WO2020018913A1 - Compositions and methods for treating disorders characterized by a defect in gpr56 expression or activity - Google Patents

Compositions and methods for treating disorders characterized by a defect in gpr56 expression or activity Download PDF

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Publication number
WO2020018913A1
WO2020018913A1 PCT/US2019/042618 US2019042618W WO2020018913A1 WO 2020018913 A1 WO2020018913 A1 WO 2020018913A1 US 2019042618 W US2019042618 W US 2019042618W WO 2020018913 A1 WO2020018913 A1 WO 2020018913A1
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Prior art keywords
gpr56
polypeptide
microglia
mice
disease
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PCT/US2019/042618
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French (fr)
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Xianhua PIAO
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Children's Medical Center Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Definitions

  • Autism spectrum disorders are class of neurological and developmental disorders that begin early in childhood and typically last throughout a person's life. ASDs include a range of conditions characterized by challenges with social skills, repetitive behaviors, speech and nonverbal communication. Autism’s most-obvious signs tend to appear between 2 and 3 years of age. In some cases, it can be diagnosed as early as 18 months and may last throughout an individuals lifetime. ASD affects 1 in 37 boys and 1 in 151 girls. Approximately one third of people with Autism remain non-verbal and
  • the present invention features compositions and methods for the treatment of ASD and other neurological diseases and disorders associated with defects in GPR56 or with undesirable increases in synapse number.
  • the invention provides a method of promoting synaptic pruning in a neuronal tissue, the method comprising contacting the tissue with an agent that activates GPR56, thereby promoting synaptic pruning.
  • the invention provides a method for treating a disease or disorder characterized by a loss or reduction in GPR56 expression or activity or an undesirable increase in synapse number, the method comprising administering to a subject in need thereof an agent that activates GPR56, thereby treating the disease or disorder.
  • the invention provides a method for treating a disease or disorder characterized by a loss or reduction in GPR56 expression or activity or an undesirable increase in synapse number, the method comprising administering to a subject in need thereof an S4 isoform of GPR56, thereby treating the disease or disorder.
  • the disease or disorder is an Autism spectrum disorder, multiple sclerosis, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis.
  • the agent is a small compound (e.g., gedunin- and vicvorin compound), polypeptide, or polynucleotide.
  • the compound is any one or more of 3-alpha-acetoxydihydrodeoxygedunin, vicvorin, 7 synthetic peptide agonist, 3- deacetylkhivorin, deoxygedunin, and l,2-Epoxygedunin.
  • the polypeptide is transglutaminase 2 (TG2) polypeptide or fragment thereof or GPR56 ligand comprising amino acids 383-404 of GPR56.
  • the GPR56 ligand comprises or consists of the amino acid sequence TYFAVLMVS or the amino acid sequence TYFAVLMVSSVEVDAVHKHYLS.
  • the agent is a GPR56 ligand that is covalently linked to a lipid or transmembrane domain.
  • the N- terminus or C-terminus of the GPR56 ligand is covalently linked to the lipid or
  • the polypeptide is a TG2 polypeptide or fragment thereof comprises amino acids 465-687 of TG2.
  • the polypeptide is a TG2 polypeptide or fragment thereof that forms one or more beta barrel domains.
  • the disease or disorder is an Autism spectrum disorder, multiple sclerosis, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis.
  • TG2 polypeptide is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_004604 and having GPR56 binding activity.
  • An exemplary TG2 polypeptide sequence is provided below.
  • Tgm2 Transglutaminase 2 ( Tgm2 ) nucleic acid molecule
  • Tgm2 Transglutaminase 2 ( Tgm2 ) nucleic acid molecule
  • An exemplary Tgm2 nucleic acid molecule sequence is provided at NCBI Accession No. NM_0046l3 and is shown below.
  • G protein-coupled receptor 56 (GPR56) polypeptide is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No.
  • NP 004604 and having RhoA and/or mammalian target of rapamycin (mTOR) pathway signaling activity.
  • mTOR mammalian target of rapamycin pathway signaling activity.
  • An exemplary GPR56 polypeptide sequence is provided below.
  • a subject having a defect in GPR56 expression or activity has or is at risk for developing a disorder characterized by an undesirable increase in synapse number in a tissue of an organism.
  • a GPR56 is an S4 isofom :
  • G protein-coupled receptor 56 (GPR56) nucleic acid molecule is meant a polynucleotide encoding a GPR56 polypeptide or fragment thereof.
  • An exemplary GPR56 nucleic acid molecule sequence is provided at NCBI Accession No. NM_00l 145770 and is shown below.
  • agent is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
  • alteration or“change” is meant an increase or decrease.
  • An alteration may be by as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or by 40%, 50%, 60%, or even by as much as 70%, 75%, 80%, 90%, or 100%.
  • biological sample any tissue, cell, fluid, or other material derived from an organism.
  • capture reagent is meant a reagent that specifically binds a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.
  • the terms“determining”,“assessing”,“assaying”,“measuring” and “detecting” refer to both quantitative and qualitative determinations, and as such, the term “determining” is used interchangeably herein with“assaying,”“measuring,” and the like. Where a quantitative determination is intended, the phrase“determining an amount” of an analyte and the like is used. Where a qualitative and/or quantitative determination is intended, the phrase“determining a level” of an analyte or“detecting” an analyte is used.
  • Detect refers to identifying the presence, absence or amount of the analyte to be detected.
  • detectable label is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • an effective amount is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • fragment is meant a portion of a protein or nucleic acid that is substantially identical to a reference protein or nucleic acid. In some embodiments the portion retains at least 50%, 75%, or 80%, or more preferably 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid described herein.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • the term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • modifications for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • microglia is meant an immune cell of the central nervous system
  • myelin is meant a fatty white substance surrounding the axon of nerve cells and forming an electrically insulating layer. Myelination is the process by which the myelin is produced.
  • oligodendrocyte is meant a glial cell that forms the myelin sheath of axons in the central nervous system. Oligodendrocytes differentiate from oligodendrocyte precursor cells in the central nervous system.
  • increasing proliferation is meant increasing cell division of a cell in vivo or in vitro.
  • the terms“prevent,”“preventing,”“prevention,”“prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • subject refers to an animal which is the object of treatment, observation, or experiment.
  • a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non human primate, murine, bovine, equine, canine, ovine, or feline.
  • reference is meant a standard of comparison or control condition.
  • the reference is a GPR56 polypeptide or nucleic acid molecule.
  • Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g ., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g.,
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g, formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • hybridization time the concentration of detergent, e.g ., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA
  • concentration of detergent e.g ., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA).
  • ssDNA denatured salmon sperm DNA
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate,
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
  • a BLAST program may be used, with a probability score between e 3 and e 100 indicating a closely related sequence.
  • binds is meant a compound (e.g ., peptide) that recognizes and binds a molecule (e.g., polypeptide), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample.
  • a compound e.g ., peptide
  • molecule e.g., polypeptide
  • the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as 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 otherwise clear from context, all numerical values provided herein are modified by the term about.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 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
  • the terms“comprises,”“comprising,”“containing,”“having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean“includes,” “including,” and the like;“consisting essentially of’ or“consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • FIG. 1 provides a schematic diagram showing that GPR56 mediates tripartite signaling among extracellular matrix, microglia and oligodendrocyte during myelination.
  • FIG. 2A provides a series of sections throught the corpus callosum of
  • Opr 56 II II II ;I > dgfr a( "r KR and ( ⁇ pr5fJ l 11 ;Pdgfr a 'reER mice following cuprizone feeding and recovery and a graph.
  • Scale bar 250 pm.
  • FIG. 2B is a graph showing that the percentage of remyelinated corpus callosum displayed significant decrease in myelination at 7 DR and 10 DR between
  • FIG. 3 A is a schematic diagram showing the strategy for focal lysolecithin injection.
  • FIG. 3B is a schematic diagram showing the site of injection.
  • FIG. 3C provides representative transmission electron micrograph (TEM) images from the corpus colosum (CC) of Tgm2 ⁇ ;Cx3cr lCre and Tgm2 ⁇ ;Cx3cr lCre + mice.
  • TEM transmission electron micrograph
  • FIG. 3E is a scatter plot displaying g-ratio values in the CC of Tgm2 fl/il ;Cx3crlCre and Tgm2 II II ;( 'x3crK 're mice, which have a microglia-derived deletion of transglutaminase 2 (encoded by Tgm2).
  • FIG. 4 is a schematic diagram illustrating the hypothesis that microglia GPR56 is a molecular target of maternal immune activation (MIA) during pregnancy and inflammatory activation during early childhood.
  • MIA maternal immune activation
  • FIG. 5 A and 5B provides two graphs showing that Poly I:C induces rapid down- regulation of Gpr56 expression.
  • FIG. 6A includes sections through cerebral cortex of mouse models of autism (right hand panels) vs. control mice (left hand panels).
  • Mouse models of autism shown were either induced using prenatal valproate, VP A) or were the result of a single-gene mutation identified in human patients (Neuroligin-3, NL-3 R451C). These slices show decreased parvalbumin positive (PV+) intemeurons in the cerebral cortex. This is one of the pathologies associated with autism.
  • FIG. 6B is a graph showing quantitation.
  • FIG. 7 shows synaptic density in brains of autistic children vs. control children.
  • FIG. 8A shows representative images showing parvalbumin positive intemeuron density in cerebral cortex of a control brain relative to the brain of a microglial GPR56 knockout mouse.
  • FIG. 8B is a graph quantitating parvalbumin positive interneuron density in wild-type and microglial GPR56 knockout mice.
  • FIG. 9A shows the co-localization of Vglut2/Homerl at synapses in wild-type and microglial GPR56 knockout mice.
  • FIG. 9B is a graph quantitating synaptic density in in wild-type and microglial GPR56 knockout mice.
  • FIG. 10A shows the results of behavioral testing of wild-type and microglial GPR56 conditional knockout mice.
  • Gpr56 knockout mice (cKO) manifest autistic behavior in the form of a marble burying obsession.
  • FIG. 10B shows that Gpr56 knockout mice (cKO) manifest autistic behavior in the form of anxiety in an open field behavioral assay.
  • FIG. 10C shows that Gpr56 knockout mice (cKO) manifest autistic behavior in the form impaired social behavior.
  • FIG. 11 provides a graphical representation showing the presence of single nucleotide polymorphisms (SNP) in GPR56 in Alzheimer’s disease (AD) patients.
  • SNP single nucleotide polymorphisms
  • FIG. 12 is a table indicating the position of SNPs in GPR56, which correlate with neurological defects including multiple sclerosis, Alzheimer’s disease, Autism, and
  • FIG. 13 is a schematic diagram illustrating agents that could increase the expression or activity of GPR56 for use as therapeutics in neurological disorders characterized by a defect in GPR56 expression or activity.
  • FIG. 14A-L shows that microglial GPR56 is required for synaptic refinement in dLGN during development.
  • FIG. 14A provides an RNAscope showing that Gpr56 transcripts co-localize with microglia in controls, but not in conditional knock out (CKO), and is absent in all cell types in the global KO. Scale bar, 20 pm.
  • FIG. 14B provides a representative image of vGlut2 and Homerl staining in dLGN at P10. The outline indicates the dLGN core, and the dotted boxes show where synapses are quantified. Scale bar, 200 pm.
  • FIG. 14C provides a representative 3D-reconstructed super-resolution images of vGlut2 and Homerl staining of P8 dLGN. Shading represents surface rendering of vGlut2 + presynaptic terminals. Darker spots represent Homerl + post-synapses within the distance of 300nm from green surface.
  • FIG. 14E provides confocal images of vGlut2 labeling retinal ganglion cell (RGC) presynaptic terminals and Homerl for post- synapses in the dLGN of CKO and controls at P10. Overlapped vGlut2 and Homerl are quantified as synapses, indicated by white circles. Scale bar, 5 pm.
  • FIG. 14G provides a Western blot of vGlut2 using microdissected dLGN tissue from P30 mice.
  • FIG. 14J provides a representative image of vGlutl and Homerl staining in dLGN at P10. Dotted boxes show where synapses are quantified in the upper part of the dLGN core (outline). Scale bar, 200 pm.
  • FIG. 14K provides representative images of vGlutl + presynaptic terminals and Homerl + postsynapses in the dLGN of CKO and controls at P10. Scale bar, 5 pm.
  • FIG. 14N-A -14N-H show that cellular properties remain unchanged upon microglial Gpr56 deletion. CKO microglia do not show significant difference compared to controls.
  • FIG. 14N-A provides images of microglia stained by anti-Ibal in dLGN at P5.
  • FIG. 14N-C provides representative images of Ibal and CD68 double staining.
  • FIG. 14N-D provides quantification of the percentage of CD68 positive microglia.
  • FIG. 14N-E provides images of individual microglia.
  • FIG. 14N-F provides quantification of coverage area of each microglia between CKO and controls.
  • N 35 (Ctrl),
  • FIG. 14N-G provides images depicting concentric circles upon manually outlined microglia at 1.25 pm intervals for Sholl analysis.
  • FIG. 14N-H provides A Sholl analysis that shows no significant change in arbor complexity in CKO.
  • FIG. 140 shows retinogeniculate synapses in CKO and controls using SIM.
  • Top panels show original images taken by SIM.
  • Middle panels show 3D rendered images after processing in Imaris.
  • FIG. 14P-A - 14P-D show that deleting microglial Gpr56 has no effect on RGC density in P5 retina.
  • FIG. 14P-A shows whole mount retinal staining of RGC using Brn3a antibody.
  • FIG. 14P-B provide representative images of RGC and microglia staining using Bm3 and Ibal antibodies in retina.
  • FIG. 14P-C shows quantification of RGC density in Ctrl and CKO.
  • FIG. 14P-D shows quantification of retinal microglia density in Ctrl and CKO.
  • FIG. 15 shows that microglial GPR56 is required for synapse refinement in the hippocampus.
  • FIG. 15A provides representative images showomg hippocampus with vGlut2 and Homerl immunostaining. White box outlines the region of interest and yellow box shows the regions where confocal images were taken.
  • FIG. 15B provides confocal images of synaptic immunostaining in CA1 striatum lacunosum-moleculare at PlO. Scale bar, 5 pm.
  • FIG. 15C and D show quantification of synapse density in CA1 striatum lacunosum- moleculare in iCKO versus control at P10 (C), and CKO versus control at P21 (D).
  • FIG. 15E provides representative images showing hippocampus with vGlutl and Homerl immunostaining.
  • FIG. 15F provides confocal images of synaptic immunostaining in CA1 striatum radiatum at P10.
  • FIG. 15G and 15H show quantification of synapse density in CA1 striatum radiatum in iCKO versus control at P10 (G), and CKO versus control at P21 (H).
  • FIG. 16A-E shows that microglial Gpr56 deficiency leads to reduced engulfment of RGC inputs, and impaired retinogeniculate circuit organization and function.
  • FIG. 16A provides a schematic representation of in vivo engulfment assay. CTB594 dyes are injected into both eyes, and anterogradely trace RGC projections to dLGN.
  • FIG. 16A provides representative surface rendered microglia from P5 dLGN of CKO or controls in which RGC inputs were labeled with CTB-594. Scale bar, 20pm.
  • FIG. 16C provides a quantification of the percentage of engulfed RGC inputs in controls and CKO microglia. More than 10 microglia cells are analyzed in each individual mouse brain.
  • FIG. 16D provides a diagram of RGC labeling for testing eye-specific segregation at P30.
  • FIG. 16E provides a CTB-labeled dLGN shows reduced eye-segregation at P30 in cKO mice.
  • the left column shows contralateral dLGN labeled with CTB488 (green), and the middle one is ipsilateral dLGN with CTB647 (magenta).
  • FIG. 16F provides a histogram distribution chart of R-value for all pixels within dLGN represents the degree of eye-specific segregation. A greater R-value means a bigger difference of ipsi-to-contraleteral fluorescence intensity. The narrower distribution of cKO in the dotted box indicates reduced segregation.
  • FIG. 16H provides a schematic diagram of electrophysiol ogical recording in a parasagittal dLGN.
  • FIG. 16J provides a Western blot of NMDAR1 using
  • FIG. 16K provides a Quantification of
  • FIG. 16M provides a Western blot of GluRl with microdissected dLGN tissue.
  • Fig. 160-A-160-D shows that microglial GPR56 Deficiency leads to impaired eye- specific segregation at P10.
  • Fig. 160-A shows in the left two columns contra- and ipsi- lateral RGC inputs labeled by CTB488 and CTB594, respectively. The middle two columns are binary images of contra- and ipsi-lateral LGN. The right images show a greater overlap between contralateral RGC inputs and ipsilateral inputs.
  • Fig. 160-C provides images of superior colliculus after 24 hours anterograde labeling of RGC by CTB488 and CTB647.
  • Fig. 160-D shows the variance of R-values from caudal LGN to cranial LGN.
  • FIG. 17A-17F shows that GPR56 GAIN domain binds to PS, and Gpr56 S4 variant is essential for synaptic refinement in dLGN development.
  • FIG. 17A provides a diagram that shows GPR56 protein structure, consisting of a PLL, a GAIN domain, and a 7TM. A full length N-terminal fragment (NTF) contains PLL and GAIN domains.
  • FIG. 17B provides a diagram of membrane lipid strip showing the lipid composition for each dot.
  • FIG. 17C shows that GAS6, NTF-hFc, and GAIN-hFc bind to several specific lipids, whereas hFc does not.
  • DAG 1, 2-Diacyl glycerol
  • PA phosphatidic acid
  • PS phosphatidylserine
  • PE phosphatidyl- ethanol- amine
  • PC phosphatidylcholine
  • PG phosphatidylglycerol
  • PI phosphati- dylinositol
  • PI(4)P phosphatidylinositol 4-phosphate
  • PI(4,5)P2 phospha- tidylinositol 4,5- bisphosphate
  • PI(3,4,5)P3 phosphatidylinositol 3,4,5-tris- phosphate.
  • FIG. 17D provides a flow chart showing the experimental design.
  • FIG. 17E provides a Flow cytometry analysis shows that only GAIN domain binds to PS.
  • FIG. 17F depicts the results of flow cytometry demonstrating that only GAIN domain is able to compete off Annexin V binding to PS.
  • FIG. 17G provides representative images of vGlut2/Homerl staining in P10 mouse dLGN of control, Gpr56 null , and Gpr56 S4 brains.
  • FIG. 17G and 17H shows relative vGlut2/Homerl synapse density in dLGN.
  • FIG. 17J-A - 17J-E relates to Gpr56 S4 isoform.
  • FIG. 17J-A and B provide diagrams showing the genomic structure of different Gpr56 variants. Solid boxes indicating exons that are transcribed.
  • FIG. 17J-C and D are standard curves of qPCR using a series of cDNA dilution.
  • FIG. 17J-E is a graph showing that only S4 transcripts were present in Gpr56 S4 mouse microglia.
  • FIG. 17K shows that a deletion of GPR56 S4 isoform did not result in more severe cortical ectopia.
  • FIG. 17 (top) provides representative images of Nissl staining of Gpr56 S4 and Gpr56 null El 6.5 neocortex. Arrows point out cortical ectopias.
  • Fig. 18A-E show that a microglial Gpr56 deficiency impairs engulfment of PS + RGC Inputs.
  • Fig. 18A provides a drawing depicting the experimental procedure where CTB488 and CTB647 were intraocularly injected, followed by intracranial injection of PSvue550 to dLGN border.
  • Fig. 18B provides a schematic diagram shows the timeline of procedures for ex vivo imaging and in vivo engulfment analysis.
  • Fig. 18C provides (top panel) representative images show PS labeling in the WT dLGN at P6 and P13. RGC inputs were labeled with CTB488. Bottom panel: Enlarged regions of the boxed region in top panels.
  • Circles indicate PS + RGC inputs. Arrows pointing to the enlarged PS + RGC inputs in the upper right hand comer. Scale bar, 5 pm.
  • Fig. 18E provides a diagram showing contra- (green) and ipsi-lateral (blue) projections overlap in P6 dLGN. Yellow box indicates the region where images were taken and analyzed.
  • F Representative images show PSVue colocalizes with contra- or ipsi-lateral RGC inputs.
  • FIG. 19A-19D shows in vivo labeling of PS by PSVue and pSIVA.
  • FIG. 19A is a diagram illustrating PS labeling by PSVue550 and RGC inputs antegrade tracing by CTB. CTB was intraoccularly injected 24 hours prior to PSVue/pSIVA injection.
  • FIG. 19B the left panel shows well-diffused PSVue into dLGN. The box indicates the region where the images were taken.
  • Right panel shows RGC inputs colocalize with PSVue signal.
  • the white box indicates the region of higher magnification image shown.
  • FIG. 19C provides a diagram showing pSIVA labeling and RGC inputs tracing by CTB.
  • FIG. 19D left panel shows pSIVA accumulated in the gap between hippocampus and LGN. Right panel shows minimal pSIVA colocalized with RGC inputs. Scale bar, 20 pm.
  • Fig. 20A-20F shows that microglia specifically engulf PS Vue-labeled PS + RGC inputs.
  • a diagram shows PSVue was injected through the hippocampus to the border of dLGN. Enlarged dLGN (white dotted line) is showed in Aii.
  • Bi A representative image of microglia from PSVue treated dLGN. Nucleus were labeled with DAPI.
  • Bii A 3D surface rendered microglia (purple) with DAPI (blue) and engulfed inputs (green) and PSVue (red).
  • FIG. 21 A and B show that microglia engulf more PS + than PS RGC inputs in dLGN.
  • FIG. 21 A provides a representative image of microglia (left) is surface rendered (middle), and RGC inputs and PSVue inside of microglia are shown in the right panel. Arrows pointing to two RGC inputs that are presented in a higher magnified insert with one being PS + and the other being PS input.
  • FIG. 22A and B indicate that a Gpr56 CKO mice shows no difference in paired pulse depression.
  • Paired pulse depression was recorded on dLGN slice from P28-P34 mice.
  • optic inputs usually demonstrate paired pulse depression, and cortical inputs show paired pulse facilitation, this data indicates that the optic tracts and not cortical inputs were stimulated
  • n 14 (Ctrl), 23 (KO) cells from 5, 7 mice.
  • P 0.694 by Student’s t-test.
  • the present invention features compositions and methods for the treatment of ASD and other neurological diseases and disorders associated with defects in GPR56 or with undesirable increases in synapse number.
  • the invention is based, at least in part, on the discovery that GPR56, a protein that functions in oligodendrocyte and interneuron development, also functions in synaptic pruning, and that defects in synaptic pruning are observed not only in autistic subjects, but in GPR56 knockout mice.
  • GPR56 a protein that functions in oligodendrocyte and interneuron development, also functions in synaptic pruning, and that defects in synaptic pruning are observed not only in autistic subjects, but in GPR56 knockout mice.
  • microglial GPR56 maintains appropriate synaptic numbers in several brain regions in time- and circuit- specific fashion.
  • PS Phosphatidylserine
  • GPR56 a specific alternatively spliced isoform of GPR56 is selectively required for microglia-mediated synaptic pruning.
  • the results presented herein provide a genetic substrate to address microglial synapse pruning in the context of other neurodevelopmental processes.
  • Microglia tissue resident macrophages of the CNS, are important for synaptic development, both promoting synapse formation and engulfing redundant synapses.
  • Immune molecules such as classical complement components and receptors, CX3CL1/CX3CR1,
  • MHC class I and PirB have been implicated in developmental synapse refinement and in synapse loss in disease models.
  • elements associated with microglial synapse refinement have been strictly limited to their expression in microglia among CNS cells.
  • Mammalian neurodevelopment involves a succession of complex and overlapping processes beginning with neurogenesis and neuronal migration, which are concurrent with microglial infiltration and morphogenesis. Subsequently, neurite arborization sets the stage for synaptogenesis, circuit establishment and refinement as well as myelination. All these processes entail cell-cell interactions, so that discovery of molecules involved in multiple processes in a cell-type specific fashion can inform our understanding how overlapping and sequential programs of intercellular signaling events are coordinately regulated.
  • microglia Originating from primitive myeloid cells in the yolk sac, microglia enter the central nervous system (CNS) at the start of brain development.
  • CNS central nervous system
  • microglia modulate neural progenitor survival by precisely timed-and- localized secretion of growth factors and the size of neural progenitor pool by clearance of dead or stressed cells.
  • GPR56 regulates neural progenitor cell proliferation, and germline deletion of Gpr56 impairs neurogenesis.
  • Gpr56 is expressed in multiple brain cell types during development, including neuroepithelial cells, intermediate progenitor cells, and first-born neurons.
  • Gpr56 message is highly expressed in young and adult microglia in both humans and mice and regulated by a microglial super enhancer.
  • GPR56 mediates tripartite signaling among ECM, microglia and oligodendrocyte during myelination. Microglia promote OPC proliferation via GPR56 signaling.
  • Tissue transglutaminase (TG2), derived from microglia, is the ligand of
  • oligodendrocyte precursor cell OPC GPR56.
  • Tissue transglutaminase TG2 derived from microglia, is the ligand of oligodendrocyte precursor cell (OPC) GPR56. Together with laminin, TG2 activates OPC GPR56 and promotes OPC proliferation and thus central nervous system myelination (FIG. 1).
  • Certain neurological disorders arise when an exogenous stressor strikes an individual having an underlying vulnerability, such as a genetic predisposition, during a critical developmental period. The exogenous stressor might include an infection that causes immune activation. It has previously been shown that GPR56 is highly expressed in microglia, and that GPR56 is downregulated in response to an inflammatory challenge. For example, two exogenous stressors, maternal immune activation (MIA) during pregnancy and inflammatory response during early childhood infection, that occur during critical developmental periods might be sufficient to induce an autism spectrum disorder in certain vulnerable individuals (FIG. 4).
  • MIA maternal immune activation
  • GPR56 a member of the adhesion G protein-coupled receptor family
  • the adhesion G protein-coupled receptor (aGPCR) GPR56/ADGRG1 is a newly identified regulator of OL development that is evolutionarily conserved in zebrafish, mice, and humans (Ackerman et al., Nat Commun 6, 6122, 2015; Giera et al., Nat Commun 6, 6121, 2015).
  • BFPP bilateral frontoparietal polymicrogyria
  • Gpr56 in OL lineage cells showed that the hypomyelination phenotype is caused specifically by deficiency for GPR56 signaling in oligodendrocyte precursors (OPCs) and immature oligodendrocytes (OLs) (Giera et al., Nat Commun 6, 6121, 2015).
  • OPCs oligodendrocyte precursors
  • OLs immature oligodendrocytes
  • Loss of Gpr56 in mice and zebrafish decreased OPC proliferation leading to a reduced number of mature myelinating OLs and fewer myelinated axons in the CNS (Ackerman et al., Nat Commun 6, 6122, 2015; Giera et al., Nat Commun 6, 6121, 2015).
  • the relevant GPR56 ligand during CNS myelination was not defined during these studies.
  • Microglial TG2 was identified as the ligand of OPC GPR56 via a combined approach utilizing molecular, cellular and developmental biology as well as unbiased proteomics (Giera et al., eLife, 2018, pii: e33385. doi: 10.7554/eLife.33385). This deorphanization is a mandatory first step in therapeutic exploitation of this novel pathway.
  • the past few years have seen aGPCRs implicated both in CNS and peripheral nervous system myelination and myelin maintenance (Kuffer et al., Nature 536 , 464-468, 2016; Langenhan et al., Nature reviews Neuroscience 77, 550-561, 2016).
  • TG2 binds laminin-l 11 (Aeschlimann et al., J Biol Chem 267, 11316-11321, 1992).
  • TG2 contingent on its crosslinking activity, together with laminin-l 11, binds to the GPR56 NTF and dissociates the NTF from the CTF, allowing the endogenous GPR56 tethered ligand to initiate G-protein signaling.
  • Downstream RhoA activation and CDK2 are then implicated in OPC progression through the cell cycle, to generate mature oligodendrocytes (OLs) for myelination or remyelination.
  • OLs oligodendrocytes
  • this microglial ligand-ECM-OPC receptor signaling triad is particularly relevant for OL development, where a complex array of factors and ECM components affect the varied stages of the process (Wheeler and Fuss,
  • Collagen III was previously identified as the ligand for neural progenitor cell-GPR56 in the developing neocortex (Luo et al., Proc Natl Acad Sci U S A 108, 12925-12930, 2011). As shown herein, collagen III was not the GPR56 ligand in OPCs, and microglia-derived TG2 was the ligand of OPC-GPR56. The results of this study highlight a unique property of adhesion GPCRs: activation by distinct ligands in different cellular and developmental contexts.
  • the signaling module of GPR56 contains multiple potential targets for therapeutic intervention, including the GPR56-CTF, which similar to other GPCRs, serves as a legitimate drug target. Given the importance of myelin formation, maintenance and repair in neurological diseases across the human life span, and the importance of synapse pruning and maintenance in ASD, these findings have the potential to provide clinical benefit for both developmental and acquired neurological diseases involving myelination and/or synapse formation/pruning.
  • Transglutaminase 2 As a member of the transglutaminase family, Transglutaminase 2 (TG2) is a versatile and multi-faceted protein that displays several diverse biological functions. In addition to the typical transamidating/crosslinking function, studies over the last decade reveal non- enzymatic functions of extracellular TG2, including promoting cell adhesion, migration, and survival. TG2 was identified as a regulator of OL development by serving as a ligand of GPR56. A direct mitogenic effect of TG2 on OPCs could be elicited. Without being bound to theory, this indicates an extracellular non-enzymatic function of TG2.
  • GPR56 a member of the adhesion G protein-coupled receptor family, is a recently identified novel regulator of oligodendrocyte development.
  • Microglia-derived transglutaminase 2 (encoded by Tgm2 ) is the GPR56 ligand for OPCs.
  • a search for the ligand of GPR56 in the developing white matter identified one or more putative ligands of GPR56 predominantly expressed in microglia. Further biotin-streptoavidin pull- down from mixed glia cells followed by mass spectrometry analysis revealed
  • TG2 transglutaminase 2
  • GPR56 transglutaminase 2
  • TG2 is predominantly expressed in microglia in the postnatal brain.
  • ECM protein laminin interacts with catalytically-active TG2 to release GPR56 NTF and activates GPR56 CTF for downstream RhoA signaling, which in turn promotes OPC proliferation.
  • microglia-specific deletion of Tgm2 leads to fewer mature oligodendrocytes (OLs) and CNS hypomyelination, phenocopying OPC-specific deficiency for GPR56.
  • Tgm2 knockout mice manifest with decreased oligodendrocyte precursor cell (OPC) proliferation, leading to fewer mature oligodendrocytes and a reduced number of myelinated axons in the corpus callosum via RhoA pathway, phenocopying the Gpr56 knockout mice.
  • OPC oligodendrocyte precursor cell
  • Recombinant TG2 stimulated OPC proliferation in a GPR56-dependent manner in vitro.
  • Recombinant TG2 rescued remyelination failure in Tgm2 knockout cerebellar slices.
  • OPC-specific deletion of Gpr56 impairs CNS remyelination after cuprizone-induced demyelination, demonstrating a function with regard to repair as well as development.
  • transglutaminase 2 (TG2, gene symbol Tgm2 ) was identified as a ligand for GPR56 during white matter development.
  • TG2 was reported to be present in oligodendrocyte precursors (OPCs) (Van Strien et al. Glia 59, 1622-1634, 2011) and astrocytes (Van Strien et al. PLoS One 6, e25037, 2011)
  • OPCs oligodendrocyte precursors
  • astrocytes Van Strien et al. PLoS One 6, e25037, 2011
  • Zhang et al. demonstrated that Tgm2 is predominantly expressed in microglia through gene expression profiling using purified glial cells and neurons (Zhang et al. JNeurosci 34, 11929-11947, 2014).
  • TG2 protein was only detected in microglia by western blot analysis. It is possible that previous reports detected the TG2 that binds to the cell surface of OPCs and astrocytes.
  • Microglia-derived TG2 promotes OPC proliferation via the RhoA pathway, providing a novel molecular link
  • the present invention provides methods of treating disease and/or disorders or symptoms thereof associated with a reduction in GPR56 expression or activity or an undesirable reduction in synaptic pruning (e.g., Autism spectrum disorder, multiple sclerosis, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis), which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising administering an agent that activates GPR56 to a subject (e.g., a mammal such as a human).
  • a subject e.g., a mammal such as a human
  • a subject e.g., a mammal such as a human
  • one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder or symptom thereof.
  • the method includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
  • the methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
  • the application prevents or treats neurological diseases, for example, characterized by decreased expression or activity of GPR56 (e.g., ASD).
  • the invention provides for the treatment of a variety of diseases and disorders associated with decreased synaptic pruning (e.g., multiple sclerosis, Alzheimer’s disease, Autism, and Amyotrophic Lateral Sclerosis). Such diseases are amenable to treatment by increased expression or activity of GPR56.
  • diseases and disorders associated with decreased synaptic pruning e.g., multiple sclerosis, Alzheimer’s disease, Autism, and Amyotrophic Lateral Sclerosis.
  • Such diseases are amenable to treatment by increased expression or activity of GPR56.
  • the invention generally features method of increasing or promoting synaptic pruning in a subject having or at risk of developing an undesirable increase in synapse number.
  • the method involves contacting a glial cell (e.g. an oligodendrocyte or oligodendrocyte precursor) of the subject with an agonist or ligand of a GPR56 polypeptide; and activating signaling via the GPR56 polypeptide, thereby increasing or promoting myelin formation.
  • a glial cell e.g. an oligodendrocyte or oligodendrocyte precursor
  • Activating ligands of GPR56 polypeptide include naturally- occuring ligands such as TG2 or tethered ligands generated by recombinant or synthetic techniques.
  • the method involves contacting a glial cell (e.g.
  • an oligodendrocyte or oligodendrocyte precursor of the subject with a nucleic acid molecule encoding a GPR56 polypeptide or a fragment thereof; and expressing the GPR56 polypeptide in the cell, thereby increasing or promoting synaptic pruning.
  • the present invention provides methods of treating diseases and/or disorders or symptoms thereof related to demyelination that comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that increases GPR56 receptor signalling, expression, or activity to a subject (e.g., a mammal such as a human).
  • a subject e.g., a mammal such as a human
  • one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder characterized by an undesirable increase in synapse number or symptom thereof.
  • the method includes the step of administering to the mammal a therapeutic amount of an amount of an agent herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that
  • the methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect (e.g., an increase in myelination). Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof.
  • Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).
  • a diagnostic test or opinion of a subject or health care provider e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like.
  • the compounds herein may be also used in the treatment of any other disorders in which myelination deficiency or loss may be implicated.
  • GPR56 Small compounds that activate GPR56 are known in the art. See, for example, Stoveken et ah, which describes two related classes of small molecules that could activate the aGPCR GPR56/ADGRG1, gedunin- and vicvorin derived natural products. The most potent compound identified was 3-alpha-acetoxydihydrodeoxygedunin, or 3-alpha-DOG.
  • Other compounds useful in the methods of the invention include vicvorin, 7 synthetic peptide agonist, 3-deacetylkhivorin, deoxygedunin, and l,2-Epoxygedunin.
  • TG2 Binding of TG2 to GPR56 results in GPR56 activation via exposure of a tethered agonist, also known as the stalk region, which is inhibited by an extracellular N-terminal domain (NTD) of GPR56.
  • NTD N-terminal domain
  • the NTD is expressed as part of GPR56, proteolytically processed, and non-covalently bound to the 7 transmembrane domains of GPR56.
  • TG2 binds the NTD domain to expose the b-strand-l 3/stalk region, that when exposed, serves as a tethered agonist to activate G protein signaling (see., e.g., Stoveken et al. Proc Natl Acad Sci U S A. 2015 May 12; 112(19): 6194-6199, which is herein incorporated by reference in its entirety).
  • the tethered agonist or GPR56 activating ligand is linked to a membrane associated moiety.
  • Methods of making such tethered ligands are known in the art (see., e.g., U.S. Patent Nos. 8,563,519; 6,864,229; 8,440,627; 8,389,480; and 8,354,378 and U.S. Patent Publ. Nos. 20020076755; 20060166274; 20080214451; 20030148449;
  • BBB Blood-Brain Barrier
  • compositions for delivery of an agent e.g., a GPR56 tethered peptide, TG2 polypeptide or fragment thereof
  • BBB blood-brain barrier
  • the blood-brain barrier (BBB) protects and regulates the homeostasis of the brain and prevents the free passage of molecules into most parts of the brain. Transport of essential molecules such as nutrients, growth factors and hormones is achieved via a series of specific transporters and receptors that regulate passage across the brain endothelial cells.
  • an agent of the invention is fused or conjugated to a BBB peptide.
  • BBB peptide sequences are known in the art and are described at least for example at the Brainpeps® database (http://brainpeps.ugent.be/; Van Dorpe et al., Brain Structure and Function, 2012, 217(3), 687-718, which are herein incorporated by reference).
  • a BBB transporter molecule as provided herein can bind to brain microvascular endothelial cells (BMVECs), e.g., human, and can cross through BMVEC in vitro or in vivo from the peripheral vasculature into the CNS vasculature. Whether a given fragment is a BBB-penetrable fragment can be tested by a variety of in vitro or in vivo assays known to persons of ordinary skill in the art. For example, the transporter molecule can be tested in the in vitro transcytosis assay, or in an in vivo assay such as a diuresis assay.
  • BMVECs brain microvascular endothelial cells
  • transporter molecule activity can be demonstrated by visualization of the transporter molecule in the CNS.
  • a tritium-labeled transporter molecule can be delivered to a subject, and then visualized in the CNS via quantitative whole body radiography.
  • the transporter molecule localizes in specific regions of the CNS, e.g., the corpus callosum, developing white matter, and the like.
  • DNA molecules obtained by any of the methods described herein or those that are known in the art can be inserted into appropriate expression vectors by techniques well known in the art.
  • a double stranded DNA can be cloned into a suitable vector by restriction enzyme linking involving the use of synthetic DNA linkers or by blunt-ended ligation.
  • DNA ligases are usually used to ligate the DNA molecules and undesirable joining can be avoided by treatment with alkaline phosphatase.
  • the invention includes vectors (e.g., recombinant plasmids) that include nucleic acid molecules (e.g., genes or recombinant nucleic acid molecules encoding genes) as described herein.
  • the term“recombinant vector” includes a vector (e.g., plasmid, phage, phasmid, virus, cosmid, fosmid, or other purified nucleic acid vector) that has been altered, modified or engineered such that it contains greater, fewer or different nucleic acid sequences than those included in the native or natural nucleic acid molecule from which the
  • a recombinant vector was derived.
  • a recombinant vector may include a nucleotide sequence encoding a GPR56 or TG2 polypeptide, or fragment thereof, operatively linked to regulatory sequences, e.g., promoter sequences, terminator sequences, and the like, as defined herein.
  • regulatory sequences e.g., promoter sequences, terminator sequences, and the like.
  • Recombinant vectors which allow for expression of the genes or nucleic acids included in them are referred to as“expression vectors.”
  • one or more DNA molecules having a nucleotide sequence encoding one or more polypeptides of the invention are operatively linked to one or more regulatory sequences, which are capable of integrating the desired DNA molecule into a prokaryotic host cell.
  • Cells which have been stably transformed by the introduced DNA can be selected, for example, by introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • a selectable marker gene can either be linked directly to a nucleic acid sequence to be expressed, or be introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins described herein. It would be apparent to one of ordinary skill in the art which additional elements to use.
  • Factors of importance in selecting a particular plasmid or viral vector include, but are not limited to, the ease with which recipient cells that contain the vector are recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.
  • the vector(s) may be introduced into an appropriate host cell by one or more of a variety of suitable methods that are known in the art, including but not limited to, for example, transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc.
  • recombinant proteins can be detected by immunoassays including Western blot analysis, immunoblot, and immunofluorescence. Purification of recombinant proteins can be carried out by any of the methods known in the art or described herein, for example, any
  • a further purification procedure that may be used for purifying proteins is affinity chromatography using monoclonal antibodies which bind a target protein.
  • monoclonal antibodies which bind a target protein.
  • crude preparations containing a recombinant protein are passed through a column on which a suitable monoclonal antibody is immobilized.
  • the protein usually binds to the column via the specific antibody while the impurities pass through. After washing the column, the protein is eluted from the gel by changing pH or ionic strength, for example.
  • the invention provides methods for identifying agents (e.g.,
  • polypeptides, polynucleotides, antibodies, including recombinant antibodies, and small compounds useful for increasing synaptic pruning and/or treating or preventing a disease or disorder characterized by an undesirable increase in synapses.
  • the use of such cells, which express GPR56 is particularly advantageous for the identification of agents that increase GPR56 expression or biological activity. Methods of observing changes in GPR56 biological activity are exploited in high throughput assays for the purpose of identifying compounds that modulate GPR56 biological activity, e.g., transcriptional regulation or protein-nucleic acid interactions. Any number of methods are available for carrying out screening assays to identify new candidate compounds that increase the expression or activity of GPR56 and/or TG2.
  • candidate compounds are added at varying concentrations to the culture medium of cultured cells expressing GPR56 and/or TG2.
  • the cell is an oligodendrocyte, oligodendrocyte precursor, or heterologous cell expressing GPR56.
  • the cell is a microglial cell or heterologous cell expressing TG2.
  • Gene expression is then measured, for example, by microarray analysis, Northern blot analysis (Ausubel et al., supra), or RT-PCR, using an appropriate hybridization probe. The level of gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate molecule.
  • a compound which increases the expression of a GPR56 and/or Tgm2 gene, or a functional equivalent thereof, is considered useful in the invention; such a molecule may be used, for example, as a therapeutic to treat a human patient having a demyelination disease or disorder.
  • the effect of candidate compounds may be measured at the level of polypeptide production using the same general approach and standard immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific for a polypeptide encoded by a GPR56 and/or Tgm2 gene.
  • immunoassays may be used to detect or monitor the expression of at least one of the polypeptides of the invention in an organism.
  • Polyclonal or monoclonal antibodies that are capable of binding to such a polypeptide may be used in any standard immunoassay format (e.g ., ELISA, Western blot, or RIA assay) to measure the level of the polypeptide.
  • a compound that promotes an increase in the expression or biological activity of the polypeptide is considered particularly useful.
  • such a molecule may be used, for example, as a therapeutic to delay, ameliorate, or treat a neoplasia in a human patient.
  • candidate compounds may be screened for those that specifically bind to a polypeptide encoded by a GPR56 and/or Tgm2 gene.
  • the efficacy of such a candidate compound is dependent upon its ability to interact with such a polypeptide or a functional equivalent thereof. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., supra).
  • a candidate compound may be tested in vitro for its ability to specifically bind a polypeptide of the invention.
  • a candidate compound is tested for its ability to increase the biological activity of a polypeptide described herein, such as a GPR56 and/or TG2 polypeptide.
  • the biological activity of a GPR56 and/or TG2 polypeptide may be assayed using any standard method, for example, a myelination assay.
  • a nucleic acid described herein e.g ., a GPR56 and/or Tgm2 nucleic acid
  • a detectable reporter is expressed in an isolated cell (e.g., mammalian) under the control of a heterologous promoter, such as an inducible promoter.
  • the cell expressing the fusion protein is then contacted with a candidate compound, and the expression of the detectable reporter in that cell is compared to the expression of the detectable reporter in an untreated control cell.
  • Products for detecting GPCR activity are commercially available including, for example, the TangoTM GPCR Assay System (Thermo Fisher Scientific, Carlsbad, Calif.).
  • a candidate compound that alters the expression of the detectable reporter is a compound that is useful for the treatment of a demyelinating disease or disorder.
  • the compound increases the expression of the reporter.
  • a candidate compound that binds to a polypeptide encoded by a GPR56 and/or Tgm2 gene may be identified using a chromatography-based technique.
  • a recombinant polypeptide of the invention may be purified by standard techniques from cells engineered to express the polypeptide (e.g, those described above) and may be immobilized on a column.
  • a solution of candidate compounds is then passed through the column, and a compound specific for the GPR56 and/or TG2 polypeptide is identified on the basis of its ability to bind to the polypeptide and be immobilized on the column.
  • To isolate the compound the column is washed to remove non-specifically bound molecules, and the compound of interest is then released from the column and collected.
  • Similar methods may be used to isolate a compound bound to a polypeptide microarray.
  • Compounds isolated by this method may, if desired, be further purified (e.g, by high performance liquid chromatography).
  • these candidate compounds may be tested for their ability to increase the activity of a GPR56 and/or TG2 polypeptide (e.g, as described herein).
  • Compounds isolated by this approach may also be used, for example, as therapeutics to treat a demyelinating disease or disorder in a human patient.
  • Compounds that are identified as binding to a polypeptide of the invention with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention.
  • any in vivo protein interaction detection system for example, any two-hybrid assay may be utilized.
  • Animal models may also be to screen candidate compounds.
  • methods of generating genetically modified animals having mutations (e.g., in GPR56) in organisms are known in the art and available to the ordinarily skilled person.
  • a CRISPR-Cas9 system is used to create a genetically modified organism (see e.g., US Patent Nos. 8,771,945 and 8,945,839, and US Patent Publication Nos. 20140170753, 20140227787, 20150184139, 20150203872, which are herein incorporated by reference in their entirety).
  • Such organisms may include any eukaryotic organism, including, without limitation, zebrafish and mice.
  • Candidate compounds may be tested for their ability to increase or promote myelination. Tissues of test organisms can be assayed in a number of ways that are routine and well known, including, without limitation, immunohistochemical staining, in situ hybridization, and electron microscopy.
  • Each of the DNA sequences listed herein may also be used in the discovery and development of a therapeutic compound for the treatment of a demyelinating disease or disorder.
  • the encoded protein upon expression, can be used as a target for the screening of drugs.
  • the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgamo or other translation facilitating sequences of the respective mRNA can be used to construct sequences that promote the expression of the coding sequence of interest. Such sequences may be isolated by standard techniques (Ausubel et ah, supra).
  • Potential antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acids, and antibodies that bind to a nucleic acid sequence or polypeptide of the invention (e.g., a GPR56 and/or TG2 polypeptide or nucleic acid molecule).
  • Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.
  • agents that modulate e.g., activate
  • GPR56 or TQ2!Tgm2 expression, biological activity, or GPR56-dependent signaling are identified from large libraries of both natural products, synthetic (or semi-synthetic) extracts or chemical libraries, according to methods known in the art.
  • these compounds increase GPR56 expression or biological activity and/or increase or promote myelination.
  • test extracts or compounds are not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modifications of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g ., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from, for example, Brandon Associates (Merrimack, N.H.), Aldrich Chemical (Milwaukee, Wis.), and Talon Cheminformatics (Acton, Ont.)
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including, but not limited to, Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.).
  • Biotics Sussex, UK
  • Xenova Slough, UK
  • Harbor Branch Oceangraphics Institute Ft. Pierce, Fla.
  • PharmaMar, U.S.A. Chembridge, Mass.
  • any library or compound may be readily modified using standard chemical, physical, or biochemical methods.
  • the efficacy of the treatment is evaluated by measuring, for example, the biological function of the treated animal (e.g., neuronal/behavioral function).
  • biological function of the treated animal e.g., neuronal/behavioral function.
  • kits for the treatment or prevention of a disease or disorder characterized by an undesirable increase in synapses includes a composition containing an effective amount of an agent that modulates (e.g., activate) GPR56 or TG2 /Tgm2 expression, biological activity, or GPR56-dependent signaling.
  • the kit includes a therapeutic or prophylactic composition for increasing or promoting myelination in a subject in need thereof.
  • the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • an agent of the invention is provided together with instructions for administering the agent to a subject having or at risk of developing a disease or disorder characterized by a deficiency or loss of myelination.
  • the instructions will generally include information about the use of the composition for the treatment or prevention of the disease or disorder.
  • the instructions include at least one of the following:
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • microglial GPR56 To study the function of microglial GPR56, an established murine model of toxin- induced demyelination was used. A cell-specifically deleted microglial Gpr56 using a Pdgfralpha-CreE- and Pdgfralpha-CreER+ driver was created.
  • mice The Gpr56fl/fl;Cx3CrlCre/+ and control (WT, Gpr56+/+;Cx3CrlCre/+) mice were crossed with PdgfraCre/ERT mice in a C57BL/6 background that were purchased from Jackson Laboratory (Bar Harbor, ME; Cat# 018280) to obtain Gpr56fi /:fl ;PdgfraCreER ⁇ and Ortd ⁇ 1 ⁇ ;Pdgfr aCreERJ mice.
  • the Gpr56 II II ;I > dpfr a( "reER ⁇ and Gpr56 fI/:fl ;PdgfrctfPreER + mice were fed cuprizone, a copper chelator, that causes rapid demyelination and gliosis, or rapid proliferation of glia subtypes.
  • the cuprizone mouse model captures several aspects of muscular sclerosis pathology.
  • One method to study de- and remyelination in the CNS involves the direct injection of the detergent lysophosphatidylcholine (lysolecithin) into the spinal cord white matter.
  • This procedure produces a well characterized demyelinating injury consisting principally of macrophage/microglial infiltration and activation, reactive astrogliosis, perturbation of axonal homeostasis/axonal injury, and oligodendrocyte precursor cells proliferation and migration.
  • the lesion predictably evolves over the period of a few weeks and is eventually capable of fully remyelinating. This method has been particularly useful in studying the events involved in de- and remyelination.
  • FIG. 3A-3D show the effects of lysolecithin injection into the corpus collosum of a mouse having a microglial specific knockout of transglutaminase 2. Interestingly, microglia- specific Tgm2 knockout mice had fewer myelinated axons in the lesion compared to control mice.
  • FIG. 5 A and 5B show that GPR56 mRNA levels are reduced following poly I:C injection.
  • Poly FC is a synthetic analog of double-stranded RNA and is a common tool to stimulate immune response in mimicking viral infection.
  • Pregnant dams were injected with poly I:C at embryomic day 12.5 and microglia were isolated from embryonic brains 24 hours later at EI3.5. qPCR showed decreased Gpr56 mRNA in mice received poly I:C, compared to control mice (FIG. 5).
  • PV+ parvalbumin positive
  • Decreased PV+ intemeurons in the cerebral cortex is one of the pathologies associated with autism.
  • Two mouse models of autism is used here:
  • VTA prenatal valproate
  • NL-3 R451C single-gene mutation identified in human patients
  • FIG. 7 shows that pediatric subjects with autism have an increased number of synapses in their brains relative to control subjects.
  • Gpr56 is deleted in microglia, fewer interneurons are observed in Bin 3 of the cerebral cortex (FIG. 8 A, 8B).
  • Microglial GPR56 regulates synaptic fomiation/pruning.
  • a statistically significant increase in synapse number is observed in microglial Gpr56 deleting mutant, which indicates a defect in synaptic pruning (FIG. 9 A and 9B).
  • the deletion of Gpr56 has behavioral consequences as shown in FIG. 10 A, 10B, and IOC.
  • Gpr56 knockout mice show behaviors that are typical of autism including obsessive behavior, which manifests in mice as obsessive marble burying (FIG. 10 A), anxiety, which manifests in the mice spending less time in the center of an open field relative to control mice (FIG. 10B), and impaired social interactions (FIG. 10C), which manifests as the mice spending the same amount of time interacting with an inanimate object as with a mouse (social target).
  • SNPs single nucleotide polymorphisms
  • FIG. 11 Increased numbers of single nucleotide polymorphisms present in GPR56 are associated with a variety of neurological defects, including multiple sclerosis, Alzheimer’s disease, Autism, and Amyotrophic Lateral Sclerosis (FIG. 12).
  • Example 5 Microglial GPR56 is necessary for the refinement of synapses
  • Adhesion G protein-coupled receptor (aGPCR) ADGRG1/GPR56 which controls several aspects of brain development in a cell type-specific manner by mediating cell-cell and cell-matrix interactions, exhibits appropriate properties as a candidate molecule to integrate microglial synapse pruning with other neurodevelopmental events.
  • GPR56 is expressed in neural progenitor cells and migrating neurons and interacts with its extracellular matrix (ECM) ligand collagen III to regulate cortical lamination .
  • ECM extracellular matrix
  • GPR56 is highly expressed in the major glia: astrocytes, oligodendrocyte lineage cells, and microglia.
  • Oligodendrocyte precursor cell (OPC) GPR56 functions together with its microglia-produced ligand tissue transglutaminase and ECM component laminin to control developmental myelination and myelin repair. Consistent with these findings, germline homozygous loss of function mutations in GPR56 cause a complex brain malformation whose phenotype includes aberrant cortical architecture and dysmyelination. This phenotype is recapitulated in genetic mouse models indicating conserved GPR56 function.
  • Gpr56 is highly expressed in microglia from embryonic to adult stages (Fig. 13B). Governed by a super-enhancer ⁇ Cell. 2014 Dec 4;159(6): 1327-40), Gpr56 is only expressed in yolk sac-derived microglia but not in microglia-like cells engrafted from fetal liver- and bone marrow-derived hematopoietic stem cells, even after long-term adaptation in the CNS in vivo. Furthermore, Gpr56 expression is promptly lost in primary cultures of microglia. Thus, Gpr56 is one of few genes that defines the microglial lineage and requires both the appropriate ontogeny and environmental cues for its expression. Motivated by the concept that cell-type-specific functions of GPR56 might coordinate multiple sequential and overlapping neurodevelopmental processes, the hypothesis that microglial GPR56 mediates synapse refinement during postnatal life was tested.
  • Microglia-specific Gpr56 conditional knockout mice were generated by crossing mice harboring a conditional Gpr56 ⁇ allele with Cx3crl-Cre transgenic mice. Though Cx3crl is a promoter for microglia, macrophages, and monocytes, Gpr56 mRNA is not present in macrophages or monocytes, and so this approach effectively deletes Gpr56 in microglia only. Gpr56 /! /! ; CX3CRl-Cre +/ ⁇ were used as conditional knockouts (CKO) and Gpr56 +/+ ;
  • microglial GPR56 mediates synapse refinement during postnatal life was tested.
  • Microglia-specific Gpr56 conditional knockout mice were generated by crossing mice harboring a conditional GprSfF ⁇ allele with Cx3crl-Cre transgenic mice.
  • Cx3crl is a promoter for microglia, macrophages, and monocytes
  • Gpr56 mRNA is not present in macrophages or monocytes, and so this approach effectively deletes Gpr56 in microglia only.
  • GprSf/ 111 CX3CRl-Cre +/ ⁇ WERE used as conditional knockouts (CKO) and Gpr56 +/+ ; CX3CRl-Cre +/ ⁇ as controls.
  • Cell-type specific deletion was confirmed by western blot (Fig. 14M) and qPCR (FIG. 14M) using microglia isolated from CKOs and their controls.
  • RNAscope in situ hybridization further showed that Gpr56 mRNA was only absent from Ibal + microglia in CKO (Fig. 15 A).
  • mice were generated that enabled inducible deletion of microglial Gpr56 by crossing (lpr56 /l /l mice with Cx3crl-CreER mouse line.
  • Ipr56 /l /l CX3CRl-CreER +/ ⁇ mice were used as inducible conditional knockouts (iCKO), and Gpr56 +/+ ; CX3CRl-CreER +/ ⁇ mice were used as controls.
  • Tamoxifen was administered to both iCKO and controls at P1-P3 and brains were analyzed at P10. Comparable increases in retinogeniculate synaptic density in both male and female iCKO mice were observed, in comparison to their age-matched controls, indicating that there is no sexual dimorphism in microglial GPR56 function (Fig. 141).
  • iCKO and CKO mice showed a quantitatively equivalent synapse phenotype at P10 (Fig. 14F and 141), indicating synaptic phenotype is a postnatal event. Additionally, this result demonstrated that it was appropriate to use CKO for most of the remaining studies.
  • the dLGN also receives vGlutl projections from neocortical layer VI, modulatory inputs which flexibly tune postsynaptic activity in target cells.
  • vGlut2 synapses there were no significant changes in the density of vGlutl + synapses at P10 (Fig. 14J-L), indicating that microglial GPR56 functions in a synapse-specific manner and does not modulate neocortical inputs in dLGN during the developmental stages investigated.
  • vGlut2+/Homerl+ Increased synapse densities (vGlut2+/Homerl+) were found at both P10 and P21 in the hippocampal striatum lacunosum-moleculare layer (Fig. 15A-D). However, no change in vGlutl+/Homerl+synapse density was observed at either P10 or P21 in hippocampus striatum radiatum layer (Fig. 15E-H). Taken together, the data demonstrate that microglial GPR56 plays an important role during synapse development in a circuit- dependent manner.
  • mice received intraocular injection of anterograde tracers at P4 and were sacrificed 24 hours later for analysis, as peak pruning occurs around P5 in the murine retinogeniculate system (Fig. 16A). Compared to controls, the amount of RGC material found inside CKO microglia was decreased by 25.7% (Fig. 16B and 16C). This change corresponded in magnitude to the increase in synapses at P5 (Fig. 14D,
  • PS is a phospholipid that largely resides on the inner leaflet of the plasma membrane under normal conditions.
  • PS extemalization serves as an“eat me” signal for clearance of apoptotic and stressed cells (PNAS October 22, 2013 110 (43) E4098-E4107) as well as outer segment membranes of retinal photoreceptors.
  • PS might flag synapses for removal, based on the observation that PS was externalized on isolated synaptosomes.
  • BAI1/ADGRB1 another aGPCR family member, recognizes PS, the hypothesis that microglial GPR56 recognizes synapses tagged for removal by binding to PS was tested.
  • GPR56 contains an extensive N- terminal fragment (NTF) followed by a classical seven-transmembrane region (Fig. 17A) Within the long NTF, there are two functional domains, termed
  • PLL pentraxin/laminin/neurexin/sex -hormone-binding-globulin-like (PLL) and GPCR
  • GAIN autoproteolysis inducing domains recombinant proteins of human immunoglobulin Fc (hFc)-tagged full-length NTF (NTF-hFc) and GAIN-hFc were engineered (Fig. 17B).
  • hFc human immunoglobulin Fc
  • NTF-hFc full-length NTF
  • Fig. 17B GAIN-hFc
  • FITC -conjugated Annexin V a known PS- binding protein (53), served as a positive control.
  • GAIN domain not the full-length NTF, bound PS in these assays.
  • hFc a competition assay, in which labeled full-length NTF, GAIN domain, or hFc were used to displace Annexin V binding, it was confirmed that the GAIN domain, but not full length NTF, competed with Annexin V for binding to PS (Fig. 17E).
  • the PLL and GAIN domains are constrained by an interdomain disulfide bond at two cysteine residues C121 and C177. It is conceivable that PLL domain blocks GAIN domain binding to PS.
  • GPR56 S4 is an alternatively spliced GPR56 isoform, that initiates at an alternative ATG start codon in exon 4, resulting in a GPR56 variant that contains only the GAIN domain in its extracellular region (FIG. 17J-A). Based on the live-cell PS binding data, GPR56 S4 may be required for microglia-mediated synaptic pruning. Supporting this hypothesis, it was found that Gpr56 S4 is the predominant microglial transcript as determined by qPCR analysis of microglia isolated from P25 WT mouse brains (FIG. 17J-B-17E).
  • the S4 isoform may not be required for cerebral cortical lamination. Consistent with this hypothesis, a comparable cortical phenotype was observed in Gpr56 S4 and Gpr56 null mice (FIG. 17K). To test whether the GPR56 S4 isoform plays a role in synaptic refinement, retinogeniculate synapses in dLGN of Gpr56 S4 and Gpr56 null mice was examined.
  • PSVue + signals may represent PS on pre-synaptic inputs from other brain regions or post-synaptic elements of dLGN neurons. Consistent with our hypothesis that microglial GPR56 regulates synaptic pruning by binding to PS, significantly more surviving PS + RGC inputs in CKO than controls (Fig. 18H and I).
  • microglia preferentially engulf PS + synapses, although they do also engulf PS synapses.
  • Fig. 5J-L we observed significantly reduced PS + RGC inputs inside CKO microglia, compared to controls.
  • engulfed PS RGC inputs were not different in CKO as compared to control microglia (Fig. 5M).
  • CKO microglia contained a low level PS + synapses, suggesting that pathways other than GPR56 can mediate in this process. Together, these results indicate that PS-tagged synapses are preferentially eliminated by microglia partly dependent on microglial GPR56.
  • Microglial Gpr56 expression is governed by a super enhancer suggesting that it might be implicated in establishing cell identity and core functions ⁇ Cell. 153 (2): 307-19 2013; Cell. 2014 Dec 4;159(6): 1327-40). This hypothesis was supported by the finding that Gpr56 expression distinguishes the transcriptomes of microglia as contrasted with hematopoietic stem cells after both cells types have engrafted the intact brain ⁇ Neuron. 2018 Jun
  • Microglial Gpr56 conditional knockout mice were generated by crossing our Gpr56 floxed mice (Giera et ah, 2015) with a Cre driver which is specific for microglia among CNS cells, Cx3crl-Cre where Cre recombinase is present throughout microglial development.
  • a Cre driver which is specific for microglia among CNS cells, Cx3crl-Cre where Cre recombinase is present throughout microglial development.
  • poly (I:C) 20mg/kg or carrier solution PBS was injected into pregnant wild type dams at E12.5 and brains were collected at developmental time points, including E14.5, E16.5 and E18.5 and P8.
  • the cerebral cortex was stratified into layers II- IV, V, and VI.
  • the laminar localization PV-positive interneurons was quantified in correspondence to their laminar position, i.e., layer II-IV, V, and VI, respectively.
  • Five animals per genotype or per treatment [Poly (I:C) vs PBS injection] were used. Images were acquired by Zeiss LSM 700 laser scanning confocal microscope and quantified blind to genotype.
  • Immunohistochemistry and microscopic imaging Brains were dissected and fixed with 4% formaldehyde in PBS overnight, then placed in sucrose solution before freezing. Brains were sectioned at 12 um on slide with a cryostat. Immunohistochemistry was performed followed by confocal microscopic analyses using a Zeiss LSM 700 laser scanning confocal microscope. All analysis was performed blind using ImageJ software.
  • mice were acclimated to the environment and then placed in a testing arena containing 20 glass marbles, which were laid out in four rows of five marbles equidistant from one another. A marble burying index was scored. Test results were analyzed blind.
  • mice All mice were handled according to protocols approved by Boston Children’s Hospital Animal Care and Lise Committee guidelines for the ethical treatment of animals.
  • mice were generated as previously described(2).
  • Cx3Crl Cre and Cx3Crl CreER are knock-in mice, replacing the coding exon of the chemokine receptor 1 (Cx3crl) gene, we crossed these mice with Gpr5f/ 111 to generate Gpr56 1111 Cx3Crl -cre(ER) as conditional knockout mice, and Gpr56 +/+ / Cx3Crl -cre(ER) +/ ⁇ as control.
  • Gpr56 /, /l mice were crossed with CMV-cre mice (JAX stock #006054) (60) to delete exons 4-6, causing a deletion of all splicing variants of Gpr56 in all tissues.
  • Mouse brains were collected following PBS perfusion and fixation with 4% PFA, and cryoprotected in 30% sucrose. OCT-embedded tissues were cryosectioned at 14 pm or 40 pm. For synapse immunostaining, l4-pm or 40-pm Sections were incubated with blocking buffer (10% Goat serum + l%BSA; 0.3% TritonX/PBS) for 2 hours and stained with primary antibodies overnight at 4°C (guinea pig anti-vGlut2, 1 : 1000, Millipore AB2251-I; guinea pig anti-vGlutl, 1 : 1000, Millipore AB5905; rabbit anti -Homer 1, 1 :250, Synaptic Systems, 160 003).
  • blocking buffer 10% Goat serum + l%BSA; 0.3% TritonX/PBS
  • Confocal microscopy images were obtained with Zeiss LSM 700 System.
  • medial dLGN slices were used.
  • three fields of view (5 serial optical sections, 0.5 pm Z-step, 101.5 pm * 101.5 pm, 1024 * 1024 pixel) were acquired in the upper part of the core region using a 63X/1.40 oil objective.
  • Colocalization of vGlut2 or vGlutl and Homerl was quantified as described( ⁇ 52). The whole process was run in the ImageJ software (NIH, Bethesda, MD). First, each channel’s background was subtracted with a rolling ball radius of 10 pixels.
  • RNAscope was performed using RNAscope® Multiplex Fluorescent Reagent Kit v2 for fixed and frozen 12um thick sections according to the manufacturer's
  • RNAscope ® Probe- Mrn -Gpr56 (Cat No. 318241) was used to detect expression of the C-terminal target region of Gpr56 followed by immunohistochemistry for Iba! (1 :400, Wako, 019-19741)
  • sections were permealized using 0.3% Triton-X 100 in PBS for 10 mins, followed by blocking with 10% goat serum, 1% BSA and 0.1% Triton-X 100 in PBS for 1 hr at RT and incubating the primary antibody in the blocking buffer overnight at 4C. Appropriate secondary antibody was used to visualize Ibal expression.
  • Threshold-independent analyisis of eye-specific segregation was performed as described before(-/2). Mice were anesthetized with isoflurane during the whole procedure. 3ul 0.2% cholera toxin-/? subunit (CTB) conjugated Alexa 488 dye (CTB488, Life Technologies, C22841) was intravitreally injected into the left eye, and CTB conjugated Alexa 647 dye (CTB647, Life Technologies, C34778) into the right eye of P30 mice. 24 hours later, mice were transcardially perfused with PBS and fixed with 4% PFA, and cryoprotected in 30% sucrose. The injection/labeling efficiency was checked by visualizing retinas and superior colliculus (Fig. S6C).
  • the variance of R-values was computed to quantify the extent of segregation. The greater variance means more segregation between ipsilateral and contralateral RGC inputs. The full code can be found here: https://github.com/TaoLi322/Microglia_GPR56_Synapse/blob/master/Eye- Seg_R- value.
  • mice For P10 mice, lul of 0.2% CTB488 and CTB594 (Life Technologies, C22842) were injected into the left or right eye, respectively. The percentage of overlapped left and right eye projections in dLGN was quantified using a multi -threshold quantitative method, as described before(-/2).
  • mice (P28-P34) were decapitated; their brain was removed and placed in a 4°C choline solution, containing: 78.3 mM NaCl, 23 mM NaHC0 3 , 23 mM glucose, 33.8 mM choline chloride, 2.3 mM KC1, 1.1 mM NaH 2 P0 3 , 6.4 mM MgCh, and 0.45 mM CaCh.
  • the two hemispheres were then separated by an angled cut (3°-5°) relative to the cerebral longitudinal fissure.
  • AMPA and NMDA currents were then determined by gradually increasing the stimulus intensity from 0.1 up to 1 mA.
  • the maximum response size was considered to be the amplitude of the response after 3 consecutive increases in stimulation intensity failed to result in a larger response, or if the response amplitude decreased with an increase in stimulation intensity.
  • AMPA responses were collected at -70 mV and NMDA responses at +40 mV, and were collected with alternate stimulations.
  • mice were anesthetized and decapitated. Mouse brains were immediately removed into ice-cold HBSS and coronally sectioned at 100 p using vibratome. DLGN were dissected out under dissection microscope, and homogenized in proteinase inhibitor-added RIPA buffer. After 30min incubation on ice, samples were centrifuged at l4,000xg for lOmin. Supernatant were collected and used for WB of NMDAR1 (anti-NRl, 1 :2000, Sigma-Aldrich 05-432) and vGlut2 (anti-vGlut2, 1 :5000, Millipore AB2251-I). For WB of GluRl (anti-GluRl, 1 : 1000, Cell signaling 13185S), supernatant were boiled for 5min before loading.
  • NMDAR1 anti-NRl, 1 :2000, Sigma-Aldrich 05-432
  • vGlut2 anti-vGlut2, 1 :5000, Mill
  • PCR forward primer is: 5’-CCATGGAAGACTTCCGCTTCTGTGGCC-3’
  • reverse primer is: 5’ -CAGATCTC AGGTGGTTGCAC AGGC AGG-3’
  • the PCR forward primer is: 5’-ATCCATGGTTATGTG TGATCTCAAGAAGGAATTGC-3’
  • the reverse primer is: 5’ -TCTAGATCTC AGGTGGTTGCAC AGGC AGG-3’.
  • PCR product was inserted into pFUSE-hIgG2-Fc2 (Invivogen, Cat.
  • pfuse-hfc2 vector between Ncol and BglII sites.
  • the above constructs were transiently transfected into HEK-293T cells (ATCC). 24 hours later, the culture media was changed to serum-reduced Opti-MEM (Gibco, 31985070). During incubation, GPR56 fusion proteins would be secreted into the culture media. This conditioned media containing fusion proteins was harvested 48-72 hours later, and concentrated as previously described (ShiHong Li et al, 2008 Journal of neuroscience). The proteins were purified using HiTrap protein A column (GE Healthcare, 17040303).
  • GPR56 NTF-hFc, GAIN domain-hFc, or hFc were labeled with Alexa Fluor 647 (AF647) using the Alexa Fluor 647 NHS Ester labeling kit (ThermoFisher Scientific, Cat #A20006) according to the manufacturer’s protocol. Briefly, 75 mM protein was incubated with 300 mM AF647 at room temperature for 4 hours, followed by 200 mM Tris to quench the reaction. The whole reaction mixture was run through a Sephadex G-50 column and protein-dye conjugate was collected. Dye labeling efficiency for each protein ranged from 1.44-1.46 moles of dye per mole of protein.
  • Ba/F3 cell line was cultured and passaged as described (66). It was maintained in RPMI-1640 media with L-Glutamine (Invitrogen, Cat #11875093) supplemented with 10% FBS, Penicillin-Streptomycin, and kept at 37°C, 5% CQ2 in a humidified incubator. Ceils were harvested and tvashed twice with ice-cold Hank’s Balanced Salt Solution (HESS) and resuspended in HBSS supplemented with 2 mM Ca 2" . Cells were treated with 1 mM calcium ionophore A23187 (Mil!ipore Sigma, Cat #C7522) at 37°C for 15 minutes, with gentle agitation every 5 minutes, followed by washing with HBSS.
  • HESS ice-cold Hank’s Balanced Salt Solution
  • mice were given an intraocular injection of CTB647 into both eyes. Twenty four hours later, mice were mounted on a neonatal mice adaptor (RWD Life Science, #68072), and given an intracranial injection of various labeling probes.
  • the stereotaxic setting was -1.5 mm anteroposterior, 1.09 mm ediolaterai and -2.15 mm dorsoventral to lambda.

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Abstract

As described below, the present invention features compositions and methods for the treatment of ASD and other neurological diseases and disorders associated with defects in GPR56 or with undesirable increases in synapse number.

Description

COMPOSITIONS AND METHODS FOR TREATING DISORDERS
CHARACTERIZED BY A DEFECT IN GPR56 EXPRESSION OR ACTIVITY
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of the following U.S. Provisional Application No.: 62/700,595, which was filed July 19, 2018, the entire contents of which are incorporated herein by reference.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH
This invention was made with government support under Grant No. ROl NS094164 and ROl NS108446 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
Autism spectrum disorders (ASD) are class of neurological and developmental disorders that begin early in childhood and typically last throughout a person's life. ASDs include a range of conditions characterized by challenges with social skills, repetitive behaviors, speech and nonverbal communication. Autism’s most-obvious signs tend to appear between 2 and 3 years of age. In some cases, it can be diagnosed as early as 18 months and may last throughout an individuals lifetime. ASD affects 1 in 37 boys and 1 in 151 girls. Approximately one third of people with Autism remain non-verbal and
approximately the same number have an intellectual disability. Developmental synaptic remodeling is important for precise neural circuitry and a failure in this process is implicated in ASDs and other neurodevelopmental disorders. Microglia prune synapses, but integration of this synapse pruning with overlapping and concurrent neurodevelopmental processes remains elusive. Currently, there is no effective therapy for ASD or for other disorders where synapse organization is disrupted.
SUMMARY OF THE INVENTION
As described below, the present invention features compositions and methods for the treatment of ASD and other neurological diseases and disorders associated with defects in GPR56 or with undesirable increases in synapse number. In one aspect, the invention provides a method of promoting synaptic pruning in a neuronal tissue, the method comprising contacting the tissue with an agent that activates GPR56, thereby promoting synaptic pruning.
In another aspect, the invention provides a method for treating a disease or disorder characterized by a loss or reduction in GPR56 expression or activity or an undesirable increase in synapse number, the method comprising administering to a subject in need thereof an agent that activates GPR56, thereby treating the disease or disorder.
In another aspect, the invention provides a method for treating a disease or disorder characterized by a loss or reduction in GPR56 expression or activity or an undesirable increase in synapse number, the method comprising administering to a subject in need thereof an S4 isoform of GPR56, thereby treating the disease or disorder. In one embodiment, the disease or disorder is an Autism spectrum disorder, multiple sclerosis, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis.
In various embodiments of the above aspects or any other aspect of the invention delineated herein, the agent is a small compound (e.g., gedunin- and khivorin compound), polypeptide, or polynucleotide. In particular embodiments, the compound is any one or more of 3-alpha-acetoxydihydrodeoxygedunin, khivorin, 7 synthetic peptide agonist, 3- deacetylkhivorin, deoxygedunin, and l,2-Epoxygedunin. In another embodiment, the polypeptide is transglutaminase 2 (TG2) polypeptide or fragment thereof or GPR56 ligand comprising amino acids 383-404 of GPR56. In particular embodiments, the GPR56 ligand comprises or consists of the amino acid sequence TYFAVLMVS or the amino acid sequence TYFAVLMVSSVEVDAVHKHYLS. In another embodiment, the agent is a GPR56 ligand that is covalently linked to a lipid or transmembrane domain. In another embodiment, the N- terminus or C-terminus of the GPR56 ligand is covalently linked to the lipid or
transmembrane domain. In another embodiment, the polypeptide is a TG2 polypeptide or fragment thereof comprises amino acids 465-687 of TG2. In another embodiment, the polypeptide is a TG2 polypeptide or fragment thereof that forms one or more beta barrel domains. In another embodiment, the disease or disorder is an Autism spectrum disorder, multiple sclerosis, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims. Definitions
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et ah, 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). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By“Transglutaminase 2 (TG2) polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_004604 and having GPR56 binding activity. An exemplary TG2 polypeptide sequence is provided below.
1 maeelvlerc dleletngrd hhtadlcrek lvvrrgqpfw ltlhfegrny easvdsltfs
61 vvtgpapsqe agtkarfplr daveegdwta tvvdqqdctl slqlttpana piglyrlsle
121 astgyqgssf vlghfillfn awcpadavyl dseeerqeyv ltqqgfiyqg sakfiknipw
181 nfgqfedgil diclilldvn pkflknagrd csrrsspvyv grvvsgmvnc nddqgvllgr
241 wdnnygdgvs pmswigsvdi lrrwknhgcq rvkygqcwvf aavactvlrc lgiptrvvtn
301 ynsahdqnsn llieyfrnef geiqgdksem iwnfhcwves wmtrpdlqpg yegwqaldpt
361 pqeksegtyc egpvpvraik egdlstkyda pfvfaevnad vvdwiqqddg svhksinrsl
421 ivglkistks vgrderedit htykypegss eereaftran hlnklaekee tgmamrirvg
481 qsmnmgsdfd vfahitnnta eeyvcrlllc artvsyngil gpecgtkyll nlnlepfsek
541 svplcilyek yrdcltesnl ikvrallvep vinsyllaer dlylenpeik irilgepkqk
601 rklvaevslq nplpvalegc tftvegaglt eeqktveipd pveageevkv rmdllplhmg
661 lhklvvnfes dklkavkgfr nviigpa
By“Transglutaminase 2 ( Tgm2 ) nucleic acid molecule” is meant a polynucleotide encoding a TGM2 polypeptide or fragment thereof. An exemplary Tgm2 nucleic acid molecule sequence is provided at NCBI Accession No. NM_0046l3 and is shown below.
1 ataagttagc gccgctctcc gcctcggcag tgccagccgc cagtggtcgc acttggaggg
61 tctcgccgcc agtggaagga gccaccgccc ccgcccgacc atggccgagg agctggtctt
121 agagaggtgt gatctggagc tggagaccaa tggccgagac caccacacgg ccgacctgtg
181 ccgggagaag ctggtggtgc gacggggcca gcccttctgg ctgaccctgc actttgaggg
241 ccgcaactac gaggccagtg tagacagtct caccttcagt gtcgtgaccg gcccagcccc
301 tagccaggag gccgggacca aggcccgttt tccactaaga gatgctgtgg aggagggtga 361 ctggacagcc accgtggtgg accagcaaga ctgcaccctc tcgctgcagc tcaccacccc
421 ggccaacgcc cccatcggcc tgtatcgcct cagcctggag gcctccactg gctaccaggg
481 atccagcttt gtgctgggcc acttcatttt gctcttcaac gcctggtgcc cagcggatgc
541 tgtgtacctg gactcggaag aggagcggca ggagtatgtc ctcacccagc agggctttat
601 ctaccagggc tcggccaagt tcatcaagaa cataccttgg aattttgggc agtttgaaga
661 tgggatccta gacatctgcc tgatccttct agatgtcaac cccaagttcc tgaagaacgc
721 cggccgtgac tgctcccgcc gcagcagccc cgtctacgtg ggccgggtgg tgagtggcat
781 ggtcaactgc aacgatgacc agggtgtgct gctgggacgc tgggacaaca actacgggga
841 cggcgtcagc cccatgtcct ggatcggcag cgtggacatc ctgcggcgct ggaagaacca
901 cggctgccag cgcgtcaagt atggccagtg ctgggtcttc gccgccgtgg cctgcacagt
961 gctgaggtgc ctgggcatcc ctacccgcgt cgtgaccaac tacaactcgg cccatgacca
1021 gaacagcaac cttctcatcg agtacttccg caatgagttt ggggagatcc agggtgacaa
1081 gagcgagatg atctggaact tccactgctg ggtggagtcg tggatgacca ggccggacct
1141 gcagccgggg tacgagggct ggcaggccct ggacccaacg ccccaggaga agagcgaagg
1201 gacgtactgc tgtggcccag ttccagttcg tgccatcaag gagggcgacc tgagcaccaa
1261 gtacgatgcg ccctttgtct ttgcggaggt caatgccgac gtggtagact ggatccagca
1321 ggacgatggg tctgtgcaca aatccatcaa ccgttccctg atcgttgggc tgaagatcag
1381 cactaagagc gtgggccgag acgagcggga ggatatcacc cacacctaca aatacccaga
1441 ggggtcctca gaggagaggg aggccttcac aagggcgaac cacctgaaca aactggccga
1501 gaaggaggag acagggatgg ccatgcggat ccgtgtgggc cagagcatga acatgggcag
1561 tgactttgac gtctttgccc acatcaccaa caacaccgct gaggagtacg tctgccgcct
1621 cctgctctgt gcccgcaccg tcagctacaa tgggatcttg gggcccgagt gtggcaccaa
1681 gtacctgctc aacctcaacc tggagccttt ctctgagaag agcgttcctc tttgcatcct
1741 ctatgagaaa taccgtgact gccttacgga gtccaacctc atcaaggtgc gggccctcct
1801 cgtggagcca gttatcaaca gctacctgct ggctgagagg gacctctacc tggagaatcc
1861 agaaatcaag atccggatcc ttggggagcc caagcagaaa cgcaagctgg tggctgaggt
1921 gtccctgcag aacccgctcc ctgtggccct ggaaggctgc accttcactg tggagggggc
1981 cggcctgact gaggagcaga agacggtgga gatcccagac cccgtggagg caggggagga
2041 agttaaggtg agaatggacc tgctgccgct ccacatgggc ctccacaagc tggtggtgaa
2101 cttcgagagc gacaagctga aggctgtgaa gggcttccgg aatgtcatca ttggccccgc
2161 ctaagggacc cctgctccca gcctgctgag agcccccacc ttgatcccaa tccttatccc
2221 aagctagtga gcaaaatatg ccccttcttg ggccccagac cccagggcag ggtgggcagc
2281 ctatgggggc tctcggaaat ggaatgtgcc cctggcccat ctcagcctcc tgagcctgtg
2341 ggtccccact cacccccttt gctgtgagga atgctctgtg ccagaaacag tgggagccct
2401 gaccttggct gactggggct ggggtgagag aggaaagacc tacattccct ctcctgccca
2461 gatgcccttt ggaaagccat tgaccaccca ccatattgtt tgatctactt catagctcct
2521 tggagcaggc aaaaaaggga cagcatgccc cttggctgga tcagggaatc cagctcccta
2581 gactgcatcc cgtacctctt cccatgactg cacccagctc caggggccct tgggacagcc
2641 agagctgggt ggggacagtg ataggcccaa ggtcccctcc acatcccagc agcccaagct
2701 taatagccct ccccctcaac ctcaccattg tgaagcacct actatgtgct gggtgcctcc 2761 cacacttgct ggggctcacg gggcctccaa cccatttaat caccatggga aactgttgtg
2821 ggcgctgctt ccaggataag gagactgagg cttagagaga ggaggcagcc ccctccacac
2881 cagtggcctc gtggttatta gcaaggctgg gtaatgtgaa ggcccaagag cagagtctgg
2941 gcctctgact ctgagtccac tgctccattt ataaccccag cctgacctga gactgtcgga
3001 gaggctgtct ggggccttta tcaaaaaaag actcagccaa gacaaggagg tagagagggg
3061 actgggggac tgggagtcag agccctggct gggttcaggt cccacgtctg gccaggcact
3121 gccttctcct ctctgggcct ttgtttcctt gttggtcaga ggagtgattg aaccagctca
3181 tctccaagga tcctctccac tccatgtttg caatgctttt atatggccca gccttgtaaa
3241 taaccacaag gtccactccc tgctccacga agccttaagc cataggccca ggatatttct
3301 gagagtgaaa ccatgactgt gaccaccttc tgtccccagc cctgtcctgg ttccttccta
3361 tgcccaggta ccacccttca gaccccagtt ctaggggaga agagccctgg acacccctgc
3421 tctacccatg agcctgcccg ctgcaatgcc tagacttccc aacagcctta gctgccagtg
3481 ctggtcacta accaacaagg ttggcacccc agctacccct tctttgcagg gctaaggccc
3541 ccaaacatag cccctgcccc ggaggaagct tggggaaccc atgagttgtc agctttgact
3601 ttatctcctg ctctttctac atgactgggc ctcccttggg ctggaagaat tggggattct
3661 ctattggagg tgagatcaca gcctccaggg ccccccaaat cccagggaag gacttggaga
3721 gaatcatgct gttgcattta gaactttctg ctttgcacag gaaagagtca cacaattaat
3781 caacatgtat attttctcta tacatagagc tctatttctc tacggtttta taaaagcctt
3841 gggttccaac caggcagtag atgtgcttct gaaccgcaag gagcaaacac tgaaataaaa
3901 tagtttattt ttcacactca aaaaaaaaaa aaaaaaa
By“G protein-coupled receptor 56 (GPR56) polypeptide” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No.
NP 004604 and having RhoA and/or mammalian target of rapamycin (mTOR) pathway signaling activity. An exemplary GPR56 polypeptide sequence is provided below.
1 mtpqsllqtt lfllsllflv qgahgrghre dfrfcsqrnq thrsslhykp tpdlrisien
61 seealtvhap fpaahpasrs fpdprglyhf clywnrhagr lhllygkrdf llsdkassll
121 cfqhqeesla qgppllatsv tswwspqnis lpsaas ftfs fhspphtaah nasvdmcelk
181 rdlqllsqf1 khpqkasrrp saapasqqlq sleskltsvr fmgdmvs fee drinatvwkl
241 qptaglqdlh ihsrqeeeqs eimeysvllp rtlfqrtkgr sgeaekrlll vdfssqalfq
301 dknssqvlge kvlgivvqnt kvanltepvv ltfqhqlqpk nvtlqcvfwv edptlsspgh
361 wssagcetvr retqtscfcn hltyfavlmv ssvevdavhk hylsllsyvg cvvsalaclv
421 tiaaylcsrr kprdytikvh mnlllavf11 dts fllsepv altgseagcr asaiflhfsi
481 ltclswmgle gynlyrlvve vfgtyvpgyl lklsamgwgf piflvtlval vdvdnygpii
541 lavhrtpegv iypsmcwird slvsyitnlg Ifslvflfnm amlatmvvqi lrlrphtqkw
601 shvltllgls lvlglpwali ffs fasgtfq lvvlylfsii tsfqgflifi wywsmrlqar
661 ggpsplksns dsarlpissg stsssri In particular embodiments, a subject having a defect in GPR56 expression or activity has or is at risk for developing a disorder characterized by an undesirable increase in synapse number in a tissue of an organism.
In other embodiments, a GPR56 is an S4 isofom :
>sp | Q9Y653-5 | AGRG1 HUMAN Isoform 5 of Adhesion G-protein coupled receptor G1 OS=Homo sapiens_OX=9606 GN=ADGRG1
MCELKRDLQLLSQFLKHPQKASRRPSAAPASQQLQSLESKLTSVRFMGDMVSFEEDRINATVWKLQPTAGLQDLH
IHSRQEEEQSEIMEYSVLLPRTLFQRTKGRSGEAEKRLLLVDFSSQALFQDKNSSQVLGEKVLGIWQNTKVANL
TEPWLTFQHQLQPKNVTLQCVFWVEDPTLSSPGHWSSAGCETVRRETQTSCFCNHLTYFAVLMVSSVEVDAVHK
HYLSLLSYVGCWSALACLVTIAAYLCSRVPLPCRRKPRDYTIKVHMNLLLAVFLLDTSFLLSEPVALTGSEAGC
RASAIFLHFSLLTCLSWMGLEGYNLYRLWEVFGTYVPGYLLKLSAMGWGFPIFLVTLVALVDVDNYGPIILAVH
RTPEGVIYPSMCWIRDSLVSYITNLGLFSLVFLFNMAMLATMWQILRLRPHTQKWSHVLTLLGLSLVLGLPWAL
IFFSFASGTFQLWLYLFSIITSFQGFLIFIWYWSMRLQARGGPSPLKSNSDSARLPISSGSTSSSRI
By“G protein-coupled receptor 56 (GPR56) nucleic acid molecule” is meant a polynucleotide encoding a GPR56 polypeptide or fragment thereof. An exemplary GPR56 nucleic acid molecule sequence is provided at NCBI Accession No. NM_00l 145770 and is shown below.
1 agacaggcgg agcctcacct ggggctgccc gccagcccag acaagctcag actgggtgcc
61 tgtggccctg ggaggaggtg gaaggggagg agcaggccac acaggcacag gccggtgagg
121 gacctgccca gacctggagg gtctcgctct gtcacacagg ctggagtgca gtggtgtgat
181 cttggctcat cgtaacctcc acctcccggg ttcaagtgat tctcatgcct cagcctcccg
241 agtagctggg attacaggtg gtgacttcca agagtgactc cgtcggagga aaatgactcc
301 ccagtcgctg ctgcagacga cactgttcct gctgagtctg ctcttcctgg tccaaggtgc
361 ccacggcagg ggccacaggg aagactttcg cttctgcagc cagcggaacc agacacacag
421 gagcagcctc cactacaaac ccacaccaga cctgcgcatc tccatcgaga actccgaaga
481 ggccctcaca gtccatgccc ctttccctgc agcccaccct gcttcccgat ccttccctga
541 ccccaggggc ctctaccact tctgcctcta ctggaaccga catgctggga gattacatct
601 tctctatggc aagcgtgact tcttgctgag tgacaaagcc tctagcctcc tctgcttcca
661 gcaccaggag gagagcctgg ctcagggccc cccgctgtta gccacttctg tcacctcctg
721 gtggagccct cagaacatca gcctgcccag tgccgccagc ttcaccttct ccttccacag
781 tcctccccac acggccgctc acaatgcctc ggtggacatg tgcgagctca aaagggacct
841 ccagctgctc agccagttcc tgaagcatcc ccagaaggcc tcaaggaggc cctcggctgc
901 ccccgccagc cagcagttgc agagcctgga gtcgaaactg acctctgtga gattcatggg
961 ggacatggtg tccttcgagg aggaccggat caacgccacg gtgtggaagc tccagcccac
1021 agccggcctc caggacctgc acatccactc ccggcaggag gaggagcaga gcgagatcat 1081 ggagtactcg gtgctgctgc ctcgaacact cttccagagg acgaaaggcc ggagcgggga
1141 ggctgagaag agactcctcc tggtggactt cagcagccaa gccctgttcc aggacaagaa
1201 ttccagccaa gtcctgggtg agaaggtctt ggggattgtg gtacagaaca ccaaagtagc
1261 caacctcacg gagcccgtgg tgctcacttt ccagcaccag ctacagccga agaatgtgac
1321 tctgcaatgt gtgttctggg ttgaagaccc cacattgagc agcccggggc attggagcag
1381 tgctgggtgt gagaccgtca ggagagaaac ccaaacatcc tgcttctgca accacttgac
1441 ctactttgca gtgctgatgg tctcctcggt ggaggtggac gccgtgcaca agcactacct
1501 gagcctcctc tcctacgtgg gctgtgtcgt ctctgccctg gcctgccttg tcaccattgc
1561 cgcctacctc tgctccagga ggaaacctcg ggactacacc atcaaggtgc acatgaacct
1621 gctgctggcc gtcttcctgc tggacacgag cttcctgctc agcgagccgg tggccctgac
1681 aggctctgag gctggctgcc gagccagtgc catcttcctg cacttctccc tgctcacctg
1741 cctttcctgg atgggcctcg aggggtacaa cctctaccga ctcgtggtgg aggtctttgg
1801 cacctatgtc cctggctacc tactcaagct gagcgccatg ggctggggct tccccatctt
1861 tctggtgacg ctggtggccc tggtggatgt ggacaactat ggccccatca tcttggctgt
1921 gcataggact ccagagggcg tcatctaccc ttccatgtgc tggatccggg actccctggt
1981 cagctacatc accaacctgg gcctcttcag cctggtgttt ctgttcaaca tggccatgct
2041 agccaccatg gtggtgcaga tcctgcggct gcgcccccac acccaaaagt ggtcacatgt
2101 gctgacactg ctgggcctca gcctggtcct tggcctgccc tgggccttga tcttcttctc
2161 ctttgcttct ggcaccttcc agcttgtcgt cctctacctt ttcagcatca tcacctcctt
2221 ccaaggcttc ctcatcttca tctggtactg gtccatgcgg ctgcaggccc ggggtggccc
2281 ctcccctctg aagagcaact cagacagcgc caggctcccc atcagctcgg gcagcacctc
2341 gtccagccgc atctaggcct ccagcccacc tgcccatgtg atgaagcaga gattcggcct
2401 cgtcgcacac tgcctgtggc ccccgagccc ggcccagccc caggccagtc agccgcagac
2461 tttggaaagc ccaacgacca tggagagatg ggccgttgcc atggtggacg gactcccggg
2521 ctgggctttt gaattggcct tggggactac tcggctctca ctcagctccc acgggactca
2581 gaagtgcgcc gccatgctgc ctagggtact gtccccacat ctgtcccaac ccagctggag
2641 gcctggtctc tccttacaac ccctgggccc agccctcatt gctgggggcc aggccttgga
2701 tcttgagggt ctggcacatc cttaatcctg tgcccctgcc tgggacagaa atgtggctcc
2761 agttgctctg tctctcgtgg tcaccctgag ggcactctgc atcctctgtc attttaacct
2821 caggtggcac ccagggcgaa tggggcccag ggcagacctt cagggccaga gccctggcgg
2881 aggagaggcc ctttgccagg agcacagcag cagctcgcct acctctgagc ccaggccccc
2941 tccctccctc agccccccag tcctccctcc atcttccctg gggttctcct cctctcccag
3001 ggcctccttg ctccttcgtt cacagctggg ggtccccgat tccaatgctg ttttttgggg
3061 agtggtttcc aggagctgcc tggtgtctgc tgtaaatgtt tgtctactgc acaagcctcg
3121 gcctgcccct gagccaggct cggtaccgat gcgtgggctg ggctaggtcc ctctgtccat
3181 ctgggccttt gtatgagctg cattgccctt gctcaccctg accaagcaca cgcctcagag
3241 gggccctcag cctctcctga agccctcttg tggcaagaac tgtggaccat gccagtcccg
3301 tctggtttcc atcccaccac tccaaggact gagactgacc tcctctggtg acactggcct
3361 agggcctgac actctcctaa gaggttctct ccaagccccc aaatagctcc aggcgccctc
3421 ggccgcccat catggttaat tctgtccaac aaacacacac gggtagattg ctggcctgtt 3481 gtaggtggta gggacacaga tgaccgacct ggtcactcct cctgccaaca ttcagtctgg
3541 tatgtgaggc gtgcgtgaag caagaactcc tggagctaca gggacaggga gccatcattc
3601 ctgcctggga atcctggaag acttcctgca ggagtcagcg ttcaatcttg accttgaaga
3661 tgggaaggat gttcttttta cgtaccaatt cttttgtctt ttgatattaa aaagaagtac
3721 atgttcattg tagagaattt ggaaactgta gaagagaatc aagaagaaaa ataaaaatca
3781 gctgttgtaa tcacctagca aactggcgta age
By“agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By“alteration” or“change” is meant an increase or decrease. An alteration may be by as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or by 40%, 50%, 60%, or even by as much as 70%, 75%, 80%, 90%, or 100%.
By "biologic sample" is meant any tissue, cell, fluid, or other material derived from an organism.
By "capture reagent" is meant a reagent that specifically binds a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.
As used herein, the terms“determining”,“assessing”,“assaying”,“measuring” and “detecting” refer to both quantitative and qualitative determinations, and as such, the term “determining” is used interchangeably herein with“assaying,”“measuring,” and the like. Where a quantitative determination is intended, the phrase“determining an amount” of an analyte and the like is used. Where a qualitative and/or quantitative determination is intended, the phrase“determining a level” of an analyte or“detecting” an analyte is used.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
By "fragment" is meant a portion of a protein or nucleic acid that is substantially identical to a reference protein or nucleic acid. In some embodiments the portion retains at least 50%, 75%, or 80%, or more preferably 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid described herein.
The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. By "microglia" is meant an immune cell of the central nervous system
By“myelin” is meant a fatty white substance surrounding the axon of nerve cells and forming an electrically insulating layer. Myelination is the process by which the myelin is produced.
By "oligodendrocyte" is meant a glial cell that forms the myelin sheath of axons in the central nervous system. Oligodendrocytes differentiate from oligodendrocyte precursor cells in the central nervous system.
By "increasing proliferation" is meant increasing cell division of a cell in vivo or in vitro.
As used herein, the terms“prevent,”“preventing,”“prevention,”“prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
The term“subject” or“patient” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non human primate, murine, bovine, equine, canine, ovine, or feline.
By "reference" is meant a standard of comparison or control condition. In certain embodiments, the reference is a GPR56 polypeptide or nucleic acid molecule.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences ( e.g ., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g.,
Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g, formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g ., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate,
1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include
substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e 3 and e 100 indicating a closely related sequence.
By“specifically binds” is meant a compound ( e.g ., peptide) that recognizes and binds a molecule (e.g., polypeptide), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample.
As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as 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 otherwise clear from context, all numerical values provided herein are modified by the term about.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 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.
Any compounds, compositions, or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
As used herein, the singular forms“a”,“an”, and“the” include plural forms unless the context clearly dictates otherwise. Thus, for example, reference to“a biomarker” includes reference to more than one biomarker.
Unless specifically stated or obvious from context, as used herein, the term“or” is understood to be inclusive.
The term“including” is used herein to mean, and is used interchangeably with, the phrase“including but not limited to.”
As used herein, the terms“comprises,”“comprising,”“containing,”“having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean“includes,” “including,” and the like;“consisting essentially of’ or“consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides a schematic diagram showing that GPR56 mediates tripartite signaling among extracellular matrix, microglia and oligodendrocyte during myelination.
FIG. 2A provides a series of sections throught the corpus callosum of
Opr 56II II;I>dgfr a( "r KR and (}pr5fJl 11 ;Pdgfr a 'reER mice following cuprizone feeding and recovery and a graph. Representative images of Black-Gold myelin staining of the corpus callosum of Opr 56^^ ;Pdgfr dreER and Opr 56)111 ;Iydgfr 'reER mice after cuprizone feeding for 6 weeks followed by recovery for 3 d (3 DR), 7 d (7 DR) and 10 d (10 DR) (dotted line outlines quantified region). Scale bar, 250 pm.
FIG. 2B is a graph showing that the percentage of remyelinated corpus callosum displayed significant decrease in myelination at 7 DR and 10 DR between
Opr 56ll ll;I1dgfr a( re R and OprS 1111 ;Pdgfrd re R mice. * P = 0.0285 (7 DR), N = 4 (ere); N = 4 (cre+); * P = 0.0416 (10 DR), N = 3 (ere); N = 4 (cre+), unpaired /-test.
FIG. 3 A is a schematic diagram showing the strategy for focal lysolecithin injection. FIG. 3B is a schematic diagram showing the site of injection.
FIG. 3C provides representative transmission electron micrograph (TEM) images from the corpus colosum (CC) of Tgm2^ ;Cx3cr lCre and Tgm2^ ;Cx3cr lCre+ mice.
FIG. 3D is a graph showing the percentage of myelinated axons in the corpus collosum (CC) of Tgm2^ ;Cx3cr lCre and lg' m2,l ,l;( x3crK 're mice. * P = 0.0493; N= 4 per genotype; unpaired /-test.
FIG. 3E is a scatter plot displaying g-ratio values in the CC of Tgm2fl/il;Cx3crlCre and Tgm2II II;( 'x3crK 're mice, which have a microglia-derived deletion of transglutaminase 2 (encoded by Tgm2).
FIG. 4 is a schematic diagram illustrating the hypothesis that microglia GPR56 is a molecular target of maternal immune activation (MIA) during pregnancy and inflammatory activation during early childhood.
FIG. 5 A and 5B provides two graphs showing that Poly I:C induces rapid down- regulation of Gpr56 expression.
FIG. 6A includes sections through cerebral cortex of mouse models of autism (right hand panels) vs. control mice (left hand panels). Mouse models of autism shown were either induced using prenatal valproate, VP A) or were the result of a single-gene mutation identified in human patients (Neuroligin-3, NL-3 R451C). These slices show decreased parvalbumin positive (PV+) intemeurons in the cerebral cortex. This is one of the pathologies associated with autism. FIG. 6B is a graph showing quantitation.
FIG. 7 shows synaptic density in brains of autistic children vs. control children.
FIG. 8A shows representative images showing parvalbumin positive intemeuron density in cerebral cortex of a control brain relative to the brain of a microglial GPR56 knockout mouse.
FIG. 8B is a graph quantitating parvalbumin positive interneuron density in wild-type and microglial GPR56 knockout mice.
FIG. 9A shows the co-localization of Vglut2/Homerl at synapses in wild-type and microglial GPR56 knockout mice.
FIG. 9B is a graph quantitating synaptic density in in wild-type and microglial GPR56 knockout mice.
FIG. 10A shows the results of behavioral testing of wild-type and microglial GPR56 conditional knockout mice. Gpr56 knockout mice (cKO) manifest autistic behavior in the form of a marble burying obsession. FIG. 10B shows that Gpr56 knockout mice (cKO) manifest autistic behavior in the form of anxiety in an open field behavioral assay.
FIG. 10C shows that Gpr56 knockout mice (cKO) manifest autistic behavior in the form impaired social behavior.
FIG. 11 provides a graphical representation showing the presence of single nucleotide polymorphisms (SNP) in GPR56 in Alzheimer’s disease (AD) patients.
FIG. 12 is a table indicating the position of SNPs in GPR56, which correlate with neurological defects including multiple sclerosis, Alzheimer’s disease, Autism, and
Amyotrophic lateral Sclerosis
FIG. 13 is a schematic diagram illustrating agents that could increase the expression or activity of GPR56 for use as therapeutics in neurological disorders characterized by a defect in GPR56 expression or activity.
FIG. 14A-L shows that microglial GPR56 is required for synaptic refinement in dLGN during development. FIG. 14A provides an RNAscope showing that Gpr56 transcripts co-localize with microglia in controls, but not in conditional knock out (CKO), and is absent in all cell types in the global KO. Scale bar, 20 pm. FIG. 14B provides a representative image of vGlut2 and Homerl staining in dLGN at P10. The outline indicates the dLGN core, and the dotted boxes show where synapses are quantified. Scale bar, 200 pm. FIG. 14C provides a representative 3D-reconstructed super-resolution images of vGlut2 and Homerl staining of P8 dLGN. Shading represents surface rendering of vGlut2+ presynaptic terminals. Darker spots represent Homerl+ post-synapses within the distance of 300nm from green surface.
Gray spots represent Homer l+ post-synapses out of 300nm distance. Scale bar, 2 pm. FIG. 14D provides a quantification of Homerl + spots adjacent to vGlut2 surface within lOOnm, 200nm, and 300nm in CKO and controls at P8. N = 3. FIG. 14E provides confocal images of vGlut2 labeling retinal ganglion cell (RGC) presynaptic terminals and Homerl for post- synapses in the dLGN of CKO and controls at P10. Overlapped vGlut2 and Homerl are quantified as synapses, indicated by white circles. Scale bar, 5 pm. FIG. 14F provides a time course of dLGN synapse density between WT and controls. N = 3 for P0, N = 4 for P5, N=3 for P10. FIG. 14G provides a Western blot of vGlut2 using microdissected dLGN tissue from P30 mice. FIG. 14H provides a Quantification of vGlut2 expression in cKO mice and controls. N = 4, *p = 0.018. FIG. 141 provides a quantification of synapse density in the dLGN of both male and female iCKO mice at P10. N = 3 for male, N = 4 for female. FIG.
14J provides a representative image of vGlutl and Homerl staining in dLGN at P10. Dotted boxes show where synapses are quantified in the upper part of the dLGN core (outline). Scale bar, 200 pm. FIG. 14K provides representative images of vGlutl+ presynaptic terminals and Homerl+ postsynapses in the dLGN of CKO and controls at P10. Scale bar, 5 pm. FIG. 14L provides a quantification of synapse density (vGlutl/homerl) at P10 in the dLGN of CKO mice and controls. N=3 (WT), N=5 (CKO), p = 0.053. *p<0.05, **r<0.01, ***p<0.00l, Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001 using Student’s t-test (H and L) or two- way ANOVA followed by Bonferroni’s post hoc test (D, F and I). Data are presented as mean ± SD. FIG. 14M shows verification of CKO mice by WB and qPCR. On the left, a Western blot shows that GPR56 was detected in control microglia, but not in Gpr56 null or CKO microglia. On the right, QPCR detects a high level of Gpr56 transcripts in control microglia, compared to CKO microglia. N=3, **P<0.0l by Student’s t test. Data are presented as mean ± SEM.
FIG. 14N-A -14N-H show that cellular properties remain unchanged upon microglial Gpr56 deletion. CKO microglia do not show significant difference compared to controls. FIG. 14N-A provides images of microglia stained by anti-Ibal in dLGN at P5. FIG. 14N-B shows quantification of microglia density between CKO and controls. N = 3 (Ctrl), N = 4 (CKO), p = 0.462. FIG. 14N-C provides representative images of Ibal and CD68 double staining. FIG. 14N-D provides quantification of the percentage of CD68 positive microglia.
N = 3. FIG. 14N-E provides images of individual microglia. FIG. 14N-F provides quantification of coverage area of each microglia between CKO and controls. N = 35 (Ctrl),
N = 22 (CKO) microglia from 3, 4 mice, respectively p = 0.685, Student’s t-test. FIG. 14N-G provides images depicting concentric circles upon manually outlined microglia at 1.25 pm intervals for Sholl analysis. FIG. 14N-H provides A Sholl analysis that shows no significant change in arbor complexity in CKO. N = 3, F (1, 156) = 0.88, P = 0.35 by two-way ANOVA with Bonferroni’s post hoc test. Data are presented as mean ± SEM.
FIG. 140 shows retinogeniculate synapses in CKO and controls using SIM. Top panels show original images taken by SIM. Middle panels show 3D rendered images after processing in Imaris. Bottom panels show Homerl spots that are within the distance of 300nm from vGlut2 surface. Scale bar = 5 pm.
FIG. 14P-A - 14P-D show that deleting microglial Gpr56 has no effect on RGC density in P5 retina. FIG. 14P-A shows whole mount retinal staining of RGC using Brn3a antibody. FIG. 14P-B provide representative images of RGC and microglia staining using Bm3 and Ibal antibodies in retina. FIG. 14P-C shows quantification of RGC density in Ctrl and CKO. FIG. 14P-D shows quantification of retinal microglia density in Ctrl and CKO.
N=3, p = 0.187, unpaired student’s t-test. Data are presented as mean ± SD.
Fig. 15 shows that microglial GPR56 is required for synapse refinement in the hippocampus. FIG. 15A provides representative images showomg hippocampus with vGlut2 and Homerl immunostaining. White box outlines the region of interest and yellow box shows the regions where confocal images were taken. FIG. 15B provides confocal images of synaptic immunostaining in CA1 striatum lacunosum-moleculare at PlO. Scale bar, 5 pm. FIG. 15C and D show quantification of synapse density in CA1 striatum lacunosum- moleculare in iCKO versus control at P10 (C), and CKO versus control at P21 (D). At P10, N(Ctrl) = 4, N(iCKO) = 3, *p = 0.02. At P21, N = 8, **p = 0.005. FIG. 15E provides representative images showing hippocampus with vGlutl and Homerl immunostaining.
White box outlines the region of interest, and yellow box shows the regions where confocal images were taken. FIG. 15F provides confocal images of synaptic immunostaining in CA1 striatum radiatum at P10. FIG. 15G and 15H show quantification of synapse density in CA1 striatum radiatum in iCKO versus control at P10 (G), and CKO versus control at P21 (H). At P10, N = 5, p = 0.83. At P21, N = 5, p = 0.89. *p<0.05, Student’s t-test. All data are presented as mean ± SD.
FIG. 16A-E shows that microglial Gpr56 deficiency leads to reduced engulfment of RGC inputs, and impaired retinogeniculate circuit organization and function. FIG. 16A provides a schematic representation of in vivo engulfment assay. CTB594 dyes are injected into both eyes, and anterogradely trace RGC projections to dLGN. FIG. 16A provides representative surface rendered microglia from P5 dLGN of CKO or controls in which RGC inputs were labeled with CTB-594. Scale bar, 20pm. FIG. 16C provides a quantification of the percentage of engulfed RGC inputs in controls and CKO microglia. More than 10 microglia cells are analyzed in each individual mouse brain. N = 4, *p = 0.039, mean ± SEM. FIG. 16D provides a diagram of RGC labeling for testing eye-specific segregation at P30. FIG. 16E provides a CTB-labeled dLGN shows reduced eye-segregation at P30 in cKO mice. The left column shows contralateral dLGN labeled with CTB488 (green), and the middle one is ipsilateral dLGN with CTB647 (magenta). The right column represents the dLGN pseudocolored according to the R-value for each pixel (R = log(Fipsi/F contra)). Scale bar, 200 pm. FIG. 16F provides a histogram distribution chart of R-value for all pixels within dLGN represents the degree of eye-specific segregation. A greater R-value means a bigger difference of ipsi-to-contraleteral fluorescence intensity. The narrower distribution of cKO in the dotted box indicates reduced segregation. FIG. 16G provides The variance of R distributions in P30 control and cKO mice. N = 4 (Ctrl), N = 6 (CKO), **p = 0.004, mean ± SD. FIG. 16H provides a schematic diagram of electrophysiol ogical recording in a parasagittal dLGN. FIG. 161 provides a Maximal NMDAR mediated currents measured in the dLGN of P28-P34 mice, *p = 0.03, N = 14 (Ctrl), 23 (CKO) cells from 5, 7 mice, respectively. Mean ± SEM. FIG. 16J provides a Western blot of NMDAR1 using
microdissected dLGN tissue from P30 mice. FIG. 16K provides a Quantification of
NMDAR1 expression in cKO mice and control. N = 3, **p = 0.007, mean ± SD. FIG. 16L provides a maximal AMPAR mediated currents measured in the dLGN of P28-P34 mice. P=0.302, N = 13 (Ctrl), 17 (CKO) cells from 5, 7 mice, respectively. Mean ± SEM. FIG. 16M provides a Western blot of GluRl with microdissected dLGN tissue. FIG. 16N provides a quantification of GluRl expression in CKO mice and controls. N = 3, p = 0.40, mean ± SD. All data were analyzed by student’s t-test, *P<0.05, **P < 0.01.
Fig. 160-A-160-D shows that microglial GPR56 Deficiency leads to impaired eye- specific segregation at P10. Fig. 160-A shows in the left two columns contra- and ipsi- lateral RGC inputs labeled by CTB488 and CTB594, respectively. The middle two columns are binary images of contra- and ipsi-lateral LGN. The right images show a greater overlap between contralateral RGC inputs and ipsilateral inputs. Fig. 160-B shows quantification of the percentage of overlapped contra- and ipsi-lateral RGC inputs on multiple thresholds in CKO and controls at P10. N=4, ****p<0.000l, two way ANOVA with Bonferroni’s post hoc test. Fig. 160-C provides images of superior colliculus after 24 hours anterograde labeling of RGC by CTB488 and CTB647. Fig. 160-D shows the variance of R-values from caudal LGN to cranial LGN.
FIG. 17A-17F shows that GPR56 GAIN domain binds to PS, and Gpr56 S4 variant is essential for synaptic refinement in dLGN development. FIG. 17A provides a diagram that shows GPR56 protein structure, consisting of a PLL, a GAIN domain, and a 7TM. A full length N-terminal fragment (NTF) contains PLL and GAIN domains. FIG. 17B provides a diagram of membrane lipid strip showing the lipid composition for each dot. FIG. 17C shows that GAS6, NTF-hFc, and GAIN-hFc bind to several specific lipids, whereas hFc does not. DAG, 1, 2-Diacyl glycerol; PA, phosphatidic acid; PS, phosphatidylserine; PE, phosphatidyl- ethanol- amine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PI, phosphati- dylinositol; PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phospha- tidylinositol 4,5- bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-tris- phosphate. FIG. 17D provides a flow chart showing the experimental design. Briefly, Ba/F3 cells are treated with A23187 to externalize PS. For the binding assay, Alexa Fluor 647-conjugated hFc, GAIN-hFc, or NTF- hFc were incubated with PS -externalized Ba/F3 cells. For the competition assay, FITC- conjugated Annexin V was added to compete with the binding of PS to Alexa Fluor 647- conjugated hFc, GAIN-hFc, or NTF-hFc. FIG. 17E provides a Flow cytometry analysis shows that only GAIN domain binds to PS. FIG. 17F depicts the results of flow cytometry demonstrating that only GAIN domain is able to compete off Annexin V binding to PS. FIG. 17G provides representative images of vGlut2/Homerl staining in P10 mouse dLGN of control, Gpr56 null , and Gpr56 S4 brains. FIG. 17G and 17H shows relative vGlut2/Homerl synapse density in dLGN. N=l0 (Ctrl), N=3 ( Gpr56 S4 ), N=6 ( Gpr56 null), ***p < 0.001, one-way ANOVA with turkey’s post-hoc test. FIG. 171 shows relative vGlut2/Homerl synapse density of CKO dLGN at P10. N=3, **p = 0.003. *p < 0.05, **p < 0.01, ***p < 0.001, student’s t-test. Data are presented as mean ± SD.
FIG. 17J-A - 17J-E relates to Gpr56 S4 isoform. FIG. 17J-A and B provide diagrams showing the genomic structure of different Gpr56 variants. Solid boxes indicating exons that are transcribed. FIG. 17J-C and D are standard curves of qPCR using a series of cDNA dilution. FIG. 17J-E is a graph showing that only S4 transcripts were present in Gpr56 S4 mouse microglia.
FIG. 17K shows that a deletion of GPR56 S4 isoform did not result in more severe cortical ectopia. FIG. 17 (top) provides representative images of Nissl staining of Gpr56 S4 and Gpr56 null El 6.5 neocortex. Arrows point out cortical ectopias. FIG. 17 (bottom) provides quantification of cumulative ectopia size. N = 6, P = 0.64, Student’s t-test.
Fig. 18A-E show that a microglial Gpr56 deficiency impairs engulfment of PS+RGC Inputs. Fig. 18A provides a drawing depicting the experimental procedure where CTB488 and CTB647 were intraocularly injected, followed by intracranial injection of PSvue550 to dLGN border. Fig. 18B provides a schematic diagram shows the timeline of procedures for ex vivo imaging and in vivo engulfment analysis. Fig. 18C provides (top panel) representative images show PS labeling in the WT dLGN at P6 and P13. RGC inputs were labeled with CTB488. Bottom panel: Enlarged regions of the boxed region in top panels. Circles indicate PS+ RGC inputs. Arrows pointing to the enlarged PS+ RGC inputs in the upper right hand comer. Scale bar, 5 pm. Fig. 18D provides a quantification of the percentage of PS+ RGC inputs in total RGC inputs at P6 and Pl3. N = 4, 0.0001. Fig. 18E provides a diagram showing contra- (green) and ipsi-lateral (blue) projections overlap in P6 dLGN. Yellow box indicates the region where images were taken and analyzed. (F) Representative images show PSVue colocalizes with contra- or ipsi-lateral RGC inputs. (G) Quantification of the percentage of PS+ inputs in total inputs. N=3, *p = 0.03. (H) CTB traced RGC inputs were labeled by PSvue at P6 in control and CKO in vivo. White circles indicate PS+ RGC inputs. (I) Quantification of PS+ RGC input percentage in total inputs. N = 5, *p = 0.02. (J) Representative surface rendering images of engulfed PS+ and PS RGC inputs by microglia in control and CKO. (K) Quantification of engulfed PS+ RGC inputs in control and CKO is calculated as: Volume of engulfed RGC inputs / Volume of microglia cell. (L) The percentage of engulfed PS+ RGC inputs in total engulfed inputs in control and CKO. (M) Quantification of engulfed PS RGC inputs in control and CKO. N=4 (Ctrl), N=5(CKO), **p = 0.005 in (K), p = 0.80 in (L), **p = 0.003 in (M). * P < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t-test. Data are presented as mean ± SD.
FIG. 19A-19D shows in vivo labeling of PS by PSVue and pSIVA. FIG. 19A is a diagram illustrating PS labeling by PSVue550 and RGC inputs antegrade tracing by CTB. CTB was intraoccularly injected 24 hours prior to PSVue/pSIVA injection. FIG. 19B the left panel shows well-diffused PSVue into dLGN. The box indicates the region where the images were taken. Right panel shows RGC inputs colocalize with PSVue signal. The white box indicates the region of higher magnification image shown. FIG. 19C provides a diagram showing pSIVA labeling and RGC inputs tracing by CTB. FIG. 19D left panel shows pSIVA accumulated in the gap between hippocampus and LGN. Right panel shows minimal pSIVA colocalized with RGC inputs. Scale bar, 20 pm.
Fig. 20A-20F shows that microglia specifically engulf PS Vue-labeled PS+ RGC inputs. (Ai) A diagram shows PSVue was injected through the hippocampus to the border of dLGN. Enlarged dLGN (white dotted line) is showed in Aii. (Bi) A representative image of microglia from PSVue treated dLGN. Nucleus were labeled with DAPI. (Bii) A 3D surface rendered microglia (purple) with DAPI (blue) and engulfed inputs (green) and PSVue (red). (Biii) Engulfed RGC inputs and PSVue inside of microglia are enlarged from the white boxed region in Bii. Magnified insert shows PS+ RGC inputs. (Ci) 5-TAMRA, the fluorophore component of PSVue550, was used as a negative control. (Cii) Enlarged dLGN from Ci. (Di) A representative image of microglia from 5-TAMRA treated dLGN. Very few 5-TAMRA punctate were observed. (Dii) A surface rendered microglia (purple) with DAPI (blue) and engulfed inputs (green) and 5-TAMRA (red). (Dili) Engulfed RGC inputs and 5-TAMRA are enlarged. Magnified insert shows RGC inputs do not overlap with 5-TAMRA. (E) Quantification of engulfed PSvue and 5-TAMRA dyes by microglia. N=4 (PSvue), N=3 (5- TAMRA), **p = 0.001. (F) Quantification of engulfed PSvue-positive or 5-TAMRA-positve RGC inputs dyes by microglia. N=4 (PSvue), N=3 (5-TAMRA), **p = 0.006 by Student’s t- test. Data are presented as mean ± SD.
FIG. 21 A and B show that microglia engulf more PS+ than PS RGC inputs in dLGN. FIG. 21 A provides a representative image of microglia (left) is surface rendered (middle), and RGC inputs and PSVue inside of microglia are shown in the right panel. Arrows pointing to two RGC inputs that are presented in a higher magnified insert with one being PS+ and the other being PS input. FIG. 21B provides a quantification of the percentage of engulfed PS+ and PS RGC inputs in total engulfed inputs. N=4, unpaired Student’s t-test, ***p < 0.001, data are presented as mean ± SD.
FIG. 22A and B indicate that a Gpr56 CKO mice shows no difference in paired pulse depression. Paired pulse depression was recorded on dLGN slice from P28-P34 mice. Given that optic inputs usually demonstrate paired pulse depression, and cortical inputs show paired pulse facilitation, this data indicates that the optic tracts and not cortical inputs were stimulated n = 14 (Ctrl), 23 (KO) cells from 5, 7 mice. P = 0.694 by Student’s t-test.
DETAILED DESCRIPTION OF THE INVENTION
As described below, the present invention features compositions and methods for the treatment of ASD and other neurological diseases and disorders associated with defects in GPR56 or with undesirable increases in synapse number.
The invention is based, at least in part, on the discovery that GPR56, a protein that functions in oligodendrocyte and interneuron development, also functions in synaptic pruning, and that defects in synaptic pruning are observed not only in autistic subjects, but in GPR56 knockout mice. As reported herein below, microglial GPR56 maintains appropriate synaptic numbers in several brain regions in time- and circuit- specific fashion.
Phosphatidylserine (PS) on pre-synaptic elements binds GPR56 in a domain-specific manner and microglia-specific deletion of Gpr56, leads to increased PS+ excitatory synapses.
Remarkably, a specific alternatively spliced isoform of GPR56 is selectively required for microglia-mediated synaptic pruning. The results presented herein provide a genetic substrate to address microglial synapse pruning in the context of other neurodevelopmental processes. GPR56
Microglia, tissue resident macrophages of the CNS, are important for synaptic development, both promoting synapse formation and engulfing redundant synapses. Immune molecules such as classical complement components and receptors, CX3CL1/CX3CR1,
MHC class I and PirB have been implicated in developmental synapse refinement and in synapse loss in disease models. To date, elements associated with microglial synapse refinement have been strictly limited to their expression in microglia among CNS cells. Mammalian neurodevelopment involves a succession of complex and overlapping processes beginning with neurogenesis and neuronal migration, which are concurrent with microglial infiltration and morphogenesis. Subsequently, neurite arborization sets the stage for synaptogenesis, circuit establishment and refinement as well as myelination. All these processes entail cell-cell interactions, so that discovery of molecules involved in multiple processes in a cell-type specific fashion can inform our understanding how overlapping and sequential programs of intercellular signaling events are coordinately regulated.
Originating from primitive myeloid cells in the yolk sac, microglia enter the central nervous system (CNS) at the start of brain development. In recent years, it has become clear that microglial physiology is dominated by their functions during neurodevelopment. In the developing neocortex, microglia modulate neural progenitor survival by precisely timed-and- localized secretion of growth factors and the size of neural progenitor pool by clearance of dead or stressed cells. It was shown that GPR56 regulates neural progenitor cell proliferation, and germline deletion of Gpr56 impairs neurogenesis. Interestingly, Gpr56 is expressed in multiple brain cell types during development, including neuroepithelial cells, intermediate progenitor cells, and first-born neurons. Furthermore, Gpr56 message is highly expressed in young and adult microglia in both humans and mice and regulated by a microglial super enhancer.
GPR56 mediates tripartite signaling among ECM, microglia and oligodendrocyte during myelination. Microglia promote OPC proliferation via GPR56 signaling.
Tissue transglutaminase (TG2), derived from microglia, is the ligand of
oligodendrocyte precursor cell (OPC) GPR56. Tissue transglutaminase (TG2), derived from microglia, is the ligand of oligodendrocyte precursor cell (OPC) GPR56. Together with laminin, TG2 activates OPC GPR56 and promotes OPC proliferation and thus central nervous system myelination (FIG. 1). Certain neurological disorders arise when an exogenous stressor strikes an individual having an underlying vulnerability, such as a genetic predisposition, during a critical developmental period. The exogenous stressor might include an infection that causes immune activation. It has previously been shown that GPR56 is highly expressed in microglia, and that GPR56 is downregulated in response to an inflammatory challenge. For example, two exogenous stressors, maternal immune activation (MIA) during pregnancy and inflammatory response during early childhood infection, that occur during critical developmental periods might be sufficient to induce an autism spectrum disorder in certain vulnerable individuals (FIG. 4).
The molecular mechanisms underlying OL development and CNS myelination are only beginning to be elucidated, and GPR56, a member of the adhesion G protein-coupled receptor family, is a recently identified novel regulator of OL development (Ackerman et al ., Nat Commun 6, 6122, 2015; Giera et al. Nat Commun 6, 6121, 2015). The adhesion G protein-coupled receptor (aGPCR) GPR56/ADGRG1 is a newly identified regulator of OL development that is evolutionarily conserved in zebrafish, mice, and humans (Ackerman et al., Nat Commun 6, 6122, 2015; Giera et al., Nat Commun 6, 6121, 2015). Loss of function mutations in GPR56 cause a devastating human brain malformation termed bilateral frontoparietal polymicrogyria (BFPP) that comprises a constellation of structural brain defects including CNS hypomyelination (Piao et al., Annals of neurology 58, 680-687, 2005; Piao et al., Science 303, 2033-2036, 2004). Conditional deletion of Gpr56 in OL lineage cells showed that the hypomyelination phenotype is caused specifically by deficiency for GPR56 signaling in oligodendrocyte precursors (OPCs) and immature oligodendrocytes (OLs) (Giera et al., Nat Commun 6, 6121, 2015). Loss of Gpr56 in mice and zebrafish decreased OPC proliferation leading to a reduced number of mature myelinating OLs and fewer myelinated axons in the CNS (Ackerman et al., Nat Commun 6, 6122, 2015; Giera et al., Nat Commun 6, 6121, 2015). However, the relevant GPR56 ligand during CNS myelination was not defined during these studies.
Microglial TG2 was identified as the ligand of OPC GPR56 via a combined approach utilizing molecular, cellular and developmental biology as well as unbiased proteomics (Giera et al., eLife, 2018, pii: e33385. doi: 10.7554/eLife.33385). This deorphanization is a mandatory first step in therapeutic exploitation of this novel pathway. The past few years have seen aGPCRs implicated both in CNS and peripheral nervous system myelination and myelin maintenance (Kuffer et al., Nature 536 , 464-468, 2016; Langenhan et al., Nature reviews Neuroscience 77, 550-561, 2016). Although OPC-bound GPR56 was shown to be required for developmental CNS myelination (Ackerman et al., Nat Commun 6 , 6122, 2015; Giera et al., Nat Commun 6 , 6121, 2015; Salzman et al., Neuron 97, 1292-1304, 2016), the relevant ligand remained undefined and the role of GPR56 in myelin repair was not addressed.
It is known that laminin regulates oligodendrogenesis and CNS myelination by interacting with integrins and dystroglycan (Colognato et al., Nat Cell Biol 4, 833-841, 2002; Colognato et al., Development 734, 1723-1736, 2007; Colognato et al., The Journal of cell biology 767, 365-375, 2004; Colognato and Tzvetanova, Developmental neurobiology 77, 924-955, 2011). In this study, a new tripartite signaling module is presented - microglial TG2, ECM laminin, and GPR56 on OPCs - that generates regulatory inputs during OL development and CNS myelination. It was previously reported that TG2 binds laminin-l 11 (Aeschlimann et al., J Biol Chem 267, 11316-11321, 1992). TG2, contingent on its crosslinking activity, together with laminin-l 11, binds to the GPR56 NTF and dissociates the NTF from the CTF, allowing the endogenous GPR56 tethered ligand to initiate G-protein signaling. Downstream RhoA activation and CDK2 are then implicated in OPC progression through the cell cycle, to generate mature oligodendrocytes (OLs) for myelination or remyelination. Without being bound by theory, this microglial ligand-ECM-OPC receptor signaling triad is particularly relevant for OL development, where a complex array of factors and ECM components affect the varied stages of the process (Wheeler and Fuss,
Experimental neurology 283 , 512-530, 2016).
Notably, subtle dyshomeostasis could plausibly disrupt brain development (Sharon et al., Cell 167 , 915-932, 2016). Loss of function mutations in GPR56 manifest a pronounced CNS hypomyelinaiton in humans (Piao et al., Annals of neurology 58, 680-687, 2005; Piao et al., Science 303, 2033-2036, 2004; Giera et al., Nat Commun 6, 6121, 2015), although both Gpr56 and Tgm2 deletion lead to a mild decrease in OL production. Without being bound by theory, this indicates that OL development is a very tightly regulated process and a minor-in- degree deviations from typical development can be phenotypically catastrophic. Perinatal white matter injury (PWMI) is another particularly salient example of derailed
neurodevelopment, carries profound consequences and is characterized by heightened OPC proliferation with impaired OL maturation. Microglia upregulate Tgm2 from nil shortly after birth to high levels by 8 weeks of age (Matcovitch-Natan et al., Science 353, aad8670, 2016) with a further increase of >50-fold upon inflammatory challenge (Emy et al., Nat Neurosci 18, 965-977, 2015), potentially driving supraphysiological OPC proliferation. These studies showed that either Gpr56- or 7gro2-deficient mice exhibited normal g-ratios despite reduced oligodendrocytes (OLs) and myelinated axons. Without being bound by theory, this suggests that myelination by residual mature OLs was unimpaired, indicating a highly selective effect on OPC proliferation. Understanding the molecular regulation of perinatal dysregulation of myelin formation has the potential to accelerate rational intervention to ameliorate neurodevelopmental aberration as seen in PWMI.
Collagen III was previously identified as the ligand for neural progenitor cell-GPR56 in the developing neocortex (Luo et al., Proc Natl Acad Sci U S A 108, 12925-12930, 2011). As shown herein, collagen III was not the GPR56 ligand in OPCs, and microglia-derived TG2 was the ligand of OPC-GPR56. The results of this study highlight a unique property of adhesion GPCRs: activation by distinct ligands in different cellular and developmental contexts.
The signaling module of GPR56 contains multiple potential targets for therapeutic intervention, including the GPR56-CTF, which similar to other GPCRs, serves as a legitimate drug target. Given the importance of myelin formation, maintenance and repair in neurological diseases across the human life span, and the importance of synapse pruning and maintenance in ASD, these findings have the potential to provide clinical benefit for both developmental and acquired neurological diseases involving myelination and/or synapse formation/pruning.
Transglutaminase 2 (TG2)
As a member of the transglutaminase family, Transglutaminase 2 (TG2) is a versatile and multi-faceted protein that displays several diverse biological functions. In addition to the typical transamidating/crosslinking function, studies over the last decade reveal non- enzymatic functions of extracellular TG2, including promoting cell adhesion, migration, and survival. TG2 was identified as a regulator of OL development by serving as a ligand of GPR56. A direct mitogenic effect of TG2 on OPCs could be elicited. Without being bound to theory, this indicates an extracellular non-enzymatic function of TG2.
Recent literature suggests that microglia have physiological roles during the development of the central nervous system (CNS) myelination. However, the underlying molecular mechanism remains elusive. GPR56, a member of the adhesion G protein-coupled receptor family, is a recently identified novel regulator of oligodendrocyte development. Microglia-derived transglutaminase 2 (encoded by Tgm2 ) is the GPR56 ligand for OPCs. A search for the ligand of GPR56 in the developing white matter identified one or more putative ligands of GPR56 predominantly expressed in microglia. Further biotin-streptoavidin pull- down from mixed glia cells followed by mass spectrometry analysis revealed
transglutaminase 2 (TG2), a known binding partner of GPR56, as one of the top ligand candidates.
TG2 is predominantly expressed in microglia in the postnatal brain. In vitro, ECM protein laminin interacts with catalytically-active TG2 to release GPR56 NTF and activates GPR56 CTF for downstream RhoA signaling, which in turn promotes OPC proliferation. In vivo, microglia-specific deletion of Tgm2 leads to fewer mature oligodendrocytes (OLs) and CNS hypomyelination, phenocopying OPC-specific deficiency for GPR56. Tgm2 knockout mice manifest with decreased oligodendrocyte precursor cell (OPC) proliferation, leading to fewer mature oligodendrocytes and a reduced number of myelinated axons in the corpus callosum via RhoA pathway, phenocopying the Gpr56 knockout mice. Recombinant TG2 stimulated OPC proliferation in a GPR56-dependent manner in vitro. Recombinant TG2 rescued remyelination failure in Tgm2 knockout cerebellar slices. OPC-specific deletion of Gpr56 impairs CNS remyelination after cuprizone-induced demyelination, demonstrating a function with regard to repair as well as development. Thus, transglutaminase 2 (TG2, gene symbol Tgm2 ) was identified as a ligand for GPR56 during white matter development.
Although TG2 was reported to be present in oligodendrocyte precursors (OPCs) (Van Strien et al. Glia 59, 1622-1634, 2011) and astrocytes (Van Strien et al. PLoS One 6, e25037, 2011), Zhang et al. demonstrated that Tgm2 is predominantly expressed in microglia through gene expression profiling using purified glial cells and neurons (Zhang et al. JNeurosci 34, 11929-11947, 2014). Consistent with this observation, TG2 protein was only detected in microglia by western blot analysis. It is possible that previous reports detected the TG2 that binds to the cell surface of OPCs and astrocytes. Microglia-derived TG2 promotes OPC proliferation via the RhoA pathway, providing a novel molecular link on how microglia regulate OL development and CNS myelination.
Methods of Treatment
The present invention provides methods of treating disease and/or disorders or symptoms thereof associated with a reduction in GPR56 expression or activity or an undesirable reduction in synaptic pruning (e.g., Autism spectrum disorder, multiple sclerosis, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis), which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising administering an agent that activates GPR56 to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
The increased expression or activity of GPR56 in an oligodendrocyte or precursor thereof, and/or the increased expression or activity of TG2, which is an activating ligand of GPR56, promotes synaptic pruning. The application prevents or treats neurological diseases, for example, characterized by decreased expression or activity of GPR56 (e.g., ASD).
Accordingly, the invention provides for the treatment of a variety of diseases and disorders associated with decreased synaptic pruning (e.g., multiple sclerosis, Alzheimer’s disease, Autism, and Amyotrophic Lateral Sclerosis). Such diseases are amenable to treatment by increased expression or activity of GPR56.
The invention generally features method of increasing or promoting synaptic pruning in a subject having or at risk of developing an undesirable increase in synapse number.
Therapies provided by the invention include small molecule, polypeptide therapies and polynucleotide therapies. In on embodiment, the method involves contacting a glial cell (e.g. an oligodendrocyte or oligodendrocyte precursor) of the subject with an agonist or ligand of a GPR56 polypeptide; and activating signaling via the GPR56 polypeptide, thereby increasing or promoting myelin formation. Activating ligands of GPR56 polypeptide include naturally- occuring ligands such as TG2 or tethered ligands generated by recombinant or synthetic techniques. In another embodiment, the method involves contacting a glial cell (e.g. an oligodendrocyte or oligodendrocyte precursor) of the subject with a nucleic acid molecule encoding a GPR56 polypeptide or a fragment thereof; and expressing the GPR56 polypeptide in the cell, thereby increasing or promoting synaptic pruning. The present invention provides methods of treating diseases and/or disorders or symptoms thereof related to demyelination that comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an agent that increases GPR56 receptor signalling, expression, or activity to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder characterized by an undesirable increase in synapse number or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of an agent herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect (e.g., an increase in myelination). Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which myelination deficiency or loss may be implicated.
Small Compounds
Small compounds that activate GPR56 are known in the art. See, for example, Stoveken et ah, which describes two related classes of small molecules that could activate the aGPCR GPR56/ADGRG1, gedunin- and khivorin derived natural products. The most potent compound identified was 3-alpha-acetoxydihydrodeoxygedunin, or 3-alpha-DOG. Other compounds useful in the methods of the invention include khivorin, 7 synthetic peptide agonist, 3-deacetylkhivorin, deoxygedunin, and l,2-Epoxygedunin.
Tethered Ligands
Binding of TG2 to GPR56 results in GPR56 activation via exposure of a tethered agonist, also known as the stalk region, which is inhibited by an extracellular N-terminal domain (NTD) of GPR56. The NTD is expressed as part of GPR56, proteolytically processed, and non-covalently bound to the 7 transmembrane domains of GPR56. TG2 binds the NTD domain to expose the b-strand-l 3/stalk region, that when exposed, serves as a tethered agonist to activate G protein signaling (see., e.g., Stoveken et al. Proc Natl Acad Sci U S A. 2015 May 12; 112(19): 6194-6199, which is herein incorporated by reference in its entirety).
In various embodiments, the tethered agonist or GPR56 activating ligand is linked to a membrane associated moiety. Methods of making such tethered ligands are known in the art (see., e.g., U.S. Patent Nos. 8,563,519; 6,864,229; 8,440,627; 8,389,480; and 8,354,378 and U.S. Patent Publ. Nos. 20020076755; 20060166274; 20080214451; 20030148449;
20070179090; 20090270322; and US20100137207, which are herein incorporated by reference in their entireties).
Blood-Brain Barrier (BBB) Transport
This disclosure provides compositions for delivery of an agent (e.g., a GPR56 tethered peptide, TG2 polypeptide or fragment thereof) across the blood-brain barrier (BBB) using a transporter molecule that can cross brain endothelial cells while associated with the agent. The blood-brain barrier (BBB) protects and regulates the homeostasis of the brain and prevents the free passage of molecules into most parts of the brain. Transport of essential molecules such as nutrients, growth factors and hormones is achieved via a series of specific transporters and receptors that regulate passage across the brain endothelial cells. In one embodiment an agent of the invention is fused or conjugated to a BBB peptide. BBB peptide sequences are known in the art and are described at least for example at the Brainpeps® database (http://brainpeps.ugent.be/; Van Dorpe et al., Brain Structure and Function, 2012, 217(3), 687-718, which are herein incorporated by reference).
In certain aspects, a BBB transporter molecule as provided herein can bind to brain microvascular endothelial cells (BMVECs), e.g., human, and can cross through BMVEC in vitro or in vivo from the peripheral vasculature into the CNS vasculature. Whether a given fragment is a BBB-penetrable fragment can be tested by a variety of in vitro or in vivo assays known to persons of ordinary skill in the art. For example, the transporter molecule can be tested in the in vitro transcytosis assay, or in an in vivo assay such as a diuresis assay.
In certain aspects, transporter molecule activity can be demonstrated by visualization of the transporter molecule in the CNS. For example, a tritium-labeled transporter molecule can be delivered to a subject, and then visualized in the CNS via quantitative whole body radiography. In certain aspects, the transporter molecule localizes in specific regions of the CNS, e.g., the corpus callosum, developing white matter, and the like.
Recombinant Polypeptide Expression
In order to express the polypeptides of the invention, DNA molecules obtained by any of the methods described herein or those that are known in the art, can be inserted into appropriate expression vectors by techniques well known in the art. For example, a double stranded DNA can be cloned into a suitable vector by restriction enzyme linking involving the use of synthetic DNA linkers or by blunt-ended ligation. DNA ligases are usually used to ligate the DNA molecules and undesirable joining can be avoided by treatment with alkaline phosphatase.
Therefore, the invention includes vectors (e.g., recombinant plasmids) that include nucleic acid molecules (e.g., genes or recombinant nucleic acid molecules encoding genes) as described herein. The term“recombinant vector” includes a vector (e.g., plasmid, phage, phasmid, virus, cosmid, fosmid, or other purified nucleic acid vector) that has been altered, modified or engineered such that it contains greater, fewer or different nucleic acid sequences than those included in the native or natural nucleic acid molecule from which the
recombinant vector was derived. For example, a recombinant vector may include a nucleotide sequence encoding a GPR56 or TG2 polypeptide, or fragment thereof, operatively linked to regulatory sequences, e.g., promoter sequences, terminator sequences, and the like, as defined herein. Recombinant vectors which allow for expression of the genes or nucleic acids included in them are referred to as“expression vectors.”
In some of the molecules of the invention described herein, one or more DNA molecules having a nucleotide sequence encoding one or more polypeptides of the invention are operatively linked to one or more regulatory sequences, which are capable of integrating the desired DNA molecule into a prokaryotic host cell. Cells which have been stably transformed by the introduced DNA can be selected, for example, by introducing one or more markers which allow for selection of host cells which contain the expression vector. A selectable marker gene can either be linked directly to a nucleic acid sequence to be expressed, or be introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins described herein. It would be apparent to one of ordinary skill in the art which additional elements to use. Factors of importance in selecting a particular plasmid or viral vector include, but are not limited to, the ease with which recipient cells that contain the vector are recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.
Once the vector(s) is constructed to include a DNA sequence for expression, it may be introduced into an appropriate host cell by one or more of a variety of suitable methods that are known in the art, including but not limited to, for example, transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc.
After the introduction of one or more vector(s), host cells are usually grown in a selective medium, which selects for the growth of vector-containing cells. Expression of recombinant proteins can be detected by immunoassays including Western blot analysis, immunoblot, and immunofluorescence. Purification of recombinant proteins can be carried out by any of the methods known in the art or described herein, for example, any
conventional procedures involving extraction, precipitation, chromatography and
electrophoresis. A further purification procedure that may be used for purifying proteins is affinity chromatography using monoclonal antibodies which bind a target protein. Generally, crude preparations containing a recombinant protein are passed through a column on which a suitable monoclonal antibody is immobilized. The protein usually binds to the column via the specific antibody while the impurities pass through. After washing the column, the protein is eluted from the gel by changing pH or ionic strength, for example.
Screening
Accordingly, the invention provides methods for identifying agents (e.g.,
polypeptides, polynucleotides, antibodies, including recombinant antibodies, and small compounds) useful for increasing synaptic pruning and/or treating or preventing a disease or disorder characterized by an undesirable increase in synapses. The use of such cells, which express GPR56 is particularly advantageous for the identification of agents that increase GPR56 expression or biological activity. Methods of observing changes in GPR56 biological activity are exploited in high throughput assays for the purpose of identifying compounds that modulate GPR56 biological activity, e.g., transcriptional regulation or protein-nucleic acid interactions. Any number of methods are available for carrying out screening assays to identify new candidate compounds that increase the expression or activity of GPR56 and/or TG2. In one example, candidate compounds are added at varying concentrations to the culture medium of cultured cells expressing GPR56 and/or TG2. In various embodiments, the cell is an oligodendrocyte, oligodendrocyte precursor, or heterologous cell expressing GPR56. In other embodiments, the cell is a microglial cell or heterologous cell expressing TG2. Gene expression is then measured, for example, by microarray analysis, Northern blot analysis (Ausubel et al., supra), or RT-PCR, using an appropriate hybridization probe. The level of gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate molecule. A compound which increases the expression of a GPR56 and/or Tgm2 gene, or a functional equivalent thereof, is considered useful in the invention; such a molecule may be used, for example, as a therapeutic to treat a human patient having a demyelination disease or disorder.
In another example, the effect of candidate compounds may be measured at the level of polypeptide production using the same general approach and standard immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific for a polypeptide encoded by a GPR56 and/or Tgm2 gene. For example, immunoassays may be used to detect or monitor the expression of at least one of the polypeptides of the invention in an organism. Polyclonal or monoclonal antibodies that are capable of binding to such a polypeptide may be used in any standard immunoassay format ( e.g ., ELISA, Western blot, or RIA assay) to measure the level of the polypeptide. In some embodiments, a compound that promotes an increase in the expression or biological activity of the polypeptide is considered particularly useful. Again, such a molecule may be used, for example, as a therapeutic to delay, ameliorate, or treat a neoplasia in a human patient.
In yet another working example, candidate compounds may be screened for those that specifically bind to a polypeptide encoded by a GPR56 and/or Tgm2 gene. The efficacy of such a candidate compound is dependent upon its ability to interact with such a polypeptide or a functional equivalent thereof. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., supra). In one embodiment, a candidate compound may be tested in vitro for its ability to specifically bind a polypeptide of the invention. In another embodiment, a candidate compound is tested for its ability to increase the biological activity of a polypeptide described herein, such as a GPR56 and/or TG2 polypeptide. The biological activity of a GPR56 and/or TG2 polypeptide may be assayed using any standard method, for example, a myelination assay.
In another working example, a nucleic acid described herein ( e.g ., a GPR56 and/or Tgm2 nucleic acid) is expressed as a transcriptional or translational fusion with a detectable reporter, and expressed in an isolated cell (e.g., mammalian) under the control of a heterologous promoter, such as an inducible promoter. The cell expressing the fusion protein is then contacted with a candidate compound, and the expression of the detectable reporter in that cell is compared to the expression of the detectable reporter in an untreated control cell. Products for detecting GPCR activity are commercially available including, for example, the Tango™ GPCR Assay System (Thermo Fisher Scientific, Carlsbad, Calif.). A candidate compound that alters the expression of the detectable reporter is a compound that is useful for the treatment of a demyelinating disease or disorder. Preferably, the compound increases the expression of the reporter.
In another example, a candidate compound that binds to a polypeptide encoded by a GPR56 and/or Tgm2 gene may be identified using a chromatography-based technique. For example, a recombinant polypeptide of the invention may be purified by standard techniques from cells engineered to express the polypeptide (e.g, those described above) and may be immobilized on a column. A solution of candidate compounds is then passed through the column, and a compound specific for the GPR56 and/or TG2 polypeptide is identified on the basis of its ability to bind to the polypeptide and be immobilized on the column. To isolate the compound, the column is washed to remove non-specifically bound molecules, and the compound of interest is then released from the column and collected. Similar methods may be used to isolate a compound bound to a polypeptide microarray. Compounds isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g, by high performance liquid chromatography). In addition, these candidate compounds may be tested for their ability to increase the activity of a GPR56 and/or TG2 polypeptide (e.g, as described herein). Compounds isolated by this approach may also be used, for example, as therapeutics to treat a demyelinating disease or disorder in a human patient. Compounds that are identified as binding to a polypeptide of the invention with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention. Alternatively, any in vivo protein interaction detection system, for example, any two-hybrid assay may be utilized.
Animal models may also be to screen candidate compounds. For example, methods of generating genetically modified animals having mutations (e.g., in GPR56) in organisms are known in the art and available to the ordinarily skilled person. In various embodiments, a CRISPR-Cas9 system is used to create a genetically modified organism (see e.g., US Patent Nos. 8,771,945 and 8,945,839, and US Patent Publication Nos. 20140170753, 20140227787, 20150184139, 20150203872, which are herein incorporated by reference in their entirety). Such organisms may include any eukaryotic organism, including, without limitation, zebrafish and mice. Candidate compounds may be tested for their ability to increase or promote myelination. Tissues of test organisms can be assayed in a number of ways that are routine and well known, including, without limitation, immunohistochemical staining, in situ hybridization, and electron microscopy.
Each of the DNA sequences listed herein may also be used in the discovery and development of a therapeutic compound for the treatment of a demyelinating disease or disorder. The encoded protein, upon expression, can be used as a target for the screening of drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgamo or other translation facilitating sequences of the respective mRNA can be used to construct sequences that promote the expression of the coding sequence of interest. Such sequences may be isolated by standard techniques (Ausubel et ah, supra).
Potential antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acids, and antibodies that bind to a nucleic acid sequence or polypeptide of the invention (e.g., a GPR56 and/or TG2 polypeptide or nucleic acid molecule). Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.
Test Extracts and Agents
In general, agents that modulate (e.g., activate) GPR56 or TQ2!Tgm2 expression, biological activity, or GPR56-dependent signaling are identified from large libraries of both natural products, synthetic (or semi-synthetic) extracts or chemical libraries, according to methods known in the art. Preferably, these compounds increase GPR56 expression or biological activity and/or increase or promote myelination.
Those skilled in the art will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modifications of existing compounds. Numerous methods are also available for generating random or directed synthesis ( e.g ., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from, for example, Brandon Associates (Merrimack, N.H.), Aldrich Chemical (Milwaukee, Wis.), and Talon Cheminformatics (Acton, Ont.)
Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including, but not limited to, Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art (e.g., by combinatorial chemistry methods or standard extraction and fractionation methods). Furthermore, if desired, any library or compound may be readily modified using standard chemical, physical, or biochemical methods.
Methods for Evaluating Therapeutic Efficacy
In one approach, the efficacy of the treatment is evaluated by measuring, for example, the biological function of the treated animal (e.g., neuronal/behavioral function). Such methods are standard in the art and are described, for example, in the Textbook of Medical Physiology, Tenth edition, (Guyton et ak, W.B. Saunders Co., 2000).
Kits
The invention provides kits for the treatment or prevention of a disease or disorder characterized by an undesirable increase in synapses. In one embodiment, the kit includes a composition containing an effective amount of an agent that modulates (e.g., activate) GPR56 or TG2 /Tgm2 expression, biological activity, or GPR56-dependent signaling. In another embodiment, the kit includes a therapeutic or prophylactic composition for increasing or promoting myelination in a subject in need thereof.
In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired an agent of the invention is provided together with instructions for administering the agent to a subject having or at risk of developing a disease or disorder characterized by a deficiency or loss of myelination. The instructions will generally include information about the use of the composition for the treatment or prevention of the disease or disorder. In other embodiments, the instructions include at least one of the following:
description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present invention employs, unless otherwise indicated,
conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second 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);“Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES
Example 1: GPR56 is required for remyelination in vivo
To study the function of microglial GPR56, an established murine model of toxin- induced demyelination was used. A cell-specifically deleted microglial Gpr56 using a Pdgfralpha-CreE- and Pdgfralpha-CreER+ driver was created. The Gpr56fl/fl;Cx3CrlCre/+ and control (WT, Gpr56+/+;Cx3CrlCre/+) mice were crossed with PdgfraCre/ERT mice in a C57BL/6 background that were purchased from Jackson Laboratory (Bar Harbor, ME; Cat# 018280) to obtain Gpr56fi/:fl;PdgfraCreER~ and Ortdόί1^ ;Pdgfr aCreERJ mice. RNAseq experiments using FACS sorted microglia (CD1 lb+, CD45low) from conditional knockout (CKO, Gpr56fl/fl;Cx3CrlCre/+ and control (WT, Gpr56+/+;Cx3CrlCre/+) mouse forebrain confirmed that CKO microglia completely lack Gpr56 transcripts.
The Gpr56II II;I>dpfr a( "reER~ and Gpr56fI/:fl;PdgfrctfPreER+ mice were fed cuprizone, a copper chelator, that causes rapid demyelination and gliosis, or rapid proliferation of glia subtypes. The cuprizone mouse model captures several aspects of muscular sclerosis pathology. FIG. 2 shows representative images of Black-Gold myelin staining of the corpus callosum of Gpr56fl/fl;PdgfraCreER- and Gpr56fl/fl;PdgfraCreER+ mice after cuprizone feeding for 6 weeks followed by recovery for 3 d (3 DR), 7 d (7 DR) and 10 d (10 DR)
(dotted line outlines quantified region. There is less remyelinatation in the corpus callosum at 7 DR and 10 DR in the knock-out mouse panels. The difference in remyelination is quantified in the FIG 2B.
One method to study de- and remyelination in the CNS involves the direct injection of the detergent lysophosphatidylcholine (lysolecithin) into the spinal cord white matter. This procedure produces a well characterized demyelinating injury consisting principally of macrophage/microglial infiltration and activation, reactive astrogliosis, perturbation of axonal homeostasis/axonal injury, and oligodendrocyte precursor cells proliferation and migration. The lesion predictably evolves over the period of a few weeks and is eventually capable of fully remyelinating. This method has been particularly useful in studying the events involved in de- and remyelination.
FIG. 3A-3D show the effects of lysolecithin injection into the corpus collosum of a mouse having a microglial specific knockout of transglutaminase 2. Interestingly, microglia- specific Tgm2 knockout mice had fewer myelinated axons in the lesion compared to control mice. FIG. 5 A and 5B show that GPR56 mRNA levels are reduced following poly I:C injection. Poly FC is a synthetic analog of double-stranded RNA and is a common tool to stimulate immune response in mimicking viral infection. Pregnant dams were injected with poly I:C at embryomic day 12.5 and microglia were isolated from embryonic brains 24 hours later at EI3.5. qPCR showed decreased Gpr56 mRNA in mice received poly I:C, compared to control mice (FIG. 5).
Example 2: Parvalbumin+ Interneurons are Reduced in Mouse Models of Autism
Decreased parvalbumin positive (PV+) intemeurons in the cerebral cortex is one of the pathologies associated with autism. Two mouse models of autism are used here:
prenatal valproate, VP A) or single-gene mutation identified in human patients (Neuroligin-3, NL-3 R451C). Interestingly, both of these models show fewer PV+ intemeurons present in cerebral cortex than control mice.
Decreased PV+ intemeurons in the cerebral cortex is one of the pathologies associated with autism. Two mouse models of autism is used here:
prenatal valproate, VTA) or single-gene mutation identified in human patients (Neuroligin-3, NL-3 R451C) (FIG. 6 A and 6B).
Example 3: Synaptic Defects Present in Autistic Subjects
FIG. 7 shows that pediatric subjects with autism have an increased number of synapses in their brains relative to control subjects. When Gpr56 is deleted in microglia, fewer interneurons are observed in Bin 3 of the cerebral cortex (FIG. 8 A, 8B). Microglial GPR56 regulates synaptic fomiation/pruning. A statistically significant increase in synapse number is observed in microglial Gpr56 deleting mutant, which indicates a defect in synaptic pruning (FIG. 9 A and 9B). The deletion of Gpr56 has behavioral consequences as shown in FIG. 10 A, 10B, and IOC. In particular, Gpr56 knockout mice show behaviors that are typical of autism including obsessive behavior, which manifests in mice as obsessive marble burying (FIG. 10 A), anxiety, which manifests in the mice spending less time in the center of an open field relative to control mice (FIG. 10B), and impaired social interactions (FIG. 10C), which manifests as the mice spending the same amount of time interacting with an inanimate object as with a mouse (social target). Example 4: GPR56 and Alzheimer’s disease
Increased numbers of single nucleotide polymorphisms (SNPs) are observed in GPR56 in subjects with Alzheimer’s disease (FIG. 11). SNPs present in GPR56 are associated with a variety of neurological defects, including multiple sclerosis, Alzheimer’s disease, Autism, and Amyotrophic Lateral Sclerosis (FIG. 12).
These results suggest that agents that increase GPR56 expression or activity (FIG. 13) are useful for the treatment of ASD and other neurological disorders, such as multiple sclerosis, Alzheimer’s disease, Autism, and Amyotrophic Lateral Sclerosis that are associated with defects in GPR56.
Example 5: Microglial GPR56 is necessary for the refinement of synapses
Adhesion G protein-coupled receptor (aGPCR) ADGRG1/GPR56, which controls several aspects of brain development in a cell type-specific manner by mediating cell-cell and cell-matrix interactions, exhibits appropriate properties as a candidate molecule to integrate microglial synapse pruning with other neurodevelopmental events. During embryonic brain development, GPR56 is expressed in neural progenitor cells and migrating neurons and interacts with its extracellular matrix (ECM) ligand collagen III to regulate cortical lamination . In later stages of brain development and throughout life, GPR56 is highly expressed in the major glia: astrocytes, oligodendrocyte lineage cells, and microglia.
Oligodendrocyte precursor cell (OPC) GPR56 functions together with its microglia-produced ligand tissue transglutaminase and ECM component laminin to control developmental myelination and myelin repair. Consistent with these findings, germline homozygous loss of function mutations in GPR56 cause a complex brain malformation whose phenotype includes aberrant cortical architecture and dysmyelination. This phenotype is recapitulated in genetic mouse models indicating conserved GPR56 function.
Gpr56 is highly expressed in microglia from embryonic to adult stages (Fig. 13B). Governed by a super-enhancer {Cell. 2014 Dec 4;159(6): 1327-40), Gpr56 is only expressed in yolk sac-derived microglia but not in microglia-like cells engrafted from fetal liver- and bone marrow-derived hematopoietic stem cells, even after long-term adaptation in the CNS in vivo. Furthermore, Gpr56 expression is promptly lost in primary cultures of microglia. Thus, Gpr56 is one of few genes that defines the microglial lineage and requires both the appropriate ontogeny and environmental cues for its expression. Motivated by the concept that cell-type-specific functions of GPR56 might coordinate multiple sequential and overlapping neurodevelopmental processes, the hypothesis that microglial GPR56 mediates synapse refinement during postnatal life was tested.
Microglia-specific Gpr56 conditional knockout mice were generated by crossing mice harboring a conditional Gpr56^^ allele with Cx3crl-Cre transgenic mice. Though Cx3crl is a promoter for microglia, macrophages, and monocytes, Gpr56 mRNA is not present in macrophages or monocytes, and so this approach effectively deletes Gpr56 in microglia only. Gpr56/! /!; CX3CRl-Cre+/~ were used as conditional knockouts (CKO) and Gpr56+/+;
CX3CRl-Cre+/~ as controls. Cell-type specific deletion was confirmed by western blot (FIG. 15) and qPCR (FIG. 15) using microglia isolated from CKOs and their controls. RNAscope in situ hybridization further showed that Gpr56 mRNA was only absent from Ibal+ microglia in CKO (FIG. 14A). To investigate whether deleting microglial Gpr56 affected microglial cellular properties, we asked whether there were differences between control and CKO microglial cell density, phagocytic activity (defined as %CD68+ cells), coverage area, and morphology in the dLGN of postnatal day 5 (P5) mice (Fig. 14N-A-14N-H). This
comprehensive analysis showed no differences, enabling a functional evaluation of microglial GPR56 in synapse development.
The hypothesis that microglial GPR56 mediates synapse refinement during postnatal life was tested. Microglia-specific Gpr56 conditional knockout mice were generated by crossing mice harboring a conditional GprSfF^ allele with Cx3crl-Cre transgenic mice. Though Cx3crl is a promoter for microglia, macrophages, and monocytes, Gpr56 mRNA is not present in macrophages or monocytes, and so this approach effectively deletes Gpr56 in microglia only. GprSf/111; CX3CRl-Cre+/~ WERE used as conditional knockouts (CKO) and Gpr56+/+; CX3CRl-Cre+/~ as controls. Cell-type specific deletion was confirmed by western blot (Fig. 14M) and qPCR (FIG. 14M) using microglia isolated from CKOs and their controls. RNAscope in situ hybridization further showed that Gpr56 mRNA was only absent from Ibal+ microglia in CKO (Fig. 15 A). To investigate whether deleting microglial Gpr56 affected microglial cellular properties, we asked whether there were differences between control and CKO microglial cell density, phagocytic activity (defined as %CD68+ cells), coverage area, and morphology in the dLGN of postnatal day 5 (P5) mice (Fig. 14N-A -14N- H). This comprehensive analysis showed no differences, enabling a functional evaluation of microglial GPR56 in synapse development.
To test whether microglial GPR56 is implicated in synaptic refinement, synaptic density in the dLGN, which undergoes microglia-mediated synapse refinement between P5- P10, was examined. Structured illumination microscopy (SIM) imaging in the dLGN core (Fig. 14B), to resolve juxtaposed pre- and post-synaptic structures with high resolution. Previous transmission electron microscopy (EM) studies estimated a distance of - 80nm between Homerl and the synaptic cleft and a span from 0nm-200nm between the vGlut2 vesicle pool and the pre-synaptic cleft. In this analysis of 3D reconstructions of the SIM images, a synapse we\as defined when the distance from the center of a Homerl
immunoreactive spot to a vGlut2 surface was between 100 -300 nm. Using this approach, CKO mice had a significantly increased synapse numbers compared to controls (FIG. 14 C and 14D, Fig. 140). Next a time course study was conducted using confocal imaging, which allows rapid analysis of multiple brain regions and time points. An equal retinogeniculate synapse density (vGlut2+/Homerl+) was observed at P0 in CKO mice and their controls, with a steady increase in synapses by -25% and 35% in CKO mice compared to their age- and sex-matched controls, at P5 and P10, respectively (Fig. 14E and 14F). Increased synapse density was not due to altered retinal ganglion cell (RGC) number, since CKO had comparable Bm3a+ RGC density compared to controls (Fig. 14P). Importantly, SIM and confocal analyses yielded comparable synapse density values (Fig. 14D and F), supporting the usage of confocal imaging and analyses for other brain regions and transgenic mice. Consistent with increased vGlut2+/Homerl+ synapse numbers in CKOs, Western blot analysis of microdissected dLGN from P30 animals showed increased vGlut2 protein levels in CKOs as compared to control brains (Fig. 14G and 14H).
To address whether the observed synaptic density increase was a postnatal event, animals were generated that enabled inducible deletion of microglial Gpr56 by crossing (lpr56/l /l mice with Cx3crl-CreER mouse line. (Ipr56/l /l; CX3CRl-CreER+/~ mice were used as inducible conditional knockouts (iCKO), and Gpr56+/+; CX3CRl-CreER+/~ mice were used as controls. Tamoxifen was administered to both iCKO and controls at P1-P3 and brains were analyzed at P10. Comparable increases in retinogeniculate synaptic density in both male and female iCKO mice were observed, in comparison to their age-matched controls, indicating that there is no sexual dimorphism in microglial GPR56 function (Fig. 141).
Furthermore, iCKO and CKO mice showed a quantitatively equivalent synapse phenotype at P10 (Fig. 14F and 141), indicating synaptic phenotype is a postnatal event. Additionally, this result demonstrated that it was appropriate to use CKO for most of the remaining studies.
The dLGN also receives vGlutl projections from neocortical layer VI, modulatory inputs which flexibly tune postsynaptic activity in target cells. In contrast to increased vGlut2 synapses, there were no significant changes in the density of vGlutl+ synapses at P10 (Fig. 14J-L), indicating that microglial GPR56 functions in a synapse-specific manner and does not modulate neocortical inputs in dLGN during the developmental stages investigated. Taken together, these findings demonstrate that microglial GPR56 is necessary for the refinement of retinogeniculate synapses during early development in dLGN.
To address whether microglial GPR56 affected developmental synapse refinement in other brain regions, synaptic density was examined in the hippocampus at P10 and P21. In the hippocampus, Schaffer-collateral axons from CA3 synapse on CA1 pyramidal neurons exclusively on dendritic domains in the stratum (S.) oriens and S. radiatum (35). Perforant- path axons from the entorhinal cortex form synapses on CA1 region pyramidal neurons exclusively on dendritic domains in the S. lacunosum-moleculare, a critical neural circuit for temporal associative memory. Increased synapse densities (vGlut2+/Homerl+) were found at both P10 and P21 in the hippocampal striatum lacunosum-moleculare layer (Fig. 15A-D). However, no change in vGlutl+/Homerl+synapse density was observed at either P10 or P21 in hippocampus striatum radiatum layer (Fig. 15E-H). Taken together, the data demonstrate that microglial GPR56 plays an important role during synapse development in a circuit- dependent manner.
The data so far showed significant increased excitatory inputs in CKO mice, but did not discriminate increased synapse production from decreased synapse pruning. This increase in synapse numbers may have resulted from a decrease in microglial engulfment of synapses. To test this hypothesis, the retinogeniculate system was analyzed because its early-postnatal synaptic pruning has been well-characterized and this synapse refinement process is tractable for mechanistic studies. To address the effect of microglial GPR56 deletion on synapse uptake, in vivo engulfment assays were performed. Mice received intraocular injection of anterograde tracers at P4 and were sacrificed 24 hours later for analysis, as peak pruning occurs around P5 in the murine retinogeniculate system (Fig. 16A). Compared to controls, the amount of RGC material found inside CKO microglia was decreased by 25.7% (Fig. 16B and 16C). This change corresponded in magnitude to the increase in synapses at P5 (Fig. 14D,
14F, and 141) and supports the hypothesis that reduced synapse-element engulfment by G/i/ d-deficient microglia led to the overall increase in synapses.
The functional significance of the increased synapse numbers observed with microglia-specific loss of Gpr56. During early postnatal periods, overlapping inputs from both eyes are remodeled giving rise to eye-specific segregation, resulting in the termination of ipsilateral and contralateral inputs in distinct nonoverlapping domains in the mature dLGN. Defective synaptic pruning of RGC inputs to dLGN leads to incomplete eye-specific segregation. To investigate the effects of microglial GPR56 deficiency on eye-specific segregation, , anterograde tracing of RGC inputs with fluorescent CTB were performed. A significantly larger overlap at P10 in CKO mice was observed (FIG. 160-A-160-C), persisting to P30 (Fig. 16D-G) when eye-specific segregation should be fully accomplished, suggesting improper organization of retinogeniculate projections sustained throughout development.
To evaluate whether the elevated numbers of synapses carry functional consequences, the strength of the synaptic drive from RGCs to dLGN neurons was examined by
electrophysiological recordings. Whole-cell patch-clamp recordings from dLGN neurons were conducted in acute slices from either control or CKO mice at ~P30 (Fig. 16H). When maximal AMPA and NMDA receptor-mediated currents was evoked by stimulation of the optic fibers that project to the dLGN, CKO mice displayed a significant increase in maximal NMDA receptor-mediated current (Fig. 161), consistent with an increase in overall number of retinal inputs onto dLGN relay neurons (Fig. 14C-F). Increased NMDAR1 protein levels in Western blot analysis of microdissected dLGN from P30 CKOs was observed compared to that from controls (Fig. 16J and K). Mean maximal AMPA receptor-mediated currents were comparable between CKO and control mice (Fig. 16L), consistent with a comparable AMPAR protein level on Western blot (Fig. 16M and N). Together, the data show microglial GPR56 is required for proper retinogeniculate circuit organization and function.
How do microglia use GPR56 to identify synaptic elements for removal? PS is a phospholipid that largely resides on the inner leaflet of the plasma membrane under normal conditions. PS extemalization serves as an“eat me” signal for clearance of apoptotic and stressed cells (PNAS October 22, 2013 110 (43) E4098-E4107) as well as outer segment membranes of retinal photoreceptors. PS might flag synapses for removal, based on the observation that PS was externalized on isolated synaptosomes. As BAI1/ADGRB1, another aGPCR family member, recognizes PS, the hypothesis that microglial GPR56 recognizes synapses tagged for removal by binding to PS was tested. GPR56 contains an extensive N- terminal fragment (NTF) followed by a classical seven-transmembrane region (Fig. 17A) Within the long NTF, there are two functional domains, termed
pentraxin/laminin/neurexin/sex -hormone-binding-globulin-like (PLL) and GPCR
autoproteolysis inducing (GAIN) domains recombinant proteins of human immunoglobulin Fc (hFc)-tagged full-length NTF (NTF-hFc) and GAIN-hFc were engineered (Fig. 17B). To determine whether GPR56 binds PS on live cells using flow cytometry analyses of Ba/F3, an IL-3 dependent murine pro-B cell line, that externalizes PS upon calcium ionophore A23187 treatment, a direct binding assay using Alexa Fluor 647 (AF647)-labeled full length NTF, GAIN domain, or hFc was performed (Fig. 17C). FITC -conjugated Annexin V, a known PS- binding protein (53), served as a positive control. Unexpectedly, only the GAIN domain, not the full-length NTF, bound PS in these assays (Fig. 17D). Using a competition assay, in which labeled full-length NTF, GAIN domain, or hFc were used to displace Annexin V binding, it was confirmed that the GAIN domain, but not full length NTF, competed with Annexin V for binding to PS (Fig. 17E). The PLL and GAIN domains are constrained by an interdomain disulfide bond at two cysteine residues C121 and C177. It is conceivable that PLL domain blocks GAIN domain binding to PS.
S4 is an alternatively spliced GPR56 isoform, that initiates at an alternative ATG start codon in exon 4, resulting in a GPR56 variant that contains only the GAIN domain in its extracellular region (FIG. 17J-A). Based on the live-cell PS binding data, GPR56 S4 may be required for microglia-mediated synaptic pruning. Supporting this hypothesis, it was found that Gpr56 S4 is the predominant microglial transcript as determined by qPCR analysis of microglia isolated from P25 WT mouse brains (FIG. 17J-B-17E).
The cortical phenotype of the germline Gpr56 gene-targeted mice was published, before the discovery of Gpr56 splicing variants. At that time, the data suggested that this line represented a null allele. Subsequently, it was characterized as a hypomorphic allele, represented by selective expression of the Gpr56 S4 variant (Fig. 17B-E). In the present report this genetic model is termed Gpr56 S4. To extend the investigation of Gpr56 biology, Gpr56 exons 4-6 were deleted, yielding a global null mutant termed Gpr56 null that lacked both full length GPR56 and its S4 variant (FIG. 17-J-B). Based on the fact that the PLL domain binds collagen III, which is the relevant GPR56 ligand in the developing cerebral cortex, the S4 isoform may not be required for cerebral cortical lamination. Consistent with this hypothesis, a comparable cortical phenotype was observed in Gpr56 S4 and Gpr56 null mice (FIG. 17K). To test whether the GPR56 S4 isoform plays a role in synaptic refinement, retinogeniculate synapses in dLGN of Gpr56 S4 and Gpr56 null mice was examined.
Strikingly, germline Gpr56 null phenocopied microglial Gpr56 CKO, while Gpr56 S4 phenocopied WT mice (Fig. 17F-H). Taken together, these results indicate that the GPR56 S4 isoform regulates microglia-mediated developmental synaptic refinement. To enable the analysis of microglial GPR56-mediated recognition of synaptic PS in vivo , we performed a sequential dual labeling with the PS marker PSVue (selected because of superior diffusion into dLGN as compared with pSIVA) and the anterograde tracer CTB (Fig. 18A and B Fig. 19). PSVue labeled PS-positive RGC inputs to dLGN in WT mice (Fig. 19B). In the developing dLGN, microglial engulfment of synaptic elements peaks at P5 and is dramatically decreased at P10. Dual labeling using PSVue and CTB at P6 and Pl3revealed drastically reduced PSVue-positive RGC inputs in P13 dLGN (Fig. l8C and D), supporting the use of PSVue labeling to detect pre-synaptic inputs flagged for removal.
At P6, retained ipsilateral inputs primarily overlap with contralateral inputs in a restricted region of the dLGN, where eye-specific segregation happens (Fig. 18E). Upon comparing the percentage of PS+ contralateral inputs with PS+ ipsilateral inputs in this overlapping area (the boxed regions in Fig. 18E), significantly more PS+ ipsilateral inputs weRE observed than PS+ contralateral inputs (Fig. 18F and G). These data are consistent with the proposal that PS flags RGC-dLGN synaptic elements targeted for elimination. Of note, there were occasional PS+ cells with condensed nuclei, whose morphology was consistent with that of apoptotic cells. Other PSVue+ signals may represent PS on pre-synaptic inputs from other brain regions or post-synaptic elements of dLGN neurons. Consistent with our hypothesis that microglial GPR56 regulates synaptic pruning by binding to PS, significantly more surviving PS+ RGC inputs in CKO than controls (Fig. 18H and I).
PS labeling and in vivo engulfment assays were combined to extend the
characterization of mechanisms by which microglial GPR56 to mediates synapse engulfment. It was confirmed that microglia engulf PS+ pre-synaptic elements rather than free fluorescent dye, by performing a control experiment using 5-Carboxytetramethylrhodamine (5-TAMRA), the fluorophore component of PSVue550 that does not bind PS. As shown in Fig. 20, very sparse 5-TAMRA signals in microglia were observed compared to PSVue, supporting the validity of our assay. In WT animals, we observed -73% PS+ and 27% PS RGC inputs within microglia (Fig. 21), suggesting microglia preferentially engulf PS+ synapses, although they do also engulf PS synapses. Consistent with a key role for microglial GPR56 in this process, we observed significantly reduced PS+ RGC inputs inside CKO microglia, compared to controls (Fig. 5J-L). In contrast, engulfed PS RGC inputs were not different in CKO as compared to control microglia (Fig. 5M). CKO microglia contained a low level PS+ synapses, suggesting that pathways other than GPR56 can mediate in this process. Together, these results indicate that PS-tagged synapses are preferentially eliminated by microglia partly dependent on microglial GPR56.
Microglial Gpr56 expression is governed by a super enhancer suggesting that it might be implicated in establishing cell identity and core functions {Cell. 153 (2): 307-19 2013; Cell. 2014 Dec 4;159(6): 1327-40). This hypothesis was supported by the finding that Gpr56 expression distinguishes the transcriptomes of microglia as contrasted with hematopoietic stem cells after both cells types have engrafted the intact brain {Neuron. 2018 Jun
27;98(6): 1170-1183). This report provides insight into the functional significance of this expression pattern and its epigenetic regulation by showing that a microglia-specific alternatively spliced GPR56 isoform mediates circuit-specific synaptic pruning in multiple regions of the developing brain. Defective synapse pruning has been implicated in autism spectrum disorder, whereas excessive synapse removal has been linked to schizophrenia. Insight into the mechanisms underlying synapse refinement during development will help decipher neurodevelopmental disorders linked to synapse imbalance. Given GPR56 high expression in adult microglia, the data reported here suggest that microglial GPR56 plays a role in adult synapse homeostasis. Given its multifarious and cell-type specific roles in neurodevelopment {Trends Pharmacol Sci. 2019 Apr;40(4):278-293), further study of GPR56 function in synaptic pruning represents an attractive opportunity to integrate this process into other aspects of brain wiring including myelination. This understanding will promote the objective of enabling therapeutic targeting of GPR56 for combating neurodevelopmental and neurodegen erative disorders.
The results described in Examples 1-4 herein above, were carried out as follows.
Mouse models: Microglial Gpr56 conditional knockout mice were generated by crossing our Gpr56 floxed mice (Giera et ah, 2015) with a Cre driver which is specific for microglia among CNS cells, Cx3crl-Cre where Cre recombinase is present throughout microglial development. For MIA model, poly (I:C) 20mg/kg or carrier solution PBS was injected into pregnant wild type dams at E12.5 and brains were collected at developmental time points, including E14.5, E16.5 and E18.5 and P8.
Interneuron Analysis: The cerebral cortex was stratified into layers II- IV, V, and VI. The laminar localization PV-positive interneurons was quantified in correspondence to their laminar position, i.e., layer II-IV, V, and VI, respectively. Five animals per genotype or per treatment [Poly (I:C) vs PBS injection] were used. Images were acquired by Zeiss LSM 700 laser scanning confocal microscope and quantified blind to genotype.
Immunohistochemistry and microscopic imaging: Brains were dissected and fixed with 4% formaldehyde in PBS overnight, then placed in sucrose solution before freezing. Brains were sectioned at 12 um on slide with a cryostat. Immunohistochemistry was performed followed by confocal microscopic analyses using a Zeiss LSM 700 laser scanning confocal microscope. All analysis was performed blind using ImageJ software.
Behavior: A cohort of 10-15 animals per genotype (or per treatment) was tested. For the social behavior testing, a three-chamber arena apparatus was used. After one day of habituation, 8-weeks old mice were placed in the center and allowed to explore adjacent chambers containing either an inanimate object or a social object (unfamiliar to the mice). Exploration was recorded and analyzed using the Noldus tracking system and a social preference index will be calculated. The repetitive behavior testing was explored using the marble burying test. One week after the social task, mice were acclimated to the environment and then placed in a testing arena containing 20 glass marbles, which were laid out in four rows of five marbles equidistant from one another. A marble burying index was scored. Test results were analyzed blind.
Results described in Example 5 were carried out using the following methods and materials.
Animals
All mice were handled according to protocols approved by Boston Children’s Hospital Animal Care and Lise Committee guidelines for the ethical treatment of animals.
GprSfJ1^ mice were generated as previously described(2). The Cx3Crl-cre (B6J.B6N(Cg)- Cx3crltml l(cre)Jungl , #025524) and Cx3Crl-creER (B6.l29P2(Cg)- ( 'x3criin2 licn' / /<,/ y/"7WganJ, #021160) mice were obtained from Jackson Laboratories.
Considering both Cx3CrlCre and Cx3CrlCreER are knock-in mice, replacing the coding exon of the chemokine receptor 1 (Cx3crl) gene, we crossed these mice with Gpr5f/111 to generate Gpr561111 Cx3Crl -cre(ER) as conditional knockout mice, and Gpr56+/+/ Cx3Crl -cre(ER)+/~ as control. To generate Gpr56 null mice, Gpr56/, /l mice were crossed with CMV-cre mice (JAX stock #006054) (60) to delete exons 4-6, causing a deletion of all splicing variants of Gpr56 in all tissues.. Immunohistochemistry
Mouse brains were collected following PBS perfusion and fixation with 4% PFA, and cryoprotected in 30% sucrose. OCT-embedded tissues were cryosectioned at 14 pm or 40 pm. For synapse immunostaining, l4-pm or 40-pm Sections were incubated with blocking buffer (10% Goat serum + l%BSA; 0.3% TritonX/PBS) for 2 hours and stained with primary antibodies overnight at 4°C (guinea pig anti-vGlut2, 1 : 1000, Millipore AB2251-I; guinea pig anti-vGlutl, 1 : 1000, Millipore AB5905; rabbit anti -Homer 1, 1 :250, Synaptic Systems, 160 003). For microglia staining, 40-pm Sections were incubated with blocking buffer (l%BSA; 0.2% TritonX/PBS) for 2 hours followed by incubation with primary antibodies overnight at 4°C and with fluorophore-conjugated secondary antibodies for 2 hours at room temperature.
Synapse quantification
Confocal microscopy images were obtained with Zeiss LSM 700 System. For synapse quantification in dLGN, medial dLGN slices were used. In each dLGN, three fields of view (5 serial optical sections, 0.5 pm Z-step, 101.5 pm * 101.5 pm, 1024 * 1024 pixel) were acquired in the upper part of the core region using a 63X/1.40 oil objective. Colocalization of vGlut2 or vGlutl and Homerl was quantified as described(<52). The whole process was run in the ImageJ software (NIH, Bethesda, MD). First, each channel’s background was subtracted with a rolling ball radius of 10 pixels. Then thresholding was properly applied to each channel to distinguish synaptic puncta from background, and generate two new binary images. To detect the overlay of pre- and post-synaptic puncta, a logical operation“AND” was performed between these two images. Lastly, the overlaid puncta were counted as synapse using the“analyze particles” function. The full code can be found at:
https://github.com/TaoLi322/Microglia_GPR56_Synapse/blob/master/Synapse- quantification. All images were acquired and analyzed blindly.
For super-resolution imaging of synapse, a modified version of the protocol in Hong et al. (2017) (63) was employed. In brief, imaging was performed using a Zeiss ELYRA PS1 structured illumination microscopy (SIM). Tissue were mounted with Prolong Gold
(Invitrogen P36934) and covered by High Precision Cover Glass (1.5H, Azer scientific). Sections were imaged at 90 nm Z-step using a 100X oil-immersed objective lens. Images were processed using the Zen image software (Carl Zeiss), and then inputted into Imaris for 3D rendering and analysis. The vGlut2 channel was processed using surface rendering function, and Homerl channel was processed with the spot function. A MatLab program “spot to surface” was applied to count the number of adjacent Homerl spots to vGlut2 surface (< 100 nm, 200 nm and 300 nm distance from spot centers to surface).
RNAscope
RNAscope was performed using RNAscope® Multiplex Fluorescent Reagent Kit v2 for fixed and frozen 12um thick sections according to the manufacturer's
instructions. RNAscope® Probe- Mrn -Gpr56 (Cat No. 318241) was used to detect expression of the C-terminal target region of Gpr56 followed by immunohistochemistry for Iba! (1 :400, Wako, 019-19741) In short, after signal amplification step for Gpr56 , sections were permealized using 0.3% Triton-X 100 in PBS for 10 mins, followed by blocking with 10% goat serum, 1% BSA and 0.1% Triton-X 100 in PBS for 1 hr at RT and incubating the primary antibody in the blocking buffer overnight at 4C. Appropriate secondary antibody was used to visualize Ibal expression.
Eye specific segregation
Threshold-independent analyisis of eye-specific segregation was performed as described before(-/2). Mice were anesthetized with isoflurane during the whole procedure. 3ul 0.2% cholera toxin-/? subunit (CTB) conjugated Alexa 488 dye (CTB488, Life Technologies, C22841) was intravitreally injected into the left eye, and CTB conjugated Alexa 647 dye (CTB647, Life Technologies, C34778) into the right eye of P30 mice. 24 hours later, mice were transcardially perfused with PBS and fixed with 4% PFA, and cryoprotected in 30% sucrose. The injection/labeling efficiency was checked by visualizing retinas and superior colliculus (Fig. S6C). Brains were cryosectioned coronally at 60 pm and mounted with Fluoromount-G (SouthernBiotech). Since R-value varies much from cranial to caudal LGN(43), we chose three to four dLGN (420 to 600 pm from caudal LGN) as indicated in (Fig. S6D) for analysis. 16-bit images were digitally acquired at 20X with a CCD camera (Nikon eclipse Ti). The same exposure time and gains were used for each channel across all samples. For P30 mice, images were analyzed in a threshold-independent way. First, background fluorescence values were calculated from non-LGN regions, and used to be subtracted from images. Then images were normalized by adjusting the contrast to 5% saturation. For each pixel, R-value was computed as R = logio(Fipsi/ FCOntra), where Fipsi or F contra is the fluorescence intensity of the pixel in ipsilateral or contralateral channel, respectively. Those pixels whose values equal to zero were excluded. Over 99.9% of R- values ranged between -3 to 2, and they were plotted as a distribution graph. The variance of R-values was computed to quantify the extent of segregation. The greater variance means more segregation between ipsilateral and contralateral RGC inputs. The full code can be found here: https://github.com/TaoLi322/Microglia_GPR56_Synapse/blob/master/Eye- Seg_R- value.
For P10 mice, lul of 0.2% CTB488 and CTB594 (Life Technologies, C22842) were injected into the left or right eye, respectively. The percentage of overlapped left and right eye projections in dLGN was quantified using a multi -threshold quantitative method, as described before(-/2).
LGN slice preparation and electrophysiology
Mice (P28-P34) were decapitated; their brain was removed and placed in a 4°C choline solution, containing: 78.3 mM NaCl, 23 mM NaHC03, 23 mM glucose, 33.8 mM choline chloride, 2.3 mM KC1, 1.1 mM NaH2P03, 6.4 mM MgCh, and 0.45 mM CaCh. The two hemispheres were then separated by an angled cut (3°-5°) relative to the cerebral longitudinal fissure. The medial aspect of the right hemisphere was then glued onto an angled (20°) agar block, and 250 pm LGN slices were cut in ice cold, choline solutions using a vibratome (Leica VT1200S) based upon previously described protocols Slices were allowed to recover at 33° C for 30 minutes in choline solution and then for an additional 30-50 minutes in artificial cerebral spinal fluid (ACSF). ACSF contained: 125 mM NaCl, 25 mM NaHC03, 25 mM glucose, 2.5 mM KC1, 1.25 mM NaH2P03, 1 mM MgCh, and 2 mM CaCl2. All solutions were continuously supplied with oxygen (95% 02/5% C02).
Whole-cell voltage-clamp recordings of neurons from the dLGN were conducted using a Multiclamp 700B amplifier, digitized with a Digidata 1440A, collected with Clampex 10.2, and analyzed using Clampfit 10.2 (Axon Instruments). The slice containing dLGN was perfused with oxygenated ACSF, supplemented with 50 pM picrotoxin. Neurons were then patched using glass electrodes (3-4 MW) filled with 35 mM CsF, 100 mM CsCl, 10 mM EGTA, 10 mM HEPES, and 0.1 mM D600 (methoxyverapamil hydrochloride). Internal solution has a pH of 7.32 with CsOH. ACSF filled glass pipettes were placed in the optic tract, and these stimulating electrodes were moved repeatedly until reaching the location that gave the largest postsynaptic response. The maximum AMPA and NMDA currents were then determined by gradually increasing the stimulus intensity from 0.1 up to 1 mA. The maximum response size was considered to be the amplitude of the response after 3 consecutive increases in stimulation intensity failed to result in a larger response, or if the response amplitude decreased with an increase in stimulation intensity. AMPA responses were collected at -70 mV and NMDA responses at +40 mV, and were collected with alternate stimulations. To confirm that the optic tracts and not cortical inputs were stimulated, paired pulse data with a 50 ms interstimulus interval were collected, given that optic inputs usually demonstrate paired pulse depression, and cortical inputs paired pulse facilitation. No difference in paired pulse depression was seen between control and conditional knockout mice (Fig. 22A and B, p = 0.694, Student’s t-test).
Western blot of dLGN
Mice (P30) were anesthetized and decapitated. Mouse brains were immediately removed into ice-cold HBSS and coronally sectioned at 100 p using vibratome. DLGN were dissected out under dissection microscope, and homogenized in proteinase inhibitor-added RIPA buffer. After 30min incubation on ice, samples were centrifuged at l4,000xg for lOmin. Supernatant were collected and used for WB of NMDAR1 (anti-NRl, 1 :2000, Sigma-Aldrich 05-432) and vGlut2 (anti-vGlut2, 1 :5000, Millipore AB2251-I). For WB of GluRl (anti-GluRl, 1 : 1000, Cell signaling 13185S), supernatant were boiled for 5min before loading.
In vivo engulfment
In vivo engulfment assay was carried out as previously described!/ 7). Briefly, P4 mice were given an intraocular injection of 0.5m1 0.5% CTB594 into each eye 24 hours prior brain collection. 40pm cryosectioned were obtained for the assay. 2-3 sections of medial dLGN were chosen from each mouse for further free-floating immunostainmg with Ibal antibody. Images were taken on a UltraView Vox spinning disk confocal microscope at 60X with 0.2pm z-step or on a Zeiss LSM700 confocal microscope at 63X with 0.2 pm z-step. ImageJ (NUT) and Imaris (Bitplane) were used for image processing. 3D reconstruction of microglia and RGC inputs were created by surface rendering in Imaris. To evaluate microglial engulfment ability, the percentage of engulfment was calculated as: volume of internalized RGC inputs / volume of each microglia. Generation of GPR56 fusion proteins and recombinant GAS6
Human IgG Fc tagged constructs were generated by PCR. For GPR56 NTF-hFc, the PCR forward primer is: 5’-CCATGGAAGACTTCCGCTTCTGTGGCC-3’, the reverse primer is: 5’ -CAGATCTC AGGTGGTTGCAC AGGC AGG-3’ . For GPR56 GAIN-hFc, the PCR forward primer is: 5’-ATCCATGGTTATGTG TGATCTCAAGAAGGAATTGC-3’, the reverse primer is: 5’ -TCTAGATCTC AGGTGGTTGCAC AGGC AGG-3’. Then PCR product was inserted into pFUSE-hIgG2-Fc2 (Invivogen, Cat. pfuse-hfc2) vector between Ncol and BglII sites. For the expression of GPR56 fusion proteins, the above constructs were transiently transfected into HEK-293T cells (ATCC). 24 hours later, the culture media was changed to serum-reduced Opti-MEM (Gibco, 31985070). During incubation, GPR56 fusion proteins would be secreted into the culture media. This conditioned media containing fusion proteins was harvested 48-72 hours later, and concentrated as previously described (ShiHong Li et al, 2008 Journal of neuroscience). The proteins were purified using HiTrap protein A column (GE Healthcare, 17040303).
Flow cytometry
GPR56 NTF-hFc, GAIN domain-hFc, or hFcwere labeled with Alexa Fluor 647 (AF647) using the Alexa Fluor 647 NHS Ester labeling kit (ThermoFisher Scientific, Cat #A20006) according to the manufacturer’s protocol. Briefly, 75 mM protein was incubated with 300 mM AF647 at room temperature for 4 hours, followed by 200 mM Tris to quench the reaction. The whole reaction mixture was run through a Sephadex G-50 column and protein-dye conjugate was collected. Dye labeling efficiency for each protein ranged from 1.44-1.46 moles of dye per mole of protein.
Ba/F3 cell line was cultured and passaged as described (66). It was maintained in RPMI-1640 media with L-Glutamine (Invitrogen, Cat #11875093) supplemented with 10% FBS, Penicillin-Streptomycin, and kept at 37°C, 5% CQ2 in a humidified incubator. Ceils were harvested and tvashed twice with ice-cold Hank’s Balanced Salt Solution (HESS) and resuspended in HBSS supplemented with 2 mM Ca2". Cells were treated with 1 mM calcium ionophore A23187 (Mil!ipore Sigma, Cat #C7522) at 37°C for 15 minutes, with gentle agitation every 5 minutes, followed by washing with HBSS. To measure direct binding, cells were resuspended in 0 9% Nad and incubated with 1 mM protein- Alexa Fluor 647 conjugate at room temperature for 2 hours. After binding, cells were washed once and resuspended in HBSS, followed by flow cytometry on an LSRFortessa (BD Biosciences). For the competition assay, ceils were treated with A23187 as above and washed with HBSS. Next, cells were resuspended in 100 mI_ staining buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and incubated with 1 mM AF647 conjugated NTF, GAIN, or hFc and 50 nM Annexin V-FITC for 1 hour at room temperature. Cells were washed once with HBSS, followed by flow cytometry on an LSRFortessa (BD Biosciences). Ba/F3 cell line was a gift from the Ma alis laboratory at Massachusetts institute of Technology.
In vivo phosphatidylserine labeling
P5 mice were given an intraocular injection of CTB647 into both eyes. Twenty four hours later, mice were mounted on a neonatal mice adaptor (RWD Life Science, #68072), and given an intracranial injection of various labeling probes. The stereotaxic setting was -1.5 mm anteroposterior, 1.09 mm ediolaterai and -2.15 mm dorsoventral to lambda. 2m1 PSVue(20pM in TES buffer) or pSIVAfl :2, 2mM, lOrnM, 20mM Ca2+ in HBSS) were injected into the gap between hippocampus and dLGN via a 33G needle attached Hamilton syringe at 0.33pl/min (see Supplemental figure 5). Six or 24 hours later, brains were collected freshly, and sectioned at 180mhi in ice-cold TES buffer or ImM Ca2 HBSS on the vibratome (Leica VT1000 S). Two to three sections with medial dLGN were chosen, and directly mounted on homemade framed (concave, 200mhi depth) glass slides. The images were taken immediately within an hour on a Zeiss LSM700 confocal microscope at 63X.
Statistical Analysis
For all quantification, images were acquired blindly to genotype before quantification. All data are shown as mean ± SD or mean ± SEM as indicated in figure legends. Asterisks indicate significance: ****R<0.0001, ***P<0.00l, **P<0.0l, *P<0.05. All effects of genotype were analyzed by Student’s t-test, multiple student’s t-test, one-way ANOVA, or two-way ANOVA (Graph Pad Software, Inc).
Other Embodiments
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:
1. A method of promoting synaptic pruning in a neuronal tissue, the method comprising contacting the tissue with an agent that activates GPR56, thereby promoting synaptic pruning.
2. A method for treating a disease or disorder characterized by a loss or reduction in GPR56 expression or activity or an undesirable increase in synapse number, the method comprising administering to a subject in need thereof an agent that activates GPR56, thereby treating the disease or disorder.
3. The method of claim 1 or 2, wherein the agent is a small compound, polypeptide, or polynucleotide.
4. The method of claim 3, wherein the small compound is a gedunin- and khivorin compound.
5. The method of claim 4, wherein the compound is selected from the group consisting of 3- alpha-acetoxydihydrodeoxygedunin, khivorin, 7 synthetic peptide agonist, 3- deacetylkhivorin, deoxygedunin, and l,2-Epoxygedunin
6. The method of claim 3, wherein the polypeptide is transglutaminase 2 (TG2) polypeptide or fragment thereof or GPR56 ligand comprising amino acids 383-404 of GPR56
7. The method of claim 6, wherein the GPR56 ligand comprises the amino acid sequence TYFAVLMVS.
8. The method of claim 6, wherein the GPR56 ligand comprises the amino acid sequence TYFAVLMVS SVEVD AVHKHYLS .
9. The method of any one of claims 1-4, wherein the agent is a GPR56 ligand that is covalently linked to a lipid or transmembrane domain.
10. The method of claim 8, wherein the N-terminus or C-terminus of the GPR56 ligand is covalently linked to the lipid or transmembrane domain.
11. The method of claim 1 or 2, wherein the polypeptide is a TG2 polypeptide or fragment thereof comprises amino acids 465-687 of TG2.
12. The method of claim 11, wherein the polypeptide is a TG2 polypeptide or fragment thereof that forms one or more beta barrel domains.
13. The method of claim 1 or 2, wherein the disease or disorder is an Autism spectrum disorder, multiple sclerosis, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis
14. A method of promoting synaptic pruning in a neuronal tissue, the method comprising contacting the tissue with an S4 isoform of GPR56, thereby promoting synaptic pruning.
15. A method for treating a disease or disorder characterized by a loss or reduction in GPR56 expression or activity or an undesirable increase in synapse number, the method comprising administering to a subject in need thereof an S4 isoform of GPR56, thereby treating the disease or disorder.
16. The method of claim 15, wherein the disease or disorder is an Autism spectrum disorder, multiple sclerosis, Alzheimer’s disease, and Amyotrophic Lateral Sclerosis.
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CN113999873A (en) * 2021-12-31 2022-02-01 北京市疾病预防控制中心 Construction method and application of genetically modified non-human animal

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WO2017172945A1 (en) * 2016-03-31 2017-10-05 Children's Medical Center Corporation Compositions and methods for oligodendrocyte development

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WO2017172945A1 (en) * 2016-03-31 2017-10-05 Children's Medical Center Corporation Compositions and methods for oligodendrocyte development

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DATABASE UniProtKB [online] 15 March 2004 (2004-03-15), "Adhesion G-protein coupled receptor G1", Database accession no. AGRG1_HUMAN *
STOVEKEN ET AL.: "Gedunin- and Khivorin- Derivatives are Small-Molecule Partial Agonists for Adhesion G Protein-Coupled Receptors GPR56/ADGRG1 and GPR114/ADGRG5", MOLECULAR PHARMACOLOGY, vol. 93, no. 5, May 2018 (2018-05-01), pages 477 - 488, XP055676790, DOI: 10.1124/mol.117.111476 *

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