WO2022177967A1 - Remyelination therapy by targeting adgrg1 and its effectors in oligodendrocytes - Google Patents

Abstract

ADGRG1 activity was found to promote myelination by its activity in pre-myelinating oligodendrocytes. Interventions made at various points along the ADGRG1-mediated myelination process can promote myelination. ADGRG1 activators, RhoA activators, ROCK activators, and cofilin inhibitors promote myelination. These myelinating agents may be applied in methods of treating demyelinating conditions, such as multiple sclerosis.

Description

Title: Remyelination Therapy By Targeting ADGRGl and Its Effectors in Oligodendrocytes

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS: This application claims the benefit of priority to United States Provisional Patent Application Serial Number 63/150,052 entitled “Remyelination Therapy By Targeting ADGRG1 and Its Effectors in Oligodendrocytes,” filed February 16, 2021, the contents of which are hereby incorporated by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT: This invention was made with government support under grant number NS094164 awarded by The National Institutes of Health. The government has certain rights in the invention.

[0003] Background of the Invention

[0004] In brain development and function, the deposition and maintenance of the myelin sheath around the axons by specialized glia named oligodendrocytes (OLs) is critical to central nervous system (CNS) function. OLs follow a step-wise differentiation process to create and maintain the myelin sheath. During myelination, OL precursor cells (OPCs) expand by locally proliferating, typically exhibiting a bipolar morphology during the search for axons to myelinate. Next, OPCs differentiate into pre-myelinating OLs (pmOLs), where process branching becomes much more elaborate and complex. At this stage, pmOLs are responsible for the ensheathment of axons, a critical intermediate step that precedes the deposition of myelin. After ensheathment, pmOLs further differentiate into mature myelinating OLs (mOLs), spirally wrapping around the axon while simultaneously extending myelin along the axon longitudinally.

[0005] Demyelination is a pathological process that underlies various conditions, such as multiple sclerosis (MS), congenital and acquired white matter defects in premature infants and neonates, and other conditions. In demyelinating conditions, defects in myelin sheath formation and/or maintenance of the myelin sheath occur. In many such conditions, for example, in MS, autoimmune activity is implicated in the demyelination processes.

Currently approved therapeutics for demyelinating diseases focus on preventing the immune system from causing further damage to myelin. However, to date, no therapeutic has been approved to directly target the OLs in order to stimulate remyelination. This is largely due to the fact that the molecular mechanisms of myelination are currently not well understood. Accordingly, there is a need in the art for an improved understanding of how OLs coordinate and achieve the myelination process.

[0006] The G-protein coupled receptor (GPCR) ADGRG1 (also known as GPR56) is known to have a role in myelination. Previous studies have shown the importance of ADGRG1 in neurodevelopment, including in cortical lamination, neurulation, synaptogenesis, and myelination. Previous studies of ADGRG1 in myelination have largely focused on this receptor in the context of oligodendrocyte precursor cells (OPCs), finding reduced proliferation of OPCs when the Adgrgl gene is knocked out. Much research focus has been placed on enhancing OPC proliferation as a restorative measure. However, recent single-cell RNA-sequencing data revealed that the highest expression of Adgrgl is in pmOLs, a stage of OL development that occurs after proliferation of OPCs. Furthermore, a recent study has demonstrated that it is the surviving OLs that remyelinate the so-called “shadow plaques” or demyelinated lesions rather than newly generated OPCs. Therefore, it is clear that therapies targeting OLs must address and enhance the function of the surviving OLs, rather than focusing only on the proliferation of OPCs Accordingly, there is a strong need in the art for an understanding of how ADGRG1 functions in OLs to achieve myelination and maintenance of myelin sheath integrity. Additionally, there is a need in the art for more effective treatments for demyelinating conditions such as MS. These needs are met, and other benefits are provided, by the inventions disclosed herein

[0007] Summary of the Invention

[0008] The inventors of the present disclosure have derived numerous insights into the mechanisms of myelination, as described herein. The inventors of the present disclosure have elucidated various mechanisms of myelination and remyelination and have developed therapeutic methods and compositions for the treatment of demyelinating conditions.

[0009] In a first aspect, the inventors of the present disclosure have determined that ADGRGl controls actin polymerization and subsequent myelin formation, specifically at the pmOL stage of axon ensheathment, and that activation of ADGRGl in pmOLs significantly enhances remyelination. Accordingly, in one implementation, the scope of the invention encompasses activators of ADGRG1 for use in a method or promoting myelination in a subject, for example, activators of ADGRG1 for use in a method of treating a demyelinating condition in a subject.

[0010] In another aspect, the inventors of the present disclosure have elucidated the intracellular signaling mechanisms by which ADGRG1 exerts its effects on myelination in pmOLs. As demonstrated herein, ADGRG1 signals through G0112/13 and RhoA, then through Rho-associated protein kinase (ROCK), LIM Idnase (LIMK), and finally through cofilin in order to regulate the F-actin polymerization that is essential to initiation of myelination. Furthermore, the inventors of the present disclosure have demonstrated that interventions at various points along the ADGRG1 signaling pathway can promote myelination. Specifically myelination can be promoted by the activation of RhoA, activation of ROCK, and the inhibition of cofilin.

[0011] Accordingly, the scope of the invention encompasses methods of promoting myelination, for example, treating a demyelinating condition, wherein the method encompasses applying an intervention in the downstream ADGRG1 signaling pathway to promote myelination processes. In one implementation, the scope of the invention encompasses activators of RhoA for use in a method of promoting myelination in a subject, for example, activators of RhoA for use in a method of treating a demyelinating condition in a subject. In one implementation, the scope of the invention encompasses activators of ROCK for use in a method of promoting myelination in a subject, for example, activators of ROCK for use in a method of treating a demyelinating condition in a subject. In one implementation the scope of the invention encompasses inhibitors of cofilin for use in a method of promoting myelination in a subject, for example, inhibitors of cofilin for use in a method of treating a demyelinating condition in a subject.

[0012] BRIEF DESCRIPTIN OF THE DRAWINGS

[0013] Fig. 1. Fig. 1 depicts downstream signaling from ADGRG1 that contributes to myelin formation. Signaling through ADGRG1 leads to Gai2_/i3 activation and subsequent RhoA, ROCK, and LIMK activity. LIMK serves to inhibit cofilin which leads to subsequent actin polymerization. Various points of this pathway can be targeted (ovals) to promote remyelination.

[0013] Fig. 2. Fig. 2 depicts effects of interventions applied to downstream effectors of ADGRGl on axon myelination in Shiverer mouse cerebellar slices co-cultured with OPCs from control or Adgrgl knockout (cKO) mice, 14 days following microinjection of transplanted OPC’s, quantified by immunostaining for axons (NF200) and myelin (MBP). Treatments were added four days following transplantation of OPCs: vehicle control (DMSO); RhoA Inhibitor I (RI); Rock inhibitor Y27632; LIMK inhibitor (Pyrl); RhoA Activator II (RA); Rock activator Calpeptin (Calp); and cofilin inhibitor Cucurbitacin E (CCB E).

[0014] Fig. 3. Fig. 3 depicts the effects of interventions on ADGRG1 downstream effectors on myelin sheath length. The frequency distribution of MBP-positive sheaths was observed on microfibers after 14 days being cultured with OPC’s from ADGRGl knockout mice (cKO). Gaussian regression paired with a log transformation of sheath lengths was applied. Treatments were added at day four: Vehicle control (DMSO); RhoA Activator II (RA); Rock activator Calpeptin; and cofilin inhibitor Cucurbitacin E (CCB E).

[0015] Fig. 4A and 4B. Fig 4A and 4B depict the effect of interventions on ADGRGl downstream effectors on myelination by OLs from ADGRG1 knockout mice (cKO). Data were derived from measurements of MBP stained sheaths produced by OLs, after 14 days of culture of OPC’s from cKO mice on microfibers. Treatments were added at day four: Vehicle control (DMSO); Vehicle control (DMSO); RhoA Activator II (RA); Rock activator Calpeptin; and cofilin inhibitor Cucurbitacin E (CCB E). Fig. 4A: Effects of treatments on the number of sheaths produced per OL, wherein frequency is depicted by oval width. Fig. 4B: Effects of treatments on the distribution of sheath length per cell.

[0016] DETAILED DESCRIPTION OF THE INVENTION.

[0017] In a general method, the scope of the invention encompasses the administration of a therapeutically effective amount of a myelinating agent to a subject to promote myelination in the nervous system of the subject. “Promoting myelination” may encompass any myelination of axons, for example, enhancing myelination of developing axons, maintenance of myelin sheath integrity, or remyelinating demyelinated axons. In a primary implementation, the methods of the invention are applied in the treatment of a demyelinating condition, such as MS. The various elements of the general method and specific implementations thereof are described next. [0018] Demyelinating Conditions. The methods and compositions disclosed herein are directed to the treatment of demyelinating conditions. A “demyelinating condition” encompasses any condition wherein a subject has damaged myelin or impaired myelination capacity, for example, impaired myelin formation in a developmental context (e.g. congenital defects) or a disease state wherein the loss or damage of myelin occurs. In a primary implementation, the demyelinating condition is a condition of the central nervous system. In an alternative implementation, the demyelinating condition is a condition of the peripheral nervous system.

[0019] In a primary embodiment, the demyelinating condition is Multiple Sclerosis, referred to commonly and herein as “MS.” MS is the most common chronic autoimmune disease of the central nervous system. The disease is characterized by the formation of lesions, also called plaques, in the CNS as well as neuroinflammation, and the destruction of the myelin sheaths of axons. The cause of MS is generally believed to be autoimmune attack on myelin antigens and other components of the CNS involved in myelin deposition, maintenance, and remyelination. These immune and inflammatory processes result in thinning or loss of myelin and the eventual breakdown of axons and neuron dysfunction. Impaired remyelination capacity in MS subjects further contributes to the progression and pathology of the disease.

[0020] The methods of the invention are directed to treatment of MS in all forms and stages. The treatment methods of the invention may be applied at any stage of MS. In one implementation, the methods are preventative, applied to subjects at risk of MS. In one implementation, the methods are applied in the pre-clinical MS stage, in which the initial processes of the disease are initiated. The treatment methods of the invention may be applied during the relapsing-remitting (RRMS) clinical stage, which encompasses periodic episodes of neurologic dysfunction. The treatment methods of the invention may also be applied during the progressive clinical stage, during which substantial and progressive neurodegeneration is occurring.

[0021] In other implementations, the methods of the invention are applied to a demyelinating condition of the CNS other than MS. In various embodiments, the demyelinating condition may be any of: bilateral frontoparietal polymicrogyria, Devic’s disease; acute disseminated encephalomyelitis; various congenital leukodystrophic disorders; central nervous system neuropathy; central pontine myelinolysis; myelinoclastic disorder; or progressive multifocal leukoencephalopathy; white matter injury associated with prematurity and hypoxic-ischemic encephalopathy.

[0022] The compositions and methods of the invention may also be applied to treat demyelinating conditions of the peripheral nervous system. In peripheral nerves, myelination and remyelination processes are similar to those of the CNS and are mediated by Schwann cells instead of OLs. Various causative factors such as infection, genetic defects, and autoimmune factors disrupt Schwann cell function and result in demyelination of axons in the peripheral nerves. In one embodiment, the demyelinating condition of the PNS is Peripheral demyelinating disease (PDD), encompassing a suite of disorders wherein PNS demyelination occurs. In one embodiment, the demyelinating condition of the PNS is selected from the group consisting of Guillain-Barre Syndrome, Chronic inflammatory demyelinating polyradiculoneuropathy, Anti-Myelin Associated Glycoprotein (MAG) neuropathy, Polyneuropathy, Organomegaly, Endocrinopathy, M protein, and Skin changes (POEMS), and Charcot Marie Tooth disease.

[0023] Subjects. The various inventions disclosed herein are directed to promoting myelination, for example, in the context of treating a demyelinating condition, in a subject in need of treatment therefor. The subject may be any animal species. In a primary embodiment, the subject is a human, for example, a human patient. In other implementations, the subject is a non-human animal, for example, a veterinary subject, pet, livestock, or test animal. Exemplary non-human animals include mice, rats, pigs, horses, cows, dogs, cats, non-human primates and others.

[0024] The subject may be a subject having a demyelinating disease, a subject suffering from any form of demyelination, or a subject at risk of a demyelinating disease or at risk of any form of demyelination.

[0025] In a primary embodiment, the subject is a subject at risk of, or having, any form of MS. Subjects at risk of MS include, for example, subjects with genetic predisposition to MS, subjects with a family history of MS, and subjects exposed to environmental MS risk factors. Subjects may be subjects having one or more heritable genetic risk factors of MS, for example, including HLA-DRB1 *15:01 allele in the class II major histocompatibility complex, interleukin-2 receptor alphagene ( IL2RA ), and interleukin-7 receptor alpha gene (IL7R). In one embodiment the subject is a subject having one or more environmental or lifestyle factors that impart a risk of MS, including residency at or migration to higher latitudes, low Vitamin D levels or low ultraviolet radiation exposure, cigarette smoking, or exposure to infectious agents such as infectious mononucleosis or Epstein Barr Vims.

[0026] The subject may be an MS subject at any stage of MS, for example, including a subject having any of: radiologically isolated syndrome (RIS), a clinically isolated syndrome (CIS), relapsing-remitting stage MS, primary progressive MS, or secondary progressive MS.

[0027] Treatment. The various implementations of the invention are directed to the treatment of a selected demyelinating condition. As used herein, “treatment” will encompass achieving any number of therapeutic effects and outcomes with respect to the selected demyelinating condition, including, for example: promoting normal myelination; ameliorating symptoms associated with demyelination (e.g. nerve signal disruption, axonal damage, and neurodegeneration); slowing the progression of a demyelinating condition; preventing further loss of myelin; restoring lost myelin; promoting normal development and function of the CNS and/or peripheral nervous systems; and a reduction in morbidity and/or mortality associated with the demyelinating condition.

[0028] Treatment, as used herein, will further encompass prevention of an enumerated condition. For example, a preventative treatment may encompass achievement of any of: preventing or delaying the onset of a demyelinating condition in an at-risk subject; maintaining normal development and function of the CNS and/or peripheral nervous systems; or otherwise preventing onset of the demyelinating condition.

[0029] In the context of MS, treatment may encompass any treatment or prevention of MS, including inhibiting the progression of MS; decreasing the severity and/or frequency of relapse; slowing or amelioration of MS symptoms, such as improving measures such Expanded Disability Status Scale score (EDSS), Multiple Sclerosis Severity Score, Multiple Sclerosis Functional Composite score (MSFC), global brain atrophy, grey matter atrophy, white matter atrophy, and retinal axonal degeneration.

[0030] Pharmaceutical Compositions. The methods and compositions of the invention encompass the administration of various agents for the promotion of myelination or the treatment of a demyelinating condition, which agents will be referred to as “myelinating agents.” The myelinating agents may be formulated in what will be termed “pharmaceutical compositions.” As used herein, the pharmaceutical composition will comprise one or more myelinating agents and may further comprise any number of additional compositions of matter, including excipients, carriers, diluents, release formulations, drug delivery or drug targeting vehicles, as well as additional active therapeutic agents.

[0031 ] The pharmaceutical compositions of the invention may comprise one or more myelinating agents in combination with drug delivery compositions. Drug delivery compositions encompass any moieties, materials, or other compositions of matter that facilitate the delivery of the myelinating agent to a selected target, such as a compartment of the body or to a specific cell type. In some implementations, the drug delivery composition facilitates targeting of the myelinating agent to the CNS, PNS, or other selected target compartment of the nervous system. In some implementations, the delivery composition comprises a composition of matter that facilitates crossing of the blood brain barrier (BBB). In some implementations, the delivery compositions comprise a composition of matter that targets the myelinating agent to: glial cells; to oligodendrocytes; or to specific oligodendrocyte types including OPCs, pmOLs, and myelinating OLs. The pharmaceutical compositions comprising one or more myelinating agents in combination with one or drug delivery agents, may encompass any form of combination, including functionalization of the myelinating agent with the delivery composition, conjugation of the myelinating agent to the delivery composition; admixture of the myelinating agent with the delivery composition; encapsulation or infusion of the myelinating agent within the delivery composition, or any other combination.

[0032] In one implementation, the delivery compositions comprise materials that facilitate the myelinating agent’s crossing of the BBB. For example, the delivery composition may comprise ligands that facilitated transcytosis across the BBB through brain endothelial cells to the basolateral side, such as anti-transferrin receptor antibodies or antigen-binding fragments thereof, for example 0X26 antibodies, Angiopep2 or like polypeptides, ApoE proteins and mimetics thereof; diphtheria toxin, and surfactants. Additional targeting moieties include BBB-crossing peptides, such as those described in Van Dorpe et al., Brainpeps: The blood-brain barrier peptide database, Brain Structure and Function, 2012, 217(3), 687-718.

[0033] In one implementation, the delivery compositions comprise carriers to which the myelinating agent is conjugated, encapsulated within, or otherwise combined with to facilitate crossing of the BBB and/or delivery to target cells, e.g. glial cells. Exemplary carriers include: liposomes; extracellular vesicles or synthetic mimetics thereof, such as exosomes; red blood cells modified with myelinating agents; microspheres, such as poly(lactic-co-glycolic acid) (PLGA) microspheres; and other drug delivery nanoparticles such as PLGA-PEG nanoparticles, alginate or chitosan nanoparticles, silica nanoparticles, and iron oxide nanoparticles. The carrier molecules or compositions may further be functionalized with ligands that promote crossing of the BBB, such as anti-tfR antibodies, Angiopep2 or like polypeptides, ApoE proteins and mimetics thereof; diphtheria toxin, and surfactants, as described above.

[0034] In one implementation, smyelinating agents are formulated as drug antibody conjugates, for example, comprising an antibody or antigen-binding fragment thereof targeted to OLs, for example, by targeting to OL cell surface markers, for example: CSPG4, myelin basic protein, tubulin polymerization promoting protein/p25, (Tpp/p25), phospho- taul81 (P-Taul81), and Neurofilament, light (NFL). In one embodiment, the myelinating agent is targeted to OPCs by combination with antibodies or antigen binding fragments thereof against NG-2 chondroitin sulfate proteoglycan, expressed on OPCs.

[0035] Exemplary targeting of therapeutic agents OLs has been demonstrated by use of various delivery compositions. For example, Rittchen et al., 2015. Myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with nanoparticles encapsulating leukaemia inhibitory factor (LIF), Biomaterials, 56: 78-85 describes targeting PLGA nanoparticles to OLs by use of anti- NG-2 chondroitin sulfate proteoglycan antibodies. Zhao et al., 2012. Carbohydrate-coated fluorescent silica nanoparticles as probes for the galactose/3 -sulfogalactose carbohydrate-carbohydrate interaction using model systems and cellular binding studies. Bioconjug Chem 23:1166-73 describes targeting OLs with galactose and 3-sulfogalactose-coated silica nanoparticles. Hohnholt et al., 2011. Treatment with iron oxide nanoparticles induces ferritin synthesis but not oxidative stress in oligodendroglial cells. Acta Biomater 7:3946-54 describe targeting OLs by iron oxide nanoparticles.

[0036] In one embodiment the delivery composition comprises or is incorporated within an implant, for example, a drug-eluting implant placed within the target tissue, for example, the brain parenchyma. Exemplary implants include, for example, polymeric drug-eluting wafers, injectable hydrogels, implantable hydrogel scaffolds, and other drug-eluting implants known in the art. [0037] Certain myelinating agents of the invention, for example, small molecule agents, may be formulated as a pharmaceutically acceptable salt, as known in the art.

[0038] The pharmaceutical compositions of the invention may be formulated to be compatible with the selected route of administration, as described below.

[0039] The pharmaceutical compositions of the invention may comprise combination products comprising a myelinating agent in combination with one or more additional therapeutic agents, for example, other myelinating drugs or therapeutics that act on different aspects of the selected demyelinating condition. Exemplary therapeutic agents for treatment of MS include, for example, fmgolimod, dimethyl fumarate, diroximel fumarate, teriflunomide, siponimod and cladribine.

[0040] The pharmaceutical compositions of the invention may be formulated in any number of dosage forms. Exemplary dosage forms include: liquid solutions; sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; suspensions in a liquid; emulsions; aqueous and non-aqueous, isotonic sterile injection solutions; compositions stored in a freeze-dried, lyophilized condition; and other dosage forms known in the art. In one embodiment, the composition is formulated as a dietary supplement or medicinal food, for example, compositions comprising cucurbitacin E or a derivative thereof. Dietary supplements and medicinal foods may comprise solutions, suspensions, emulsions, powders, granules, tablets, capsules, drinks, and other edible items.

[0041] Methods of Administration. The methods of the invention encompass the delivery of myelinating agents, for example, as pharmaceutical compositions, to target cells of the nervous system, for example, cells of the CNS or PNS. The methods of the invention are not limited to any particular form of delivery and may encompass any form of systemic or local delivery, for example, oral delivery, intrathecal delivery, intranasal delivery, intravenous delivery, intraperitoneal delivery, inhalation delivery, subcutaneous delivery, intraocular delivery, transmucosal delivery, and transdermal delivery.

[0042] The method of delivery may comprise systemic delivery, wherein the one or more myelinating agents is administered to a non-brain compartment of the body and wherein delivery to target cells of the CNS requires the therapeutic agent to cross the BBB. [0043] Alternatively, a myelinating agent may be administered across the BBB, for example, by transient and local disruption of the BBB, for example, by use of focused ultrasound. In one embodiment, ultrasound and microbubble delivery methods are utilized, as known in the art.

[0044] In one implementation, the myelinating agent is delivered by invasive local delivery to the brain, for example, by the implant of a drug-eluting structure, or by intraparenchymal delivery methods, such as by convection-enhanced delivery, infusion by catheter, or other methods of invasive delivery known in the art.

[0045] In one embodiment the myelinating agent is delivered by intrathecal delivery, e.g. via injection into the spinal canal or into the subarachnoid space such that the agent accesses the cerebrospinal fluid and from there transits to the CNS.

[0046] In one embodiment, the delivery is intranasal delivery, wherein myelinating agents, for example, formulated in lipophilic pharmaceutical compositions, will cross through the nasal epithelium, or pass into the brain by transneuronal pathways.

[0047] In one embodiment, the myelinating agent is administered systemically and taken up by the PNS, or is delivered to the PNS, which results in delivery to the to the CNS by retrograde neuronal transport.

[0048] The methods of the invention encompass the administration of one or more myelinating agents in a therapeutically effective amount. In one measure, a therapeutically effective amount is an amount of myelinating agent that is sufficient to induce a measurable therapeutic effect. The therapeutic effect may be the attainment of a selected physiological outcome or state, for example, increased myelin formation, reduced demyelination, increased remyelination, improvements in neuron function, and the like. Exemplary dosages may be calculated by one of skill in the art taking into account the formulation, subject body size, ADMET characteristics, active agent toxicity and tolerance, and other factors utilized in calculating effective dosages. Exemplary doses may be in the range of 1 ng to one or more grams, for example, in the range of 100 ng to 1 mg, etc., depending on the relevant factors. For example, small molecule drugs are typically administered at smaller doses, for example, 1-100 micrograms per kilogram body weight, while biologies such as proteins and cell-based agents generally require administrations at higher doses, for example, from 0.5-2.0 g/kg body weight. [0049] Administration may be chronic, periodic, or acute, and may be selected by one of skill in the art based on the therapeutic window wherein treatment is beneficial, the duration of efficacy of the delivered agents, and other factors. Exemplary treatment dosage regimens include a short course of treatments (i.e. 1-10 treatments), or ongoing administration, for example, daily administration, administration 2-4 times per week, weekly administration, monthly administration, and administration at longer intervals.

[0050] Therapeutic Methods.

[0051 ] The treatment methods of the invention are based on discoveries by the inventors of the present disclosure regarding ADGRG1 -mediated myelination. In the initial stage of myelin sheath formation, OPCs encounter axons and differentiate into pmOLs, which subsequently achieve the ensheathment of axons, a critical step that precedes myelin deposition. Ensheathment of axons is achieved by lamellar extensions from the pmOLs which wrap circumferentially around the axon to form what is ultimately the first turn of the multilayered myelin sheath. The projections of the pmOLs are facilitated by the deployment of an actin cytoskeleton, dependent on the polymerization of actin. The cytoskeleton is subsequently disassembled to facilitate the subsequent spiraling axon wrapping phase. This disassembly is achieved by the actin depolymerization proteins gelsolin and cofilin. Accordingly, regulation of actin cytoskeleton formation is key to pmOL function and is controlled by a series of regulators as follows:

ADGRG1 is expressed in pmOLs, and, as demonstrated herein, activated ADGRG1, via the G-alpha 12/13 protein, activates the small GTPase Transforming protein Ras homolog family member A (RhoA);

RhoA, in its activated form, in turn activates the Rho-associated, coiled-coil containing protein kinase (ROCK);

ROCK phosphorylates Lim domain kinase 1 (LIMK); and when phosphorylated, LEMK in turn phosphorylates cofilin, inhibiting it and increasing F-actin formation, axon ensheathment and remyelination.

[0052] Accordingly, each link in the foregoing process may be targeted by therapeutic intervention to achieve remyelination in target cells, as described below. The myelinating agents of the invention encompass compositions of matter that promote myelination by promoting any stage of the ADGRG1 -mediated myelination pathway disclosed herein. In one embodiment, the myelinating agent is an ADGRG1 activator. In one embodiment, the myelinating agent is a RhoA activator. In one embodiment, the myelinating agent is a ROCK activator. In one embodiment, the myelinating agent is a LIMK activator. In one embodiment, the myelinating agent is a cofilin inhibitor.

[0053] ADGRGl activators. In a first aspect, the inventors of the present disclosure have determined that remyelination may be enhanced by modulating the activity of ADGRGl in oligodendrocytes. ADGRGl, also known as GPR56, is a widespread G protein-coupled receptor involved in signal transduction in many cell types and biological processes. Among its many downstream functions, ADGRGl is a known regulator of the RhoA pathway, wherein Ga 3 subunit activates RhoA, which in turn regulates downstream signaling molecules. The inventors of the present disclosure have determined that ADGRGl is required for proper myelin formation during the intermediate, pre-myelinating stage of oligodendrocyte development through a discrete signaling pathway. As disclosed herein, when ADGRGl is removed from pre-myelinating oligodendrocytes, significant deficits in myelin in formation and remyelination capacity are observed. Moreover, the inventors of the present disclosure herein demonstrate that oligodendrocyte actin polymerization is dependent upon ADGRGl

[0054] Accordingly, various inventions disclosed herein are directed to methods of promoting myelination in a subject, wherein the method comprises the administration of a myelinating agent comprising an ADGRGl activator to the subject. In various embodiments, the method may encompass administration of an ADGRGl activator to glial cells of the subject, to oligodendrocytes of the subject, and administration of an ADGRGl activator to pmOLs of the subject.

[0055] In one implementation, the scope of the invention encompasses an ADGRGl activator for use in a method of promoting myelination in a subject, wherein the method comprises the administration of the ADGRGl activator to the subject. In one implementation, the scope of the invention encompasses an ADGRGl activator for use in a method of treating a demyelinating condition in a subject, wherein the method comprises the administration of the ADGRGl activator to the subject. In a related implementation, the scope of the invention encompasses a method of promoting myelination in a subject by the administration to the subject of a therapeutically effective amount of a myelinating agent comprising an ADGRG1 activator. In a related implementation, the scope of the invention encompasses the treatment of a demyelinating condition in a subject in need of treatment therefor by administration to the subject of a therapeutically effective amount of an ADGRG1 activator. In one embodiment, the scope of the invention encompasses a method of making a medicament for promoting myelination, for example, a medicament for treating a demyelinating condition, by the use of an ADGRG1 activator.

[0056] ADGRG1 activators, as used herein, encompass any composition of matter that activates, promotes the activation of, or increases the activity of ADGRG1, including agonists, activators, and enhancers of ADGRG1 signaling activity in glial cells, oligodendrocytes, and/or pmOLs. In a primary implementation, the ADGRG1 activator is a composition that promotes ADGRG1 -mediated activation of RhoA in glial cells, oligodendrocytes, and/or pmOLs.

[0057] Exemplary ADGRG1 activators include, for example: transglutaminase 2 (TG2) polypeptide or active ADGRGl-actiavting fragments thereof, for example, comprising amino acids 465-687 of TG2, or a TG2 fragment that forms one or more beta barrel domains; 3-a- DOG or ADGRG1 -activating fragments thereof; an ADGRG1 ligand comprising activating subsequences of ADGRG1, for example, comprising amino acids 383-404 of ADGRG1 (also known as P7), for example, as described in PCT International Patent Application Number WO20017172945, Compositions and Methods for Oligodendrocyte Development, by Xianhua Piao; gedunin and khivorin derivatives, as described in Stoveken et al., 2018. Gedunin- and Khivorin-Derivatives Are Small-Molecule Partial Agonists for Adhesion G Protein-Coupled Receptors GPR56/ADGRG1 and GPR114/ADGRG5, Molecular Pharmacology 93: 477-488; monobody activators, for example, as described in Salzman et al., 2017. Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region, PNAS 114: 10095-10100; and synthetic peptides mimicking a GPR56 tethered agonist peptide, for example, as described in Olaniru et al., 2018, Activation of the adhesion GPCR GPRS 6 by a synthetic tethered agonist improves islet b-cell function, Endocrine Abstracts 59: P109. In one embodiment, the ADGRGl activator is a nucleic acid construct that induces or increases ADGRGl expression levels in pmOLs, such as a CRISPR/Cas- mediated ADGRGl knock-in transformation vector or a transformation vector that expresses ADGRGl -activating ligands. [0058] RHOA Activators. In a second aspect, the inventors of the present disclosure have determined that remyelination may be enhanced by promoting the activity of RhoA in oligodendrocytes, for example, in pmOLs.

[0059] The small GTPases RhoA, also known as Transforming protein Rho A, is a known actor in various cellular processes such regulation of cytoskeletal dynamics, regulating transcription, and regulation of the cell cycle. The inventors of the present disclosure have elucidated a role for RhoA in myelination. RhoA was observed to be prominent in the processes of pmOLs and to co-localize with ADGRG1. In the pmOLs of ADGRG1 knockout animals, RhoA activity in processes was ablated, indicating that ADGRG1 mediates RhoA activation, specifically, in the processes of pmOLs. Furthermore, treatment with RhoA activators was observed to rescue myelination in adgrgl knockout animals, while RhoA inhibition reduced myelination in wild type animals.

[0060] Accordingly, various inventions disclosed herein are directed to promoting myelination, for example, treating a selected demyelinating condition in a subject, by the administration of a myelinating agent comprising a RhoA activator to the subject. In various embodiments, the method may encompass administration of a RhoA activator to oligodendrocytes of the subject; for example, administration of a RhoA activator to pmOLs of the subject.

[0061] In one implementation, the scope of the invention encompasses a myelinating agent comprising a RhoA activator for use in a method of promoting myelination in a subject, wherein the method comprises the administration of the RhoA activator to the subject. In one implementation, the scope of the invention encompasses a RhoA activator for use in a method of treating a demyelinating condition in a subject, wherein the method comprises the administration of the RhoA activator to the subject. In a related implementation, the scope of the invention encompasses a method of promoting myelination in a subject by the administration to the subject of a therapeutically effective amount of a RhoA activator. In a related implementation, the scope of the invention encompasses the treatment of a demyelinating condition in a subject in need of treatment therefor by administration to the subject of a therapeutically effective amount of a RhoA activator. In one embodiment, the scope of the invention encompasses a method of making a medicament for promoting myelination, for example, a medicament for treating a demyelinating condition, by the use of a RhoA activator. [0062] The RhoA activator may encompass any composition of matter that activates, promotes the activation of, or increases the activity of RhoA. RhoA activation, as used herein, encompasses any transformation of RhoA from its inactive form to its active form in glial cells, oligodendrocytes, and/or pmOLs. In a primary implementation, the RhoA activator is a composition that promotes RhoA-mediated activation of ROCK in glial cells, oligodendrocytes, and/or pmOLs, for example, in the processes of pmOLs.

[0063] Exemplary RhoA activators include, for example: RhoA Activator II; guanosine-5'- diphosphate; cytotoxic necrotizing factor- 1 and derivatives thereof; thrombin; calf serum; lysophosphatidic acid; calpeptin; lysophosphatidylcholine; calpeptin; colchicine; nocodazole; vinblastine; cytochalasin D; sphingosine-1 -phosphate; lysophosphatidic acid; bombesin and other RhoA activators known in the art.

[0064] ROCK Agonists. In a third aspect, the inventors of the present disclosure have determined that remyelination may be enhanced by modulating the activity of ROCK in oligodendrocytes, for example, in pmOLs.

[0065] ROCK, or Rho-associated, coiled-coil containing protein kinase, is a known downstream effector of Rho signaling and is active in a wide range of physiological processes, particularly with regards to actin dynamics. ROCK is found in two closely related forms, ROCK1 and ROCK2. The inventors of the present disclosure have demonstrated that ROCK activation is an essential link in the ADRG1 -mediated myelination process. For example, as demonstrated herein, inhibition of ROCK reduced myelination in wild-type animals, while conversely, activation of ROCK rescued myelination capacity in myelination- impaired adgrgl knockout animals.

[0066] Accordingly, various inventions disclosed herein are directed to promoting myelination in a subject, for example, treating a selected demyelinating condition in the subject, by the administration of a myelinating agent comprising a ROCK activator to the subject. In various embodiments, the method may encompass administration of ROCK activator to oligodendrocytes of the subject; for example, administration of a ROCK activator to pmOLs of the subject.

[0067] In one implementation, the scope of the invention encompasses a myelinating agent comprising a ROCK activator for use in a method of promoting myelination in a subject, wherein the method comprises the administration of the ROCK activator to the subject. In one implementation, the scope of the invention encompasses a ROCK activator for use in a method of treating a demyelinating condition in a subject, wherein the method comprises the administration of the ROCK activator to the subject. In a related implementation, the scope of the invention encompasses a method of promoting myelination in a subject by the administration to the subject of a therapeutically effective amount of a ROCK activator. In a related implementation, the scope of the invention encompasses the treatment of a demyelinating condition in a subject in need of treatment therefor by administration to the subject of a therapeutically effective amount of a ROCK activator. In one embodiment, the scope of the invention encompasses a method of making a medicament for promoting myelination, for example, a medicament for treating a demyelinating condition, by the use of a ROCK activator.

[0068] The ROCK activator may encompass any composition of matter that activates, promotes the activation of, or increases the activity of ROCK. In ROCK’s inactive form, the C-terminal region of ROCK acts as an auto-inhibitory domain that blocks ROCK’s kinase domain. Interaction with GTP-loaded RhoA removes the inhibitory region from the kinase domain, activating ROCK for kinase action on its downstream effectors. ROCK activation, as used herein, encompasses any transformation of ROCK from its inactive form to its active form or any other increase in ROCK activity. In a primary implementation, the ROCK activator is a composition that enhances, promotes, or increases ROCK phosphorylation of LIMK. In a primary implementation, the ROCK activator is a composition that activates ROCK in glial cells, oligodendrocytes, and/or pmOLs. The ROCK activator may be an activator of ROCK1, an activator of ROCK2 or and activator of both.

[0069 In various embodiments, the ROCK agonist may be calpeptin, narciclasine, or lysophosphatidic acid.

[0070] Cof!lin Inhibitors. In a fourth aspect, the inventors of the present disclosure have determined that remyelination may be enhanced by modulating the activity of cofilin. Cofilin is an actin-binding protein that regulates actin filament dynamics and is implicated in a number of cellular processes. The inventors of the present disclosure have determined that ADGRG1 promotes myelination through a signaling pathway wherein ROCK phosphorylates LIMK, which in turn phosphorylates and inhibits cofilin. Cofilin activity is regulated by its phosphorylation state, wherein phosphorylated cofilin is the less active or inactive form. Surprisingly, the inventors of the present disclosure have determined that inhibiting cofilin activity can promote remyelination, despite the need for actin disassembly (mediated by active cofilin) to proceed to the phase of myelin deposition. For example, administration of the cofilin inhibitor cucurbitacin E rescued myelination in myelination-impaired adgrgl knockout animals, while inhibition of LIMK, a repressor of cofilin, resulted in decreased myelination in wild type animals. Without being bound by any particular theory, it is believed that cofilin inhibition slows the rate of actin depolymerization and this may stabilize the actin cytoskeleton to such degree that ensheathment is more efficiently achieved by OLs.

[0071 ] Accordingly, various inventions disclosed herein are directed to promoting myelination in a subject, for example, treating a selected demyelinating condition in the subject, by the administration of a myelinating agent comprising a cofilin inhibitor to the subject. In various embodiments, the method may encompass administration of a cofilin inhibitor to oligodendrocytes of the subject; for example, administration of a cofilin inhibitor to pmOLs of the subject.

[0072] In one implementation, the scope of the invention encompasses a myelinating agent comprising a cofilin inhibitor for use in a method of promoting myelination in a subject, wherein the method comprises the administration of the cofilin inhibitor to the subject. In one implementation, the scope of the invention encompasses a cofilin inhibitor for use in a method of treating a demyelinating condition in a subject, wherein the method comprises the administration of the cofilin inhibitor to the subject. In a related implementation, the scope of the invention encompasses a method of promoting myelination in a subject by the administration to the subject of a therapeutically effective amount of a cofilin inhibitor. In a related implementation, the scope of the invention encompasses the treatment of a demyelinating condition in a subject in need of treatment therefor by administration to the subject of a therapeutically effective amount of a cofilin inhibitor. In one embodiment, the scope of the invention encompasses a method of making a medicament for promoting myelination, for example, a medicament for treating a demyelinating condition, by the use of a cofilin inhibitor.

[0073] The cofilin inhibitor may encompass any composition of matter that inhibits, decreases, or otherwise reduces the activity of cofilin or which reduces the abundance of cofilin. In a primary implementation, the cofilin inhibitor is a composition that reduces or ablates cofilin-mediated actin disassembly. In a primary implementation, the cofilin inhibitor is a composition that inhibits cofilin in glial cells, oligodendrocytes, and/or pmOLs. [0074] In one embodiment, the cofilin inhibitor is cucurbitacin E. Cucurbitacin E is a triterpene member of a broader class of compositions found in plants of the family Cucurbitaceae, which includes edible plants such as cucumbers, pumpkins, and squashes, and which also includes plants used in Traditional Chinese Medicine, including Bacopa monnieri, Cucurbita andreana, Citrullus colocynthis, and Cucurbita pepo Linnaeu.

Cucurbitacin E:

[0075] In other implementations, the cofilin inhibitor may comprises a derivative of cucurbitacin E having cofilin-inhibiting properties. Derivatives of cucurbitacin E, as used herein, encompass molecules having one or more elements of structural similarity to cucurbitacin E and which have some measure of cofilin inhibition activity. Cucurbitacin E derivatives include, for example: cucurbitacin A; cucurbitacin B; cucurbitacin C; cucurbitacin D; cucurbitacin F; cucurbitacin G; cucurbitacin H; cucurbitacin I; cucurbitacin J; cucurbitacin K; cucurbitacin L; cucurbitacin M; cucurbitacin N, cucurbitacin O; cucurbitacin P; cucurbitacin Q; cucurbitacin R; cucurbitacin S; cucurbitacin T, norcurubitacin, 23,24-dihydrocucurbitacin E; 7beta-hydroxycucurbitacin B; Prednisolone famesylate; Cucurbitacine; Isocucurbitacin B; 3-Epi-IsocucurbitacinB; Cucurbitacine (C); Picracin; 16-deoxycucurbitacin B; Prednisolone 21-all-cis-famesylate; Fabacein;

Cucurbitacin BE; 2-Epicucurbiticin B; and Diacetyl-2,16 cucurbitacine (I).

[0076] In one embodiment, the cucurbitacin E derivative comprises a composition comprising cucurbitacin E comprising hydrolysis of the acetyl group. For example, in one embodiment, the cucurbitacin E derivative comprises STRUCTURE 2:

wherein R is hydrogen or a group comprising an, ethyl, methyl, propyl, or other group.

[0077] In one embodiment, the cucurbitacin E derivative comprises a composition comprising a fluorinated cucurbitacin E. For example, in one embodiment, the cucurbitacin E derivative comprises STRUCTURE 3:

[0078] In one embodiment, the cucurbitacin E derivative comprises a composition comprising a hydrogenated cucurbitacin E. For example, in one embodiment, the cucurbitacin E derivative comprises STRUCTURE 4:

[0079] In one embodiment, the cucurbitacin E derivative comprises a composition comprising cucurbitacin E comprising one or more additions, such as alkyl additions, for example, at one or more of the ketones of the parent molecule. For example, in one embodiment, the cucurbitacin E derivative comprises STRUCTURE 5:

wherein, R’ is a group comprising any of a methyl, ethyl, propyl, butyl, phenyl group, or other group and R” is a group comprising any of a methyl, ethyl, allyl, benzyl or other group.

[0080] In one embodiment, the cucurbitacin E derivative comprises a composition comprising cucurbitacin E comprising one or more reductions of a ketone of the parent molecule, for example, the reduction comprising reduction to a hydrogen or addition of a group. For example, in one embodiment, the cucurbitacin E derivative comprises STRUCTURE 6:

[0081] In one embodiment, the cucurbitacin E derivative comprises a composition comprising cucurbitacin E comprising one reductive aminations of a ketone of the parent molecule. For example, in one embodiment, the cucurbitacin E derivative comprises STRUCTURE 7:

wherein R’” comprises a group comprising a methyl, ethyl, benzyl or other group. [0082] In one embodiment, the cucurbitacin E derivative comprises a composition comprising cucurbitacin E comprising cyclopropynation of the acetyl group. For example, in one embodiment, the cucurbitacin E derivative comprises STRUCTURE 8:

[0083] Other Cofilin Inhibitors. In other embodiments, the cofilin inhibitor may be, for example, any of tacrolimus (FK-506); sodium vanadate; SZ-3, as described in Saleh, 2019. Development of a Novel Cofilin Inhibitor for the Treatment of Hemorrhagic Brain Injury, PhD Thesis University of Toledo; JG-6, as described in Huang et al., 2014. JG6, a novel marine-derived oligosaccharide, suppresses breast cancer metastasis via binding to cofilin, Oncotarget 5: 3568-3578.

[0084] In one implementation, the cofilin inhibitor is a proteolysis targeting chimera (PROTAC). PROTACs are bifunctional constructs comprising a ligand that binds to a target protein, and a ligand that binds to a moiety that targets the cell’s degradation machinery, e.g., an E3 ligase. PROTAC thus captures the target protein and brings the target protein in spatial proximity to the E3 ligase, triggering ubiquitination and proteosomal degradation.

The targeting moiety may comprise any cofilin-binding species.

[0085] In another implementation, the cofilin inhibitor is an inhibitor of cofilin expression. Inhibitors of cofilin expression may encompass any composition which disrupts the transcription, translation, or other steps of the gene-to-protein sequence of cofilin production.

[0086] In one implementation, the cofilin expression inhibitor is an element of a CRISPR- Cas9 or like construct for the targeted knockdown the cofilin gene. In one embodiment, the cofilin expression inhibitor comprises the 5’ targeting sequence of a CRISPR guide RNA, the targeting RNA comprising, for example, a 15-25 nucleotide subsequence of the cofilin gene (either coding or non-coding strand), for example, a 17-20 nucleotide sequence, wherein the sequence is adjacent to a suitable protospacer adjacent motif (PAM site), for example, NGG, or CCN, wherein N is any nucleotide. In one embodiment, the guide sequence is present in an expression vector, such as a plasmid, which codes for the guide RNA sequence, and typically is co-expressed with an engineered Cas9 protein, for example, a Streptococcus pyognes Cas9 system (combined cRNA:tracrRNA, for example), for example, codon- optimized for expression in the target organism, for example, optimized for expression in human cells. SpCas9 variants may also be used with altered PAM site specificities, for example, the D1135E, VRQ, EQR, VRER, xCas9, SpG and SpRY variants, as known in the art. The Cas9 and guide RNA sequences may be placed under the control of a suitable promoter. In one embodiment, the promoter is a promoter for selective or preferential expression in CNS cells, such as oligodendrocytes. When expressed in the target cells, the guide RNA and Cas9 form a complex that will specifically targeted by the guiding sequence to the miRNA gene, activating Cas9 exonuclease cleavage of the targeted DNA, resulting in a double stranded break about three nucleotides upstream of the adjacent PAM site.

Subsequent non-homologous end joining (NHEJ) results in an indel mutation which disrupts the expression of the targeted gene.

[0087] In alternative implementations, the cofilin expression inhibitor may comprises other compositions for the targeted mutagenesis of the cofilin gene, for example, a zinc finger nuclease (ZNF), or transcription activator-like effector nuclease (TALEN) targeted to the cofilin gene. In another embodiment, the cofilin expression inhibitor may comprise a nucleic acid sequence which selectively interferes with transcription or processing of the targeted miRNA such as an antisense construct, short interfering RNA (siRNA), or short hairpin (shRNA) sequence.

[0088] EXAMPLES

[0089] Example 1. Pre-myelinating Oligodendrocyte ADGRG1 is a Mediator of Actin Polymerization and Myelin Formation.

[0090] Results: Using PDGFRa, a marker for OPCS, TCF7L2, a marker for pmOLs, and KLK6, a marker for mOLs, ADGRGl expression was assessed across the OL lineage in isolated and cultured OLs. Nearly all PDGFRa⁺ and TCF7L2⁺ cells were also positive for ADGRGl, while almost all KLK6⁺ cells were negative for ADGRGl. [0091 ] In order to test the effects of knocking out pmOL Adgrgl on myelination, myelin status was assessed in Adgrgl^¹; Plp-CreER^+/~ (cKO) mice compared to Adgrgl^+/+; Plp- CreER^+/~ (control) mice. 50 mg/kg tamoxifen was injected daily from P8 to PI 2 and myelin status was analyzed at P21, showing that knockout of Adgrgl results in decreased myelination in the corpus callosum. Knockout of pmOL Adgrgl did not affect OL proliferation. Similarly, apoptosis in OLs was unaffected. It was also found that pmOL and mOL numbers were unaffected. From these initial studies it became clear that the loss of myelin through ADGRG1 is related to the function of pmOLs.

[0092] Using phalloidin, a small molecule that binds specifically to F-actin , F-actin levels in the mouse corpus callosum were assayed at P21, comparing control with cKO mice. It was found that knockout of pmOL Adgrgl resulted in decreased F-actin levels. Furthermore upon closer examination, it was found that MBP, which normally overlaps and traces axons in the corpus callosum of control mice, aggregated within the cell body of oligodendrocytes in cKO mice. Using 3D reconstructions, it was observed that in control mice the MBP and F- actin signals were very dispersed, while in cKO mice the MBP and F-actin signals were withdrawn and close to the cell body. Moreover, in cKO OLs, the MBP and F-actin signals overlap and clearly define each OL. Transmission electron microscopy (TEM) was used to observe myelin on an ultrastructural level, using P21 optic nerves isolated from control and cKO mice, and there were clear qualitative differences in the thickness and number of axons myelinated; prominent hypomyelination was observed in cKO mice as well as overall reduced myelin thickness. G-ratio analyses between control and cKO optic nerves demonstrated significantly higher g-ratios in the cKO genotype, indicating a thinner myelin layer in cKO mice. Furthermore, three distinct degrees of myelination were observed among different axons: unmyelinated, incomplete, and wrapped. While unmyelinated and wrapped axons were either completely devoid of myelin or wrapped in multiple layers of myelin respectively, incomplete axons had only a single layer of myelin or in some cases, were only partially ensheathed. cKO mice had a significantly higher proportion of unmyelinated and incomplete axons. This was reflected in the decrease of fully wrapped axons in cKO mice compared to control.

[0093] Next, OPCs were immunopanned from control and cKO mice, allowed to differentiate into pmOLs, and subsequently Adgrgl was knocked out using 40HT. At 4 days in vitro, myelin and F-actin levels were assessed by immunocytochemistry for MBP and phalloidin. Overall quantification demonstrated significant decrease in the number of phalloidin-positive pmOLs between control and cKO conditions when Adgrgl was knocked out. Sholl analysis method was to understand radial complexity in both phalloidin-positive and MBP-positive signal. From these studies it was found that while control pmOL F-actin complexity is unaffected by addition of either 40HT or PI 9, cKO pmOL F-actin complexity was significantly reduced in the presence of 40HT, it is clear that knocking out Adgrgl significantly reduces both F-actin and MBP complexity.

[0094] In order to differentiate between axon-instructed and oligodendrocyte-intrinsic myelination in relation to ADGRG1 status, electrically spun microfibers were used as a platform for assessing myelin formation. Immunopanned cortical OPCs were plated onto microfiber inserts, incubating the cells with 40HT overnight to knock out Adgrgl. Next, the OLs were allowed to mature and myelinate for 14 days . Initial studies using this platform revealed noticeable qualitative and quantitative differences between control, heterozygous Adgrgl, and cKO OLs. Control OLs created long MBP-positive sheaths along the microfibers, wherein a reduced sheath length in was found in heterozygous OLs, and MBP signal was localized very close to the OL cell body in the cKO group. Orthogonal views of the microfibers again revealed that while control OLs completely ensheathed and wrapped the fibers in a complete circle, Heterozygous OLs contained both partially ensheathed as well as fully ensheathed fibers. Furthermore, cKO OLs only partially ensheathed or fail to ensheath the microfibers altogether. Overall, control and Het OLs created significantly longer sheaths than cKO OLs. Observing the myelination process over time by taking snapshots of the OLs myelinating the fibers at 1, 4, 7, and 14 days in vitro, it was found that while control OLs myelinate properly by 14 DIV, cKO OLs seem to get stuck at 4 DIV and do not properly ensheath or myelinate the fibers. These data demonstrate that ADGRG1 is required for proper myelin formation.

[0095] The next point of interest was to address the intracellular signaling mechanism by which ADGRG1 exerts its effects on myelination in pmOLs. First, it was confirmed that loss of Adgrgl in pmOLs leads to a reduction in the amount of GTP-RhoA. Next, pharmacological activators and inhibitors of RhoA (RhoA Activator II , RhoA Inhibitor I ) and ROCK (Calpeptin , Y27632), an inhibitor targeting LIMK (Pyrl ), and an inhibitor targeting cofilin (Cucurbitacin E, CCB E,) were used order to test each downstream signaling partner individually in both control and cKO pmOLs. As cofilin activity results in actin disassembly, inhibiting cofilin results in actin polymerization . As expected, DMSO treatment results in normal F-actin in control pmOLs with decreased F-actin in cKO pmOLs. However, when control pmOLs were treated with RhoA Inhibitor I, Y27632 or Pyrl, F-actin levels were dramatically reduced. Conversely, when cKO pmOLs were treated with RhoA Activator II, Calpeptin, or CCB E, F-actin levels were restored. When the complexity of phalloidin staining was assessed, it was found that inhibition of RhoA, ROCK, and LEMK significantly reduced the ability for control pmOLs to polymerize actin. On the other hand, phalloidin staining in cKO pmOLs revealed the ability for targeting RhoA, ROCK, and cofilin in order to restore F-actin complexity. This bypass of ADGRG1 was also observed in both control and cKO pmOLs in myelin organization. Data from these experiments demonstrate that ADGRGl signaling through RhoA, ROCK, LEMK, and cofilin in pmOLs is necessary for proper actin polymerization and myelin organization.

[0096] Next, the functional role of ADGRGl and its downstream signaling pathway was assessed in myelin formation. To do this, a co-culture system was used wherein ex vivo cerebellar slices from Shiverer mice were microinjected with OPCs. Shiverer mice have a large deletion in the gene for MBP resulting in a myelin deficiency in the CNS . By transplanting OPCs from either control or cKO mice, this platform provided insight into how ADGRGl contributes to the creation of new myelin in a physiological setting. 300 pm Shiverer cerebellar slices were cultured ex vivo for 7 days to allow for debris clearance. Subsequently, OPCs that were immunopanned from either control or cKO mice were transplanted into the slices, incubated with 40HT overnight, and allowed to culture for 14 days to facilitate myelination. At the end of the 14 day period, immunostaining for axons (NF200) and myelin (MBP) was performed. Transplantation was successful in both control and cKO OLs. When the percentage of axons that were myelinated was compared between groups, it was found that cKO OLs myelinate significantly less than control. Furthermore, using 3D reconstructions, it was observed that while MBP signal in the control injection group is clearly wrapped around the NF200 axon signal, cKO OLs instead sit next to the axons, failing to extend and ensheath nearby axons.

[0097] Applying the pharmacological approach used in the in vitro setup to this ex vivo model, next it was ascertained how downstream signaling from ADGRGl could affect myelin formation. The co-culture assay was run as before, but 4 days after transplantation of the OPCs into the Shiverer cerebellar slices, different treatments were added to the media. 4 days after transplantation was selected as a crucial time for treatment to mirror in vitro data showing high levels of pmOLs at that point. Results are presented in Fig. 2. Corroborating the in vitro results, it was found that control OLs myelinate significantly more than cKO OLs. Furthermore, adding RhoA Inhibitor I, Y27632, or Pyrl abrogated normal ADGRG1 function and significantly decreased myelin formation. However, adding RhoA Activator II, Calpeptin, or CCB E restored proper myelination.

[0098] To further investigate the role of ADGRGl downstream signaling pathway in intrinsic myelination, the pharmacological assays were repeated on OPCs cultured on microfibers. Again, activators and inhibitors were added to OLs at 4 days in vitro during the myelination period on microfibers. Results are demonstrated in Fig. 3 and Fig. 4A and 4B.

As before, DMSO treatment resulted in obvious differences in MBP signal and localization between control and cKO OLs. By performing a nonlinear Gaussian regression paired with a log transformation of sheath lengths, it was found that cKO OLs produced sheaths that were significantly shorter than control OLs. Furthermore, cKO OLs produced significantly fewer sheaths per cell and an overall lower average sheath length per cell. Addition of RhoA Inhibitor I, Y27632, or Pyrl to control OLs resulted in significantly shorter sheaths, fewer sheaths per cell, and decreased average sheath length per cell. Additionally, addition of RhoA Activator II, calpeptin, or CCB E to cKO OLs resulted in significantly increased sheath length, more sheaths per cell, and increased average sheath length per cell.

[0099] Next, it was investigated whether enhancing ADGRGl function stimulates recovery and remyelination after a white matter lesion. Two ADGRGl agonists, TG2 and 3-a-DOG, were added to the culture media during the 96 hour remyelination period (200 nM TG2 or 5 mM 3-a-DOG), using DMSO as a control. A comparison of control and cKO demonstrated that addition of TG2 or 3-a-DOG enhanced remyelination compared to DMSO treatment in control slices. However, when TG2 or 3-a-DOG were added to the cKO slice media, no significant difference was found when compared to the DMSO treatment.

[0100] Discussion. When Adgrgl was knocked out, specifically in pmOLs, a significant decrease in myelin content was found, indicating pmOL ADGRGl is heavily involved in myelin development. pmOLs have the highest expression of Adgrgl and are post-mitotic, indicating a function for ADGRGl that is not related to proliferation. This study has demonstrated a novel function for pmOL ADGRGl in regulating actin dynamics and ensheathment of axons. [0101] Data from the studies indicates that ADGRG1 regulates axon ensheathment through an actin-dependent mechanism. Upon loss of Adgrgl, OLs in the corpus callosum seem to be unable to extend actin and subsequently deposit myelin, leading to a condensation of polymerized actin and entrapment of myelin signal close to the cell body. Similarly, cKO pmOLs had decreased ability to polymerize actin which leads to altered arborization and process elaboration. These experiments demonstrate that ADGRG1 has a regulatory role in actin polymerization. The ultrastructural studies showed that Adgrgl is heavily involved in the regulation of ensheathment. With all of this data in hand, it is clear that ADGRG1 regulates actin polymerization which in turn affects proper axon ensheathment and subsequent myelination.

[0102] By analyzing the signaling pathway downstream of ADGRG1 and its greater effect on myelin formation, it was found that signaling from RhoA to ROCK, LIMK, and cofilin heavily influences myelin formation and that ADGRG1 regulates this pathway. By specifically targeting each member of the pathway it was demonstrated that ADGRG1 could be bypassed, and the activity of ADGRG1 could be supplemented when it was knocked out. These studies also elucidated a novel role for RhoA-dependent myelination.

[0103] Overall, the results demonstrate how targeting ADGRGl can enhance recovery after a demyelinating event, showing that the presence of ADGRG1 is necessary for proper remyelination, and that pmOLs that lack ADGRG1 also do not remyelinate properly. Meanwhile, applying agonists for ADGRGl signaling steps during the remyelination phase enhanced remyelination, demonstrating the utility of ADGRGl and its downstream effectors as effective targets for intervention.

[0104] All patents, patent applications, and publications cited in this specification are herein incorporated by reference to the same extent as if each independent patent application, or publication was specifically and individually indicated to be incorporated by reference. The disclosed embodiments are presented for purposes of illustration and not limitation. While the invention has been described with reference to the described embodiments thereof, it will be appreciated by those of skill in the art that modifications can be made to the structure and elements of the invention without departing from the spirit and scope of the invention as a whole.

Claims

CLAIMS What is claimed is:

Claim 1. An ADGRG1 activator, for use in a method of treating a demyelinating condition in a subject.

Claim 2. The ADGRG1 activator of Claim 1, wherein the demyelinating condition is multiple sclerosis.

Claim 3. The ADGRG1 activator of Claim 1, wherein the ADGRG1 activator is selected from the group consisting of transglutaminase 2 (TG2) polypeptide or active fragments thereof; a GPR56 ligand comprising activating subsequences of ADGRG1 ; P7; a gedunin derivative; a khivorin derivative; a monobody activator of ADGRG1 ; a synthetic peptide mimicking a GPR56 tethered agonist peptide; and a nucleic acid construct that induces or increases ADGRG1 expression levels in pmOLs.

Claim 4. A RhoA activator, for use in a method of treating a demyelinating condition in a subject.

Claim 5. The RhoA activator of Claim 4, wherein the demyelinating condition is multiple sclerosis.

Claim 6. The RhoA activator of Claim 4, wherein the RhoA activator is selected from the group consisting of guanosine-5'- diphosphate; cytotoxic necrotizing factor- 1; lysophosphatidic acid; lysophosphatidylcholine; calpeptin; colchicine; nocodazole; vinblastine; cytochalasin D; Sphingosine -1 -phosphate; Lysophosphatidic acid; and Bombesin.

Claim 7. A ROCK activator, for use in a method of treating a demyelinating condition in a subject.

Claim 8. The ROCK activator of Claim 7, wherein the demyelinating condition is multiple sclerosis.

Claim 9. The ROCK activator of Claim 7, wherein the ROCK activator is selected from the group consisting of narciclasine, calpeptin, and lysophosphatidic acid.

Claim 10. A cofilin inhibitor, for use in a method of treating a demyelinating condition in a subject.

Claim 11. The cofilin inhibitor of Claim 10, wherein the demyelinating condition is multiple sclerosis.

Claim 12. The cofilin inhibitor of Claim 10, wherein the cofilin inhibitor is selected from the group consisting of cucurbitacin E or a derivative thereof; tacrolimus; sodium vanadate; SZ-3; JG-6; a cofilin-targeting PROTAC construct; and an inhibitor of cofilin expression.

Claim 13. The cofilin inhibitor of Claim 12, wherein the cofilin inhibitor is cucurbitacin E.

Claim 14. A method of treating a demyelinating condition in a subject in need of treatment therefor, comprising administering to the subject a pharmaceutically effective amount of an ADGRG1 activator.

Claim 15. The method of Claim 14, wherein the demyelinating condition is multiple sclerosis.

Claim 16. The method of Claim 14, wherein the ADGRGl activator is selected from the group consisting of transglutaminase 2 (TG2) polypeptide or active fragments thereof; a GPR56 ligand comprising activating subsequences of ADGRGl ; P7; a gedunin derivative; a khivorin derivative; a monobody activator of ADGRGl ; a synthetic peptide mimicking a GPR56 tethered agonist peptide; and a nucleic acid construct that induces or increases ADGRGl expression levels in pmOLs.

Claim 17. A method of treating a demyelinating condition in a subject in need of treatment therefor, comprising administering to the subject a pharmaceutically effective amount of a RhoA activator.

Claim 18. The method of Claim 17, wherein the demyelinating condition is multiple sclerosis.

Claim 19. The method of Claim 17, wherein the RhoA activator is selected from the group consisting of guanosine-5'- diphosphate; cytotoxic necrotizing factor- 1; lysophosphatidic acid; lysophosphatidylcholine; calpeptin; colchicine; nocodazole; vinblastine; cytochalasin D; Sphingosine -1 -phosphate; Lysophosphatidic acid; and Bombesin.

Claim 20. A method of treating a demyelinating condition in a subject in need of treatment therefor, comprising administering to the subject a pharmaceutically effective amount of a ROCK activator.

Claim 21. The method of Claim 20, wherein the demyelinating condition is multiple sclerosis.

Claim 22. The method of Claim 20, wherein the ROCK activator is selected from the group consisting of narciclasine, calpeptin, and lysophosphatidic acid.

Claim 23. A method of treating a demyelinating condition in a subject in need of treatment therefor, comprising administering to the subject a pharmaceutically effective amount of a cofilin inhibitor.

Claim 24. The method of Claim 23, wherein the demyelinating condition is multiple sclerosis.

Claim 25. The method of Claim 23, wherein the cofilin inhibitor is selected from the group consisting of cucurbitacin E or a derivative thereof; tacrolimus; sodium vanadate; SZ-3; JG-6; a cofilin-targeting PROTAC construct; and an inhibitor of cofilin expression.

Claim 26. The method of Claim 25, wherein the cofilin inhibitor is cucurbitacin E.