a pharmaceutically acceptable inorganic or organic base. Suitable bases include, but are not limited to, the following: hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia; and organic amines, e.g., unsubstituted or hydroxysubstituted mono, di, or trialkylamines; dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono, bis, or tris(2-hydroxy lower alkylamines), e.g., mono, bis, or tris(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris(hydroxymethyl)methylamine; N,N-dilower alkyl-N-(hydroxy lower alkyl)amines, e.g., N,N-dimethyl-N-(2-hydroxyethyl)amine, or tri(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids, e.g., arginine, lysine, etc. The term “pharmaceutically acceptable salt” also refers to a salt prepared from a GPR17 antagonist and / or microglia inhibitor or scavenger having a basic functional group such as an amino functional group, and a pharmaceutically acceptable inorganic or organic acid. Suitable acids include, but are not limited to,: hydrogen sulfate, citric acid, acetic acid, oxalic acid, hydrochloric acid, hydrogen bromide, hydrogen iodide, nitric acid, phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0071] Pharmaceutical treatments For therapeutic use, compositions containing the agents described herein can be administered systemically. Preferred routes of administration include, for example, oral administration, or subcutaneous, intravenous, intraperitoneal, intramuscular, or intradermal injection, which provide the patient with a continuous and sustained level of the drug. Treatment of human patients or other animals is carried out using therapeutically effective amounts of the therapeutic agents identified herein in a physiologically acceptable carrier. In one embodiment, a GPR17 antagonist, microglia inhibitor, or scavenger, or both, may be formulated in a pharmaceutically acceptable buffer such as saline. Suitable carriers and their formulations are described, for example, in "Remington's Pharmaceutical Sciences" by E.W. Martin. The amount of therapeutic agent administered will vary depending on the method of administration, the patient's age and weight, and the clinical manifestations of the disease, disorder, or injury characterized by insufficient myelinization. Generally, the amount will be within the range used for other agents used to treat other conditions, diseases, or injuries characterized by insufficient myelinization. In some embodiments, a composition comprising the agents described herein (e.g., a GPR17 antagonist and / or a microglia inhibitor or scavenger) is administered in a dose effective in increasing myelin formation of target neurons. In other embodiments, a composition comprising a GPR17 antagonist and a composition comprising a microglia inhibitor or scavenger is administered in a dose effective in increasing myelin formation of target neurons. The effectiveness of the administration can be determined by methods known to those skilled in the art, or by using any assay that measures neuronal myelin formation.

[0072] Formulation of pharmaceutical compositions Administration of compositions comprising the agents described herein (e.g., GPR17 antagonists, microglia inhibitors or scavengers, or both) for the treatment of diseases, disorders or injuries characterized by insufficient myelin formation of target neurons can be carried out by any suitable means to provide a therapeutic concentration effective in combination with other components to increase or stabilize myelin formation of target neurons. The compositions are contained in any suitable amount in any suitable carrier material and are generally present in an amount of 1 to 95% by weight of the total weight of the composition. The compositions can be provided in dosage forms suitable for oral administration. In some embodiments, the compositions can be provided in dosage forms suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) routes of administration. Pharmaceutical compositions can be formulated according to conventional formulation practices (see, for example, Remington: The Science and Practice of Pharmacy (20th ed.), ed. AR Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988–1999, Marcel Dekker, New York).

[0073] Those skilled in the art will recognize that it is routine in the art to modify human doses compared to animal models; therefore, human doses can first be determined by extrapolating from the amount of the drug used in mice. Dosages may be determined based on doses for the effective treatment of conditions or injuries known in the art characterized by insufficient myelin formation of target neurons. In certain embodiments, the doses of the drugs described herein are approximately 0.1 mg to approximately 200 mg / day, approximately 0.1 mg to approximately 190 mg / day, approximately 0.1 mg to approximately 180 mg / day, approximately 0.1 mg to approximately 170 mg / day, approximately 0.1 mg to approximately 160 mg / day, approximately 0.1 mg to approximately 150 mg / day, approximately 0.1 mg to approximately 140 mg / day, approximately 0.1 mg to approximately 130 mg / day, approximately 0.1 mg to approximately 120 mg / day, approximately 0.1 mg to approximately 110 mg / day, and approximately 0. It is assumed that the daily doses will be approximately 1 mg to 100 mg, approximately 0.1 mg to 90 mg, approximately 0.1 mg to 80 mg, approximately 0.1 mg to 70 mg, approximately 0.1 mg to 60 mg, approximately 0.1 mg to 50 mg, approximately 0.1 mg to 40 mg, approximately 0.1 mg to 30 mg, approximately 0.1 mg to 20 mg, approximately 0.1 mg to 10 mg, approximately 0.1 mg to 5 mg, and approximately 0.1 mg to 1 mg. In some aspects, the dosage of the GPR17 antagonist is approximately 0.5 mg to 200 mg / day, 1 mg to 200 mg / day, 10 mg to 200 mg / day, 20 mg to 200 mg / day, 30 mg to 200 mg / day, 40 mg to 200 mg / day, 50 mg to 200 mg / day, 60 mg to 200 mg / day, 70 mg to 200 mg / day, 80 mg to 200 mg / day, and The recommended daily doses are approximately 90mg to 200mg, 100mg to 200mg, 110mg to 200mg, 120mg to 200mg, 130mg to 200mg, 140mg to 200mg, 150mg to 200mg, 160mg to 200mg, 170mg to 200mg, 180mg to 200mg, or 190mg to 200mg.

[0074] In some embodiments, the dose of the microglia inhibitor or scavenger is approximately 250 mg to approximately 350 mg per day. In some embodiments, the dose of the microglia inhibitor or scavenger is approximately 250 mg to approximately 325 mg / day, approximately 250 mg to approximately 300 mg / day, or approximately 250 mg to approximately 275 mg / day. In some embodiments, the dose of the microglia inhibitor or scavenger is approximately 275 mg to approximately 350 mg / day, approximately 300 mg to approximately 350 mg / day, or approximately 325 mg to approximately 350 mg / day.

[0075] In some embodiments, the drug is bexarotene, with a dosage of approximately 50 to approximately 150 mg / kg. In some embodiments, the drug is benztropine mesylate, with a dosage of approximately 5 to approximately 15 mg / kg. In some embodiments, the drug is clemastine fumarate, with a dosage of approximately 5 to approximately 15 mg / kg. In some embodiments, the drug is ibudilast, with a dosage of approximately 5 to approximately 15 mg / kg. In some embodiments, the drug is imidazole, with a dosage of approximately 5 to approximately 15 mg / kg. In some embodiments, the drug is montelukast, with a dosage of approximately 10 to approximately 40 mg / kg. In some embodiments, the drug is pranlukast, with a dosage of approximately 0.1 to approximately 1.0 mg / kg. In some embodiments, the drug is rapamycin, with a dosage of approximately 3 to approximately 9 mg / kg. In some embodiments, the drug is solifenacin succinate, with a dosage of approximately 10–30 mg / kg. In some embodiments, the drug is thalidomide, with a dosage of approximately 25–75 mg / kg. In some embodiments, the drug is pexidartinib (PLX 3397), with a dosage of approximately 225–350 mg / kg per day. Naturally, the dosage can be adjusted upward or downward depending on the results of the initial clinical trials and the needs of the specific patient, as is routinely done in such treatment protocols.

[0076] Pharmaceutical compositions according to aspects of this disclosure can be formulated to release an active compound (e.g., a GPR17 antagonist and a microglial scavenger) substantially immediately after administration or at any predetermined time or period after administration. The latter type of composition is generally known as controlled-release formulations and includes: (i) formulations that result in substantially constant drug concentrations in the body over a long period of time; (ii) formulations that result in substantially constant drug concentrations in the body over a long period of time after a predetermined time lag; (iii) formulations that sustain action over a predetermined period of time by maintaining a relatively constant effective level in the body while minimizing undesirable side effects associated with fluctuations in plasma levels of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by spatially positioning the controlled-release composition near intended target cells (e.g., brain cells); (v) formulations that enable convenient dosing, such as when the dose is administered orally once or twice daily; and (vi) formulations that target calcium channels and angiotensin receptors by using a carrier or chemical derivative to deliver the therapeutic agent to specific cell types (e.g., brain cells). In some applications, controlled-release formulations eliminate the need for frequent daytime administration to maintain plasma levels at therapeutic levels.

[0077] To obtain controlled release where the release rate exceeds the metabolic rate of the compound in question, one of many strategies can be employed. For example, controlled release can be achieved by appropriately selecting various formulation parameters and components, including, for instance, various types of release-controlled compositions and coatings. Thus, therapeutic agents are formulated, with appropriate excipients, into pharmaceutical compositions that release the therapeutic agent in a controlled manner upon administration. Examples include single-unit or multi-unit tablet or capsule compositions, oil-based solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

[0078] Parenteral composition Pharmaceutical compositions can be administered parenterally by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) via a conventional, non-toxic, pharmaceutically acceptable carrier and adjuvant in dosage forms, formulations, or via a suitable delivery device or implant. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, cited above.

[0079] Compositions for parenteral use may be provided in unit dosage forms (e.g., single-dose ampoules) or in vials containing multiple doses, which may contain appropriate preservatives (see below). The composition may be in the form of a solution, suspension, emulsion, infusion device, or implantation delivery device, or it may be presented as a dry powder that is prepared immediately before use with water or other suitable vehicle. Apart from the active agent that increases myelin formation of target neurons, the composition may contain appropriate parenterally acceptable carriers and / or additives. The therapeutic active agent may be encapsulated in microspheres, microcapsules, nanoparticles, liposomes, etc., for controlled release. Furthermore, the composition may contain suspending agents, solubilizers, stabilizers, pH adjusters, isotonic agents, and / or dispersants.

[0080] As described above, the pharmaceutical compositions according to the embodiments of this disclosure may be in a form suitable for sterile injection. To prepare such compositions, the GPR17 antagonist and / or microglial scavenger is dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that can be used include water, water adjusted to a suitable pH by the addition of appropriate amounts of hydrochloric acid, sodium hydroxide, or appropriate buffers, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride and dextrose solutions. The aqueous formulation may also contain one or more preservatives (e.g., methyl p-hydroxybenzoate, ethyl, or n-propyl). If one of the compounds is poorly soluble or only slightly soluble in water, a dissolution accelerator or solubilizer may be added, and the solvent may also contain 10-60% w / w propylene glycol, etc.

[0081] Controlled-release parenteral composition The controlled-release parenteral composition may be in the form of an aqueous suspension, microspheres, microcapsules, magnetic microspheres, oil solution, oil suspension, or emulsion. Alternatively, the active agent may be incorporated into a biocompatible carrier, liposome, nanoparticles, implant, or infusion device.

[0082] Materials used for the preparation of microspheres and / or microcapsules are biodegradable / bioerobic polymers such as polygalactin, poly(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine), and poly(lactic acid). Biocompatible carriers that can be used when formulating controlled-release parenteral formulations are carbohydrates (e.g., dextran), proteins (e.g., albumin), lipoproteins, or antibodies. Materials used for implants may be non-biodegradable (e.g., polydimethylsiloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid), poly(orthoester), or combinations thereof).

[0083] Solid dosage form for oral use Oral formulations include tablets containing an active ingredient (e.g., a GPR17 antagonist and a microglial scavenger) mixed with a non-toxic, pharmaceutically acceptable excipient. Such formulations are known to those skilled in the art. Examples of additives include: inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch containing potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulators and disintegrants (e.g., cellulose derivatives containing microcrystalline cellulose, starch containing potato starch, croscarmellose sodium, alginate, or alginic acid); binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, aluminum magnesium silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); lubricants, flow enhancers, and anti-tacks (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharmaceutically acceptable additives include colorants, flavorings, plasticizers, wetting agents, and buffering agents.

[0084] Tablets are uncoated in some embodiments and coated in others. Optionally, tablets may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period. Coatings can be adapted to release the active agent in a predetermined pattern (e.g., to obtain a controlled-release formulation) or to prevent the release of the active agent until after passage through the stomach (enteric coating). Coatings in some embodiments are sugar coatings, film coatings (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycol and / or polyvinylpyrrolidone), or enteric coatings (e.g., based on methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac and / or ethylcellulose). Furthermore, time-delaying materials, such as glyceryl monostearate or glyceryl distearate, may also be utilized.

[0085] In some embodiments, the solid tablet composition includes a coating adapted to protect the composition from undesirable chemical changes (e.g., chemical decomposition before drug release). In some embodiments, the coating is applied to the solid dosage form in a manner similar to that described in the Encyclopedia of Pharmaceutical Technology, cited above.

[0086] In one embodiment, the GPR17 antagonist and the microglial scavenger may be mixed together or separated within the tablet. In one example, the GPR17 antagonist is contained on the inside of the tablet, and the microglial scavenger is on the outside, resulting in a significant portion of the microglial scavenger being released prior to the release of the GPR17 antagonist. In some embodiments, the microglial scavenger is contained on the inside of the tablet, and the GPR17 antagonist is on the outside.

[0087] Oral formulations include chewable tablets or hard gelatin capsules in which the active ingredients (i.e., GPR17 antagonist and microglial scavenger) are mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), and also include soft gelatin capsules in which the active ingredients are mixed with a water or oil medium, such as peanut oil, liquid paraffin, or olive oil. Powders and granules are prepared in some embodiments using conventional methods, for example, using a mixer, fluidized bed apparatus, or spray dryer, with the components described above for tablets and capsules.

[0088] Controlled-release oral dosage form Oral drug release control compositions can be constructed to release the drug, for example, by controlling the dissolution and / or diffusion of the active substance. Dissolution or diffusion control release can be achieved by appropriately coating tablets, capsules, pellets, or granules of the compound, or by incorporating a drug-containing composition into a suitable matrix. In some embodiments, release control coatings include one or more of the above-mentioned coating materials, and / or, for example, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glyceryl palmitostearate, ethylcellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxymethacrylate, methacrylate hydrogel, 1,3-butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. In controlled-release matrix formulations, examples of matrix materials include hydrated methylcellulose, carnauba wax and stearyl alcohol, Carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbons.

[0089] In some embodiments, drug-containing controlled-release compositions are in the form of buoyant tablets or capsules (i.e., tablets or capsules that float on the stomach contents for a period of time after oral administration). To prepare buoyant tablets of such compositions, a mixture of a GPR17 antagonist and a microglial scavenger is granulated with an excipient and 20-75% w / w of hydrophilic colloid, such as hydroxyethylcellulose, hydroxypropylcellulose, or hydroxypropylmethylcellulose. The resulting granules can then be compressed into tablets. Upon contact with gastric fluid, the tablets form a substantially water-impermeable gel barrier around their surface. This gel barrier contributes to maintaining a density of less than 1, thereby allowing the tablets to remain suspended in gastric fluid.

[0090] Aspects of this disclosure provide a method for treating a disease, disorder, or injury characterized by insufficient myelinization, comprising administering a therapeutically effective dose of a pharmaceutical composition comprising a GPR17 antagonist and a microglial scavenger to a subject (e.g., a mammal such as a human). The method comprises the step of administering to a subject a sufficient dose of the GPR17 antagonist and microglial scavenger to treat the disease, condition, disorder, injury, or symptoms thereof, under conditions such that the disease, condition, disorder, injury, or symptoms thereof are treated. The treatment may also include prophylactic measures. In some aspects, the subject is a mammal, particularly a human, that has, has, is susceptible to, or is at risk of having, a disease or disorder characterized by insufficient myelinization of target neurons.

[0091] Combination therapy Optionally, a GPR17 antagonist and a microglial scavenger may be administered in combination with other standard treatments for diseases, disorders, conditions, or injuries characterized by insufficient myelin formation; such methods are known to those skilled in the art and are described by E.W. Martin in Remington's Pharmaceutical Sciences.

[0092] Kit or pharmaceutical system The composition can be constructed into a kit or pharmaceutical system for use in increasing myelin formation in target neurons. The kit or pharmaceutical system includes a carrier, such as a box, carton, or tube, which contains one or more containers, such as vials, tubes, ampoules, or bottles, sealed inside. The kit or pharmaceutical system may also include relevant instructions for using the agents of the embodiments of this disclosure. In some embodiments, the kit comprises a GPR17 antagonist and a microglial scavenger. In some embodiments, the GPR17 antagonist is montelukast or pranlukast. In some embodiments, the microglial scavenger is PLX3397.

[0093] Identification of compounds and compositions The present invention provides a method for identifying compounds and compositions that can be used to differentiate oligodendrocyte progenitor cells (OPCs) into early-differentiated oligodendrocytes, or to differentiate early-differentiated oligodendrocytes into mature myelin-forming oligodendrocytes. Neuronal injury and disease characterized by insufficient myelin formation (e.g., multiple sclerosis) can be tested using an optic nerve crush mouse model. In this model, it is necessary to injure the optic nerve, as described below or as known in the art. Candidate compounds are administered to animals. In some embodiments, oligodendrocyte differentiation is observed as a measure of the efficacy of the compound(s) or composition(s) for differentiating OPCs or early-differentiated oligodendrocytes. In some embodiments, myelin formation of regenerating neurons is monitored to determine the efficacy of the candidate compounds.

[0094] This identification method is particularly well-suited for identifying candidate therapeutic agents for multiple sclerosis. Optic nerve injury, or optic crush, is a superior model of multiple sclerosis compared to conventional methods of injecting mice with androgens to induce demyelination. Animals injected with androgens enjoy spontaneous myelin formation after discontinuation of androgen administration, a phenomenon not observed with optic crush. Mice with optic crush injury exhibit increased inflammation and a lack of spontaneous remyelin formation, both common to multiple sclerosis, leukodystrophy, neurodegenerative Alzheimer's disease, and central nervous system injuries associated with myelin formation deficiency (e.g., traumatic brain injury, spinal cord injury).

[0095] Unless otherwise indicated, the embodiments of this disclosure employ prior art in molecular biology (including recombination techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the scope of those skilled in the art. Such techniques are well described in literature such as "Molecular Cloning, A Laboratory Manual", 2nd edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides of the present invention and can therefore be considered in the construction and implementation of the present invention. Techniques particularly useful for specific embodiments will be described in the following sections.

[0096] The following examples are provided to give a complete disclosure and explanation of how the assays, screenings, and therapeutics of the present invention are constructed and used, and are not intended to limit the scope of what the inventors consider to be the invention. [Examples]

[0097] Example 1: OPC proliferation induced by injury Various manipulations enhance the intrinsic regenerative capacity of retinal ganglion cells (RGCs), enabling robust axonal regeneration after optic tract injury. However, none of these regenerated axons have been known to co-stain with antibodies against myelin-associated glycoprotein (MAG). To further evaluate the myelin formation status of such regenerated axons induced by PTEN deletion, electron microscopy analysis of injured nerves was performed 4 weeks after optic nerve crush injury in which all RGC axons were severed (Park et al., Science 322, 963-966 (2008)). As expected, many regenerated axons with morphologies indistinguishable from naive axons were found in the optic nerve distal to the lesion (Figure 1A). However, of the thousands of axons analyzed, only one regenerated axon had a thin layer of myelin (Figure 1A). Therefore, similar to the optic tract injury model, most regenerated axons do not show spontaneous myelin formation in the injured optic nerve.

[0098] Since OPCs are responsible for myelin formation in adults, we investigated how OPCs respond to injury. First, we evaluated OPC proliferation in injured optic nerves (ipsilateral to crush injury) and control optic nerves (contralateral) of PDGFRα-H2B-GFP mice, in which all OPCs express nuclear H2B-GFP. Since GFP is also expressed in less than 5% of vascular cells and leptomeningeal cells in this reporter mouse strain, optic nerve sections were co-stained with the oligodendrocyte marker Olig2 to evaluate GFP. + and Olig2 + Double-positive cells were defined as OPCs (Figures 1B-E). As shown in Figures 1C and 1D, the total number of OPCs in crushed nerves significantly increased at 1 and 2 weeks post-injury, but returned to baseline levels at 4 weeks. In contrast, intact nerves showed similar numbers of GFP at all time points. + / Olig2 +OPCs were observed (Figure 1D). To further evaluate the dynamics of OPC proliferation due to such injury, these mice were injected with BrdU at different time points after injury, but the injection was stopped 3 hours after injury in the expectation of labeling OPCs in the process at each time point (Figure 1F, 1G). As a result, OPC proliferation due to injury increased most significantly around 3-5 days after injury, and then decreased thereafter, indicating rapid but reversible OPC proliferation induced by optic nerve injury (Figure 1G). In summary, these results suggest that crush injury induces rapid and reversible OPC proliferation in the injured optic nerve.

[0099] Example 2: Dysdifferentiation of OPCs proliferating in damaged optic nerves Next, we investigated whether these proliferated OPCs could differentiate. To track the offspring of the OPCs, we used different reporter mouse strains: PDGFRα-CreER (Kang et al., Neuron 68, 668-681, 2010) crossed with Rosa26-STOP-Tomato mice, or PDGFRα-CreER / iRTM mice (Arenkiel et al., 2011). When tamoxifen was administered, Cre expression was elevated to PDGFRα + RTM is induced in OPCs, and RTM is expressed not only in these OPCs but also in their differentiated offspring. To monitor their differentiation, two well-supported markers, namely CC1 and Oligo1, were used. CC1 is a marker for all cells differentiated from OPCs. The translocation of Oligo1 from the nucleus to the cytoplasm is associated with a prominent feature of late differentiation into myelin-forming oligodendrocytes. Therefore, these immunohistochemical evaluations allow us to distinguish these cells from undifferentiated OPCs (CC1 - (and nuclear Oligo1), early differentiated OPC (CC1) + (and nuclear Oligo1) and mature oligodendrocyte (CC1) + It could be divided into three stages: the cytoplasmic oligo1 and the cytoplasmic oligo1 (Figure 1H).

[0100] Tamoxifen was injected into PDGFRα-CreER / iRTM mice to label existing OPCs immediately before optic nerve contusion (Young et al., Neuron 77, 873-885, 2013). Next, the fate of the labeled OPCs was examined by immunohistochemistry using anti-CC1 and anti-Oligo1 at 4 weeks post-injury (Figure 1I). Total RTM was reduced 4 weeks after tamoxifen introduction. + Although the number is smaller in the undamaged contralateral optic nerve, RTM + 67% of cells are CC1 + They become oligodendrocytes, and about half of them are cytoplasmic Oligo1 + This was shown (Figure 1J-L). This is consistent with the concept of continuous myelin formation in adults. However, in damaged optic nerves of the same mice, only 17% of RTM was observed. + Cells CC1 + These CC1 + Most cells possessed intranuclear Oligo1 rather than intracytoplasmic Oligo1 (Figure 1J-1O), suggesting undifferentiated OPCs. These results indicate that in damaged optic nerves, OPCs require early differentiation (CC1) to differentiate into mature oligodendrocytes. + This suggests the existence of at least two blockades: (becoming) and late maturation (possessing intracellular Oligo1). Furthermore, it has been shown that OPCs may differentiate into astrocytes during development (Levison and Goldman, Neuron 10, 201-212, 1993), but RTM expressing the astrocyte marker GFAP + No cells were observed (Figure 1P). Thus, these data suggest that, in contrast to intact optic nerves, proliferating OPCs in damaged nerves exhibit differentiation blockade, closely resembling what has been observed in lesions in patients with progressive multiple sclerosis (Kuhlmann et al., Brain 132, 1118, 2008).

[0101] Example 3: Upregulation of GPR17 due to injury contributes to early differentiation failure of OPCs. Based on in vitro and EAE models, previous studies have identified various compounds that may promote OPC proliferation and / or differentiation. However, it remains unclear which of these agents can promote myelin formation in regenerating axons. As a first step in investigating such differentiation blockade of proliferating OPCs, we screened a series of small molecule compounds to identify those that increase differentiated OPCs in damaged optic nerves (Figure 2A-2C). Candidate compounds include the M1 / M3 muscarinic receptor antagonist benztropine mesylate (Bzp); antihistamines and anticholinergics; the M1 / M3 muscarinic receptor antagonist clemastine (Clem); the M3 muscarinic receptor antagonist solifenacin (Sli); the retinoid X receptor agonist bexarotene (Bex); the anticholesterol synthesis compound imidazole (Imi); the clinically approved phosphodiesterase (PDE) inhibitor ibudilast (Ibud); and two different GPR17 antagonists, montelukast (Mon) and pranlukast (Pra). The mTOR inhibitor rapamycin (Rap) was also included in the screening because it has been shown to improve myelin formation in TSC1 knockout mice (Meikle et al., 2008).

[0102] Since all of these compounds can cross the blood-brain barrier, each compound was administered systemically to wild-type C57BL / 6 mice for four weeks after optic nerve injury. To track the differentiation of OPCs during proliferation, mice were injected with BrdU daily from day 4 to day 10 post-injury, when OPCs showed a high proliferation rate (Figure 1G). For a further three weeks (as differentiation can take 2-3 weeks), the differentiation-promoting effect of each compound was investigated in the injured optic nerve using BrdU. + Evaluation was performed by measuring the expression of the oligodendrocyte marker CC1 on OPC. As shown in Figures 2B and 2C, the three compounds, including montelukast, benztropine mesylate, and solifenacin, were evaluated by BrdU + and CC1 +It significantly increased the number of double-positive cells. Since montelukast showed the strongest effect, further research focused on this compound and its putative target, GPR17.

[0103] As a first step to verify these observations, montelukast treatment was administered to PDGFRα-CreER / iRTM mice, and the OPC and its offspring after optic nerve injury were selectively visualized, similar to Figures 1H-M (Figure 2D). After 4 weeks of montelukast treatment, RFP + 64% of cells are CC1 + In contrast, the figure was 12% in vehicle-treated mice (Figures 2E-2G). Surprisingly, these CC1 + RTM + 77% of the cells showed an intranuclear Oligo1 immune response rather than a cytoplasmic one (Figure 2E-2G). Furthermore, total RTM + Cell count increased after montelukast treatment (Figure 2F). Since cell death is associated with failure of OPC differentiation, such RTM is likely to occur. + The increase in cells may result from improved differentiation and reduced cell death. In summary, these results suggest that montelukast treatment can promote the early differentiation of OPCs, but these cells are not yet able to progress to mature oligodendrocytes.

[0104] Montelukast is a clinically approved therapeutic agent for the treatment of asthma and seasonal allergies. Mechanistically, it acts as an antagonist of leukotriene receptors, including the G protein-coupled receptor GPR17. In addition to montelukast, pranlukast (Pra), another GPR17 antagonist, also increased OPC differentiation, but not to a statistically significant degree (Figure 2C). This may be related to differences in pharmacological properties, such as blood-brain barrier permeability (Marschallinger et al., 2015). Nevertheless, these results strengthen the concept of the role of GPR17 in the initiation of OPC differentiation. Interestingly, previous studies have shown that GPR17 expression is downregulated in the adult CNS, and that myelin formation appears normal in adult GPR17 knockout mice; thus, it has been suggested that GPR17 is an intrinsic timer of OPC differentiation during development (Chen et al., 2009). Therefore, we used insight hybridization to evaluate the expression patterns of GPR17 in optic nerves under different conditions. Consistent with previous reports (Chen et al., 2009), GPR17 expression was rarely detected in intact optic nerves of adult mice (Figure 2H, 2I). However, optic nerve crush injury induced a significant upregulation of GPR17 in the injured nerve, which was detectable both one and two weeks after injury (Figure 2H, 2I).

[0105] Since montelukast can inhibit other leukotriene receptors in addition to GPR17, the effect of GPR17 gene deletion on OPC differentiation in damaged optic nerves was evaluated using a GPR17 knock-in mouse line (Chen et al., Nat. Neurosci. 12, 1398-1406, 2009). In this line, the GPR17 coding region is replaced with a histone 2b-fusion GFP (H2b-GFP) sequence. Therefore, these mice can be used for monitoring GPR17 expression (by GFP signaling in both heterozygotes and homozygotes) and loss-of-function studies (homozygotes). As expected, from day 7 post-injury, GFP signaling was observed. + (GPR17 + ) Cells GPR17 + / - Mouse and GPR17 - / - These GFP levels were significantly increased in both mice (Figures 2J-2L). + Most of these were also co-stained with anti-Oligo2, consistent with limited expression in the OPC lineage (Figure 2J). By 30 days post-injury, GPR17 + / - In mice, GFP + Only 2.3% of cells are CC1 + While it was oligodendrocyte, GPR17 - / - In mice, GFP + 61% of cells are CC1 + This was the case (Figure 2M~2R). Consistent with montelukast treatment, GPR17 - / - GFP + The majority of cells showed Oligo1 immune response signals in the nucleus rather than the cytoplasm (Figures 2S-2U for dpi28, and Figures 2V-2X for dpi7). Furthermore, GFP + The number of cells is GPR17 + / - Compared to a mouse, GPR17 - / - The levels were significantly higher in mice (Figure 2P-2R). Since a similar number of labeled cells were observed in both groups after BrdU labeling (Figure 2J, 2L), such GFP + The difference in cell number is likely due to increased differentiation and consequently reduced cell death caused by GPR17 deletion. Consistently, at 4 weeks post-injury, GPR17 + / -GPR17 compared to mice - / - In the mouse, BrdU + CC1 + A tenfold increase in cells was observed (Figure 2Y and 2Z). Therefore, similar to montelukast treatment, GPR17 knockout promoted early differentiation but did not promote late maturation of proliferating OPCs in damaged optic nerves.

[0106] Example 4: Differential effect of rapidly or sustainedly activated microglia on the proliferation and maturation of OPCs. Based on observations of the partial effect of GPR17 inhibition on OPC differentiation, we sought additional inhibitors for the later maturation stages of OPC differentiation. A key clue was the presence of CC1 with cytoplasmic Oligo1 in injured nerves (ipsilateral) and control uninjured nerves (contralateral). + The difference was in the number of cells (Figure 1M-1O); this suggests that environmental factors may contribute to this late differentiation blockade. Consistent with inflammation and chronic Wallerian degeneration, which were well-characterized in damaged optic nerves rather than undamaged ones, microglia were rapidly and persistently activated in damaged optic nerves; this is demonstrated by positive staining with anti-CD68 antibody and lack of immunoreactivity with anti-P2Y12, a marker of homeostatic microglia (Figure 3A-E). Since inflammation is suggested to regulate OPC proliferation and differentiation, we further investigated the role of microglia in OPC proliferation and differentiation in damaged optic nerves.

[0107] Using the observation that systemic administration of PLX3397, a colony-stimulating factor 1 receptor (CSF1R) inhibitor, could specifically deplete microglia in vivo (Figure 3G, 3H), PDGFRα-H2B-GFP mice were pre-treated with PLX3397 or a control for 7 days prior to optic nerve crushing, and OPC proliferation was examined 14 days post-injury by BrdU / GFP / Olig2 labeling (Figure 3I). As shown in Figures 3J and 3K, PLX3397 treatment significantly reduced the total number of OPCs, leaving almost no cells for differentiation analysis. Therefore, it appears that microglial activation is necessary for injury-induced OPC proliferation.

[0108] Since most OPC proliferation occurs in the first week after injury (Figure 1), we hypothesized that delayed PLX3397 treatment at 2-4 weeks post-injury would avoid its inhibition of proliferation and allow for evaluation of its effect on OPC differentiation. To this end, we conducted another experiment in which PLX3397 was administered from 2 to 4 weeks post-injury in PDGFRα-CreER:iRTM mice used in Figures 1G-1M and 3L. As shown in Figures 3M-3O, PLX treatment was performed on CC1 + The cells increased. The important thing is these CC1 + RTM + The fact that 78% of the cells expressed cytoplasmic Oligo1 suggests that delayed removal of microglia promoted the maturation of early-differentiated OPCs into myelin-forming oligodendrocytes.

[0109] Example 5: Combination treatment with montelukast and PLX3397 led to robust myelin formation in regenerated axons. Observations of the differential effects of GPR17 inhibition and delayed microglia removal on OPC differentiation prompted the investigation of the effects of combination treatment on myelin formation of regenerating axons. To this end, AAV expressing osteopontin / IGF1 / CNTF was injected into the vitreous humor of PDGFRα-CreER:iRTM mice to activate the intrinsic regenerative capacity of RGCs and induced optic nerve damage over two weeks. These mice were then treated with montelukast (4 weeks at dpi1-dpi28) and / or PLX3397 (2 weeks at dpi15-28). As shown in Figures 4A-4C, the combination treatment was CC1 + RTM + Dramatically increasing the number of double-positive cells, these CC1 + The majority of the cells possessed cytoplasmic Oligo1; this supported the idea that this combination treatment promoted both early and late differentiation of OPCs.

[0110] A portion of the mice from each group were subjected to electron microscopy analysis (Figures 4D-4H) and additional immunohistochemistry (Figure 4I). As shown in Figures 4D and 4H, in mice treated with either montelukast or PLX3397, a portion of the regenerated axons (approximately 20%) were myelinated. However, the myelin structures after montelukast treatment were significantly thinner than those after PLX treatment; this is consistent with the results that montelukast or PLX3397 promotes the generation of early-differentiated OPCs (capable of sheathing axons) and mature oligodendrocytes (capable of forming mature myelin), respectively. In contrast, in mice treated with the combination of treatments, the majority of the regenerated axons (60%) were myelinated (Figures 4D and 4H). Many of these myelin structures were relatively thin and possessed a large inner tongue, suggesting that new myelin formation was underway (Figure 4E). However, the nodes of Ranvier were clearly observed (Figures 4F and 4G). Consistently, the nodes of Ranvier, and sometimes semi-nodes, were observed by immunohistochemistry using well-established markers, including Caspr, a component of the paranodal axoglial junction, and Nav and ankyrin G, two components of the nodes of Ranvier (Figure 4I). It is interesting to note that most of these regenerating axons did not pass through the optic chiasm; this suggests that such myelin formation occurs before these regenerating axons form functional synapses with their functional targets. Interestingly, treatments that promoted myelin formation resulted in significantly more and longer regenerating axons (Figures 4J-4L); this is likely related to the protective effect of myelin formation on axons. In summary, these studies have established combination treatments that enable robust myelin formation of regenerating axons in damaged optic nerves with persistent inflammation.

[0111] Example 6: TNFα expression is upregulated by optic nerve damage. Sequencing data showed that TNF expression was upregulated after optic nerve injury, at 1 and 3 weeks post-injury (Figure 5A, 5B). Data from TNFR1 KO mice showed increased CC1 expression in the distal region near the crush site compared to heterozygous littermates. + A steady increase in oligodendrocytes was observed (Figure 5C). By using the BrdU pulse chase protocol, these mice showed significantly more BrdU + Olig2 + Cells were observed. This indicated increased survival and / or differentiation of cells derived from OPC progenitor cells. Less BrdU after 3 hours of BrdU labeling. + Olig2 + Since cells were shown (Figure 5C), this effect was not due to increased OPC proliferation.

[0112] When the TNF inhibitor thalidomide was administered from 2 to 4 weeks after injury, BrdU + / CC1 + / Olig2 + A steady increase in cells was observed in the distal region of the injury. Using PDGFRα-CreER / iRTM strain reporter mice, it was found that OPCs differentiated into mature myelin-forming oligodendrocytes in response to this treatment (Figure 5D). Their morphology was very different from that of other treatments, such as GPR17 antagonist treatment (Figure 5E).

[0113] In an analysis of the underlying mechanism of myelin formation defects in regenerating axons using an optic nerve injury model, it was found that OPCs showed rapid proliferation but were unable to differentiate into mature myelin-forming oligodendrocytes. Mechanistic research focused on CC1 of OPCs. +We revealed two distinct differentiation blockades mediated by remarkably different mechanisms: damage-induced GPR17, which hinders early differentiation into cells, and damage-activated microglia, which block the maturation stage to myelin-forming oligodendrocytes (possessing cytoplasmic Oligo1). While individual manipulations increased myelin formation to some extent, combined manipulations led to robust myelin formation in regenerated axons, highlighting the importance of addressing both endogenous and exogenous mechanisms simultaneously. Combined with recent advances in promoting axonal regeneration in adult CNS, these results provide important insights into addressing another major impairment toward the reconstruction of functionally meaningful neural circuits. Interestingly, the dynamics of OPCs observed in damaged optic nerves / optic tracts closely resemble those reported in lesions from patients with progressive multiple sclerosis, both of which show that proliferating OPCs are unable to differentiate into mature oligodendrocytes. Since activated microglia are dominant in damaged optic nerves and lesions of multiple sclerosis, the results reported here may have significant implications for designing myelogenesis-promoting therapeutic interventions in patients with progressive MS.

[0114] Many molecules are involved as important regulators of OPC differentiation. Surprisingly, montelukast appears to be the most powerful in promoting the early stages of OPC differentiation. While montelukast can target GPR17 and other cysteinyl leukotriene receptors, similar results observed in GPR17 knockout and montelukast-treated studies identify GPR17 as the most important target. In this regard, GPR17 is dramatically upregulated in damaged optic nerves, most so in early OPC lineage cells, but also in CC1 + It was shown to be very low in cells, consistent with previous reports (Chen et al., 2009; Fumagalli et al., 2011). However, GPR17 inhibition was found to affect the majority of these cells (GFP in transgenic mice). + ) is CC1 +This promoted differentiation into cells. Interestingly, the cell count in this lineage also increased significantly after GPR17 inhibition. This may be related to injury-related factors, as this was not observed in developmental knockout mice. Since GPR17 is activated by cysteinyl leukotrienes, pro-inflammatory factors may also activate GPR17, inhibiting the differentiation and even proliferation of OPCs expressing GPR17. It is important to note that several other molecules may also be involved in this process, as two other M1 / M3 muscarinic receptor antagonists, benztropine and solifenacin, also significantly increased OPC differentiation. Furthermore, another M1 / M3 muscarinic receptor antagonist, clemastine, and the retinoid X receptor agonist, bexarotene, also significantly increased OPC differentiation. + Although the number of cells increased, it did not reach a statistically significant difference.

[0115] The results presented herein also demonstrate the dual role of microglia in OPC dynamics: acutely activated microglia stimulate OPC proliferation, while chronically activated microglia inhibit OPC differentiation, particularly the maturation stage to myelin-forming oligodendrocytes. Indeed, a link between microglia and myelin formation has been proposed. Subsequently, it has been reported that inflammation can stimulate myelin formation by transplanted OPCs, and many studies have identified microglia as an important regulator of myelin formation. More recently, it has been shown that chemotherapy such as methotrexate leads to sustained activation of microglia, which contributes to impaired OPC differentiation.

[0116] In summary, this disclosure demonstrates that only the simultaneous manipulation of both endogenous (GPR17) and exogenous (microglia) factors can achieve robust myelin formation in regenerating axons. Future research will investigate whether such treatments enhance behavioral improvements in various injury models. Importantly, defective myelin formation is associated with neurodegenerative diseases such as multiple sclerosis, leukodystrophy, neurodegenerative Alzheimer's disease, and central nervous system injuries associated with myelin formation deficiency (e.g., traumatic brain injury, spinal cord injury). Since neuroinflammation may be found in these conditions, it would be interesting to examine the activation state of microglia and test the effects of our manipulations on these conditions.

[0117] The results reported in this specification were obtained using the following materials and methods.

[0118] Mouse strain All experimental procedures were carried out in accordance with animal protocols approved by the Institutional Animal Care and Use Committee of Boston Children's Hospital. Gpr17 transgenic mice were obtained from Dr. Richard Lu (Chen et al., 2009). Other mouse strains were obtained from Jackson Laboratory (Table 1). Experiments were started when the mice were 6-8 weeks old. Both male and female mice were randomized and assigned to different treatment groups before injury. No other specific randomization was employed in this animal experiment. Quantification was performed blindly.

[0119] antibody The following were used as primary antibodies: Rabbit anti-Oligo1 (1:50, donated by Dr. Charles D Stiles), Rabbit anti-Oligo2 (1:300, Novus Biologicals, NBP1-28667), Rat anti-PDGFRα (CD140a) (1:100, BD Bioscience, 558774), Mouse anti-CC1 (APC) (1:100, Millipore, OP80), Rat anti-BrdU (1:300, Abcam, ab6326), Mouse anti-Nav1.6 (1:50, Antibodies Incorporated, 75-026), Mouse anti-Ankyrin G (AnkG) (1:50, Antibodies Abcam, Inc., 75-146), rabbit anti-Caspr (1:1000, Abcam, ab34151), rat anti-MBP (1:300, Abcam, ab7349), mouse anti-MAG (1:100, Millipore, MAB1567), rat anti-CD68 (1:300, Bio-Rad, MCA1957), rabbit anti-Iba1 (1:500, WAKO Pure Chemicals, 019-19741), rabbit anti-P2Y12 (1:500, AnaSpec, AS-55043A), rat anti-GFAP (1:1000, ThermoFisher, 13-0300), rabbit anti-RFP (1:500, Abcam, ab34771). Secondary antibodies (Invitrogen) were produced in each goat species for the host species of the primary antibody, highly cross-adsorbed, and conjugated with the fluorescent dyes Alexa Fluor 488, Alexa Fluor 594, or Alexa Fluor 647, and used at a dilution of 1:500.

[0120] Method details Virus production All AAV virus vectors were produced at the Boston Children's Hospital Viral Core. In this study, AAV serotype 2 was used as follows: AAV2-Cre; AAV2-CNTF; AAV2-IGF1; AAV2-OPN. The titer of all viral preparations was at least 1.0 × 10⁶. 13The result was GC / mL.

[0121] Surgical treatment For all surgical procedures, mice were anesthetized with ketamine and xylazine, and buprenorphine was administered as a postoperative analgesic.

[0122] AAV virus injection As previously mentioned, intravitreal viral injection was performed two weeks prior to optic nerve crush injury to enable axonal regeneration. Briefly, a pulled glass micropipette was inserted near the peripheral retina behind the ora serrata, intentionally angled to avoid damage to the lens. Pten f / f mice were injected with 2 μl of AAV2 / 2-CAG-Cre virus (Park et al., 2008). Other mouse strains were injected with 2 μl of a combination of AAV2 / 2-CAG-CNTF, AAV2 / 2-CAG-IGF, and AAV2 / 2-CAG-OPN (1:1:1 mixture) (Bei et al., 2016).

[0123] optic nerve damage As previously described, the optic nerve was exposed within the orbit, and approximately 500 μm posterior to the optic disc was crushed for 5 seconds using a thin forceps (Dumont #5 FST). Afterward, ophthalmic ointment was applied postoperatively to protect the cornea. Robust axonal regeneration could be observed from two weeks post-crushing, using Alexa-conjugated cholera toxin subunit B labeling.

[0124] The compound was administered. PDGFRα-CreER mice were administered tamoxifen (100 mg / kg, po) for 5 days immediately before optic nerve contusion. In the OPC proliferation assay, BrdU (100 mg / kg, ip) was injected 3 hours before sample collection. In the drug screening assay, BrdU was injected daily from day 4 to day 10 after optic nerve contusion. Each compound or the corresponding vehicle was administered once daily for 4 weeks, starting from day 1 after optic nerve contusion (Table 2). As previously mentioned, the dosage and route of administration of the test compounds were as follows: bexarotene (100 mg / kg, po), benztropine mesylate (10 mg / kg, ip), clemastine fumarate (10 mg / kg, po), ibudilast (10 mg / kg, ip), imidazole (10 mg / kg, ip), montelukast (25 mg / kg, po), pranlukast (0.5 mg / kg, ip), rapamycin (6 mg / kg, ip), and solifenacin succinate (20 mg / kg, ip). Pexidartinib (PLX 3397) was mixed into the feed at a dose of 290 mg per kg of LabDiet laboratory animal feed.

[0125] Perfusion and tissue treatment For immunohistochemical staining, animals were anesthetized to an overdose and perfused transcardially with ice-cold PBS, followed by 4% paraformaldehyde (PFA, Sigma). After perfusion, the optic nerve was detached and post-fixed overnight at 4°C with 4% PFA. The tissue was immersed in 30% sucrose in PBS for 48 hours to prevent freezing. The samples were frozen using dry ice with Optimal Cutting Temperature compound (Tissue Tek), and the optic nerve was sectioned into 12 mm sections.

[0126] Immunohistochemical staining and image analysis Frozen sections (12 μm thick) were permeabilized and blocked in blocking buffer (0.5% Triton X-100 and 5% normal goat serum in PBS) at room temperature for 1 hour, followed by overlaying with primary antibody at 4°C overnight (Table 1). For BrdU staining, cell or tissue sections were denatured in 2N HCl at 37°C for 30 minutes, then neutralized in 0.1M sodium borate buffer for 10 minutes before proceeding to the standard blocking procedure. The following day, the corresponding Alexa Fluor 488-, 594-, or 647-conjugated secondary antibody was added (all secondary antibodies were purchased from Invitrogen). All stained sections were mounted in a solution containing DAPI-containing mounting fluid and sealed with glass coverslips. All immunofluorescence-labeled images were acquired using a Zeiss 700 or Zeiss 710 confocal microscope. For each biological sample, 3–5 sections of each optic nerve were imaged with a 10x or 20x objective lens for quantification. The number of positive cells was then manually quantified using the Plugins / Analyze / Cell Counter function in ImageJ software. For fluorescence intensity analysis, images were first converted to 8-bit depth in ImageJ software, and then the average intensity value was calculated using the built-in Analyze / Measure function.

[0127] Quantification of tissue clearing, imaging, and optic nerve regeneration Mice injected with cholera toxin B (CTB) tagged with a fluorescent dye were perfused with 4% paraformaldehyde. Next, the detached optic nerves were cleared for direct fluorescence imaging using a modified method from the previously published iDISCO tissue clearing method (Renier et al., 2014). This method has been tested for better preservation of CTB fluorescence during tissue clearing and for minimal optic nerve morphological changes. For dehydration, optic nerve samples were incubated in the dark for 0.5 hours in 80% tetrahydrofuran (THF, Sigma-Aldrich 360589-500ML) / H2O, then switched to 100% THF and incubated for 1 hour. The samples were then incubated in dichloromethane (DCM, Sigma-Aldrich 270997-1L) for 20 minutes (the nerves should sink to the bottom). Finally, the samples were switched to dibenzyl ether (DBE, Sigma-Aldrich 33630-250ML) until completely clear (at least 3 hours, but overnight is recommended). Clear nerves can be stored in DBE for at least one year without significant fluorescence decay in the CTB. For imaging, the processed nerves could be mounted in DBE and imaged with a Zeiss 710 confocal microscope. Z-stack scans and maximum projection of Z-stack images were used to capture all regenerating axons. For image analysis, a fluorescence intensity profile along the nerve was created using the ImageJ built-in function: Analyze / Plot Profile. A Matlab algorithm was developed to calculate the integral of fluorescence intensity over the entire length of the nerve, and the "area under the curve" was quantified from the plot profile data created in ImageJ.

[0128] Electron microscopy and morphometric analysis Mice were perfused with 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.2). Optic nerves were detached and fixed overnight with the same fixative. Subsequently, samples were processed in a Harvard EM core according to the following procedure: nerves were washed with PBS, fixed in 1% OsO4 in PBS for 1 hour, dehydrated in a stepwise ethanol series, infiltrated with propylene oxide, and embedded in Epon. Semithin sections were stained with toluidine blue, and ultrathin sections were stained with lead citrate. Ultrathin sections were imaged under a JEOL 1200EX - 80kV electron microscope. In ultrathin sections, the number of myelinated axons per nerve was analyzed at magnifications of 3,000x to 20,000x.

[0129] Insight Hybridization To evaluate the expression pattern of Gpr17, insight hybridization was performed using hybridization chain reaction (HCR) (Choi et al., 2018), employing a commercially available kit from Molecular Instruments containing a DNA probe set, DNA HCR amplifier, and different buffers. To prepare sections for insight hybridization, anesthetized mice were perfused with DEPC-PBS, followed by perfusion with 4% paraformaldehyde (PFA). The detached optic nerves were fixed overnight with 4% PFA, dehydrated in 30% sucrose / DEPC-PBS at 4°C, embedded in OCT, and 14 μm frozen sections were prepared. The tissue was permeabilized in 5% SDS at room temperature (RT) for 20 minutes and pre-hybridized in hybridization buffer at 37°C for 3 hours. The slides were then incubated overnight in pre-warmed hybridization buffer at 37°C containing probes (2.5 nM each). After hybridization, the slices were washed with washing buffer at 37°C for 1 hour, and then washed with 2x SSC in RT mode for 15 minutes. The amplification step was performed overnight in RT mode using a B3 HCR amplifier.

[0130] Quantification and statistical analysis Normality and similarity of variances were measured using STATA (version 12, College Station, TX, USA) before applying parametric tests. Two-sided Student's t-tests were used for single comparisons between two groups. The remaining data were analyzed using one-way or two-way ANOVA as appropriate. Post-hoc comparisons were performed only if the primary measure was statistically significant. P-values ​​for multiple comparisons were adjusted using Bonferroni correction. Error bars in all figures represent mean ± SEM. Mice were randomized to different litters, body weights, and sexes and assigned to different treatment groups; no other specific randomization was used in this animal study.

[0131] Other aspects From the above description, it will be clear that the present invention can be modified and altered to introduce the invention described herein to various uses and conditions. Such embodiments are also included in the following claims.

[0132] In this specification, the listing of elements in the definition of a variable element includes the definition of that variable element as any single element or as a combination (or partial combination) of the listed elements. In this specification, the description of an embodiment includes that embodiment as any single embodiment or as a combination with any other embodiment or part thereof.

[0133] All patents and publications listed herein are incorporated herein by reference to the same extent as each individual patent and publication is specifically and individually indicated as being incorporated by reference.

Claims

1. A method for increasing axon myelination, comprising the step of contacting oligodendrocyte progenitor cells (OPCs) in the presence of axons with a drug that inhibits GPR17 and / or a drug that removes and / or inhibits activated microglia, thereby increasing axon myelination.

2. A method for increasing axon myelination, comprising the step of contacting oligodendrocyte progenitor cells (OPCs) in the presence of axons with a drug that inhibits GPR17 and / or a drug that inhibits TNFα receptor 2 or TNFα, thereby increasing axon myelination.

3. A method for increasing the number and / or differentiation of OPCs, comprising the step of contacting oligodendrocyte progenitor cells (OPCs) with a drug that inhibits GPR17 and / or a drug that removes or inhibits activated microglia, thereby increasing the number and / or differentiation of OPCs.

4. A method for increasing the number and / or differentiation of OPCs, comprising the step of contacting oligodendrocyte progenitor cells (OPCs) with a drug that inhibits GPR17 and / or a drug that inhibits TNFα receptor 2 or TNFα, thereby increasing the number and / or differentiation of OPCs.

5. The method according to any one of claims 1 to 4, wherein the drug that inhibits GPR17 is montelukast or pranlukast.

6. The method according to any one of claims 1, 3, or 5, wherein the agent for removing or inhibiting activated microglia is PLX3397.

7. The method according to claim 2, 4, or 5, wherein the agent inhibiting TNFα receptor 2 or TNFα is thalidomide.

8. A method for increasing axon myelination, comprising the step of contacting oligodendrocyte progenitor cells (OPCs) in the presence of axons with a drug selected from the group consisting of benztropine mesylate, clemastine, montelukast, pranlukast, and thalidomide, thereby increasing axon myelination.

9. A method for increasing the number and / or differentiation of OPCs, comprising the step of contacting oligodendrocyte progenitor cells (OPCs) with a drug selected from the group consisting of benztropine mesylate, clemastine, montelukast, pranlukast, and thalidomide, thereby increasing the number and / or differentiation of OPCs.

10. The method according to any one of claims 1 to 9, for increasing the number of CC1 and / or Oligo1-positive OPCs.

11. The method according to claim 8 or 9, wherein the drug is montelukast, benztropine mesylate, clemastine, thalidomide, or pranlukast.

12. The method according to claim 9, wherein the drug is montelukast.

13. The method according to any one of claims 1 to 12, wherein the drugs are administered simultaneously or sequentially.

14. The method according to any one of claims 1 to 12, wherein a drug that inhibits GPR17 is administered simultaneously with a drug that removes or inhibits activated microglia.

15. The method according to any one of claims 1 to 12, wherein a drug that inhibits GPR17 is administered at least one week before a drug that removes or inhibits activated microglia.

16. The method according to any one of claims 1 to 15, wherein the drug is administered before, at the same time as, or after the injury.

17. The method according to claim 15, wherein the drug is administered several days or weeks after the injury.

18. The method according to claim 17, wherein the drug is administered one to two weeks after the injury.

19. The method according to any one of claims 1 to 17, wherein the drug is administered for at least 14 to 28 days.

20. The method according to claim 1, wherein the axon is damaged and / or demyelinates.

21. The method according to any one of claims 1 to 20, performed in vivo or in vitro.

22. A method for increasing axon myelination in a subject, comprising the step of administering to the subject an agent that inhibits GPR17 and / or an agent that removes or inhibits activated microglia, thereby increasing axon myelination.

23. A method for increasing the number and / or differentiation of OPCs in a subject, comprising the step of administering to the subject a drug that inhibits GPR17 and / or a drug that removes or inhibits activated microglia, thereby increasing the number and / or differentiation of OPCs.

24. The method according to claim 22 or 23, wherein the agent inhibiting GPR17 is montelukast or pranlukast.

25. The method according to any one of claims 22 to 24, wherein the agent for removing or inhibiting activated microglia is PLX3397.

26. A method for increasing axon myelination in a subject requiring such treatment, comprising the step of administering to the subject an agent selected from the group consisting of benztropine mesylate, clemastine, montelukast, pranlukast, and thalidomide, thereby increasing axon myelination.

27. A method for increasing the number and / or differentiation of OPCs in a subject in need, comprising the step of administering to the subject a drug selected from the group consisting of benztropine mesylate, clemastine, montelukast, pranlukast, and thalidomide, thereby increasing the number and / or differentiation of OPCs.

28. A method for treating a subject having a disease or injury associated with myelin dysplasia, comprising the step of administering to the subject an agent that inhibits GPR17 and / or an agent that removes or inhibits activated microglia.

29. The method according to claim 27 or 28, which increases the number of CC1 and / or Oligo1-positive OPCs.

30. The method according to any one of claims 27 to 29, wherein the subject has multiple sclerosis (MS), leukodystrophy, neurodegenerative Alzheimer's disease, traumatic brain injury, spinal cord injury, or optic nerve injury.

31. The method according to claim 30, wherein the leukodystrophy is adrenoleukodystrophy (ALD), Ecardi-Goutier syndrome, Alexander disease, Canavan disease, cerebral tenosynovitis xanthomatosis (CTX), globoid cell leukodystrophy (Krabbe disease), metachromatic leukodystrophy (MLD), Pelizaeus-Merzbach disease (X-linked spastic paraplegia), or childhood ataxia with central nervous system hypomyelination (CACH).

32. The method according to any one of claims 27 to 29, wherein the drugs are administered simultaneously or sequentially.

33. The method according to any one of claims 27 to 29, wherein a drug that inhibits GPR17 is administered simultaneously with a drug that removes or inhibits activated microglia.

34. The method according to any one of claims 27 to 33, wherein a drug that inhibits GPR17 is administered at least one week before a drug that removes or inhibits activated microglia.

35. The method according to any one of claims 27 to 33, wherein the drug is administered before, at the same time as, or after the injury.

36. The method according to claim 35, wherein the drug is administered several days or weeks after the injury.

37. The method according to claim 36, wherein the drug is administered one to two weeks after the injury.

38. The method according to claim 30, wherein the traumatic brain injury is a concussion.

39. The method according to any one of claims 1 to 38, wherein the oligodendrocyte precursor cell is CC1- and has Oligo1 localized in the nucleus.

40. The method according to any one of claims 1 to 39, wherein the OPC is an early differentiated oligodendrocyte that is CC1+ and has Oligo1 localized in the nucleus.

41. The method according to any one of claims 1 to 39, wherein the OPC is a differentiated oligodendrocyte that is CC1+ and has Oligo1 localized in the cytoplasm.

42. A composition comprising a GPR17 antagonist and a microglia inhibitor or scavenger or TNFα inhibitor.

43. The composition according to claim 42, wherein the GPR17 antagonist is montelukast.

44. The composition according to claim 42, wherein the microglia inhibitor or scavenger is PLX3397.

45. A method for identifying compounds that induce differentiation of oligodendrocytes or oligodendrocyte progenitor cells, A process that damages the optic nerve of a mouse; A step of applying a drug that regenerates axons to the optic nerve; A step of administering a candidate compound to a mouse in order to induce differentiation of oligodendrocyte progenitor cells; The step of administering a known microglia inhibitor or scavenger; and A step of determining the differentiation state of oligodendrocytes or oligodendrocyte progenitor cells, wherein an increase in CCl+ oligodendrocytes compared to an untreated control indicates that the candidate compound induced the differentiation of oligodendrocyte progenitor cells. The method, including the method described above.

46. A method for identifying compounds that induce differentiation of oligodendrocytes or oligodendrocyte progenitor cells, A process that damages the optic nerve of a mouse; A step of applying a drug that regenerates axons to the optic nerve; A step of administering to the mouse a compound known to induce differentiation of oligodendrocyte progenitor cells; The step of administering a predicted microglia inhibitor or scavenger; and A step of determining the differentiation state of oligodendrocytes or oligodendrocyte progenitor cells, wherein an increase in CCl+ oligodendrocytes having cytoplasmic Oligo1 compared to an untreated control indicates that the expected microglia inhibitor or scavenger effectively inhibited or removed microglial cells. The method, including the method described above.

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