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WO2005047530A2 - Method for assaying compounds that modulate the activity of protein tyrosine phosphatase receptor zeta (ptprzeta) - Google Patents

Method for assaying compounds that modulate the activity of protein tyrosine phosphatase receptor zeta (ptprzeta) Download PDF

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Publication number
WO2005047530A2
WO2005047530A2 PCT/US2004/037514 US2004037514W WO2005047530A2 WO 2005047530 A2 WO2005047530 A2 WO 2005047530A2 US 2004037514 W US2004037514 W US 2004037514W WO 2005047530 A2 WO2005047530 A2 WO 2005047530A2
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Prior art keywords
compound
ptprζ
agent
activity
bodily sample
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PCT/US2004/037514
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French (fr)
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WO2005047530A3 (en
Inventor
Steven A. Goldman
Fraser J. Sim
Karen Chandross
Jean Merrill
Sridaran Natesan
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Aventis Pharmaceuticals Inc.
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Publication of WO2005047530A2 publication Critical patent/WO2005047530A2/en
Publication of WO2005047530A3 publication Critical patent/WO2005047530A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

Definitions

  • the present invention relates to a method for determining whether a compound or agent modulates the activity of PTPR ⁇ . Such compounds may readily have applications in treating and/or preventing demylinating diseases or disorders.
  • BACKGROUND OP THE INVENTION Signals sent from Central Nervous System propagate to the periphery of the body via neurons. An action potential passes from one neuron to another, and thus the signal is transmitted.
  • glial cells such as Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system, synthesize myelin, which wraps around axons to form the myelin sheath.
  • the myelin sheath performs the same function as insulation around an electrical wire, i.e. insulation for the nerve cell.
  • the main functions of the myelin sheath are to allow for fast saltatory nerve conduction through the clustering of sodium channels at the nodes of Ranvier, to maintain the nodal regions, and for trophic support to neuronal axons.
  • the myelin sheath surrounding neuronal axons is damaged or destroyed. Consequently, the action potential passing through demyelinated axons is not quickly or efficiently transmitted. Rather, the action potential leaks from the axon, and becomes attenuated.
  • MS Multiple Sclerosis
  • peripheral sensations such as touch, vision, and balance, can be detrimentally affected. These affects are not necessarily chronic. • Rather, they can occur in "attacks" periodically.
  • the attacks are mild, while in others, the attacks can be severe and debilitating, resulting in sensory and/or motor deficits and paralysis.
  • Other demeylinating diseases or disorders include Charcot-Marie- Tooth disease, Pelizaeus-Merzbacher disease, encephalomyelitis, neuromyelitis optica, adrenoleukodystrophy, Guillian-Barre syndrome, and instances in which myelin forming glial cells are damaged, including spinal cord injury, neuropathies, and other types of nerve injuries.
  • oligodendrocyte progenitor cells are present in the CNS and around MS lesions and up to 70% of lesions show some evidence of remyelination, this appears to be a transient and incomplete process since only limited long term repair of the myelin sheath has been observed.
  • Traditional methods for treating the disease are principally concerned with controlling the number of attacks by suppressing the immune system, and/or ameliorating the effects or duration of an attack with the administration of corticosteroids. Unfortunately, these treatments merely ameliorate the progression of the disease, and neither reverse the progression of the disease, nor restore the nervous system or its condition prior to the onset of the disease.
  • PTPR ⁇ Protein Tyrosine Phosphatase Receptor zeta
  • PTPR ⁇ is also known as PTP ⁇ , RPTP ⁇ , HPTP ⁇ , and PTB18.
  • PTPR ⁇ is expressed on the surface of oligodendrocyte progenitor cells.
  • pleitrophin In the In the PTPR ⁇ pathway, pleitrophin, a growth and differentiation factor and a natural ligand for PTPR ⁇ , binds to and functionally inactivates the constitutive intracellular phosphatase activity of PTPR ⁇ , resulting in an increase in tyrosine phosphorylation of a molecule called ⁇ -catenin.
  • phosphorylated ⁇ -catenin stimulates a transient and limited number of cell divisions that lead to the initiation of differentiating pathways that result in the formation of mature oligodendrocytes.
  • modulating the activity of PTPR ⁇ or the PTPR ⁇ pathway may cause the differentiation of these immature cells into oligodendrocytes, which then may remediate the damage that occurs in a demyelinating disease or disorder through enhanced remyelination.
  • the present invention extends to a method for determining whether a compound or agent modulates activity of an PTPR ⁇ , or its pathway, comprising the steps of contacting the compound or agent with the PTPR ⁇ , and then determining whether the compound or agent has interacted with or bound to PTPR ⁇ .
  • the PTPR ⁇ may optionally be immobilized on a substrate, or alternatively, the compound or agent may be immobilized on a substrate.
  • Numerous subst ⁇ tes have applications in a method of the present invention. Particular examples include polyvinylidene difluoride and nitrocellulose, to name only a few.
  • the compound can be detectably labeled, the PTPR ⁇ , can be detectably labeled, both PTPR ⁇ and the compound can be detectably labeled, or a relevant pathway member of the PTPR ⁇ can be detectably labeled.
  • detectable labels have applications in a method of the present invention and are discussed infra. Particular examples include, but are not limited to radioactive isotopes, chemicals that fluoresce, chemicals that phosphoresce, biotin/avidin, etc. Particular examples are described infra.
  • PTPR ⁇ from a variety of sources, e.g. equine, bovine, canine, feline, human, primate, ovine, rodent, avian, etc. have applications in a method of the present invention.
  • the PTPR ⁇ is human, and comprises the amino acid sequence of FIG. 2 (SEQ ID NO:l).
  • the present invention extends to a method for determining whether a compound or agent modulates activity of an PTPR ⁇ , or its pathway, wherein one compound or agent at a time is evaluated.
  • a method of the present invention can be perfo ⁇ ned in a high throughput fashion.
  • the present invention extends to a method for determining whether a compound or agent modulates activity of a PTPR ⁇ or its pathway.
  • the compound or agent is contacted with a sample of brain white matter containing progenitor cells. This sample is then evaluated for a change in the number of oligodendrocytes, the extent of oligodendrocyte differentiation, the amount of myelin produced, and/or the extent of PTPR ⁇ activity or relevant pathway activity. Such results would be indicative that the compound or agent modulated the activity of the PTPR ⁇ .
  • progenitor cells that express the PTPR ⁇ can be either in vivo or ex vivo. Moreover, should the progenitor cell be in vivo, the step of contacting the compound or agent with the white matter or post-natal progenitor cell that expresses the PTPR ⁇ comprises administering the compound or agent to the organism.
  • the present invention extends to a method for determining whether a compound or agent modulates activity of an PTPR ⁇ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the number of oligodendrocytes that express PTPR ⁇ within the bodily sample is known; (b) measuring the number of oligodendrocytes that express PTPR ⁇ , after contact with the compound or agent. If the number oligodendrocytes that express PTPR ⁇ , in the bodily sample after contact with the compound or agent is greater than the number prior to contact, it is indicative that the compound or agent may modulate activity of the PTPR ⁇ .
  • the present invention further extends to a method for determining whether a compound or agent modulates activity of an PTPR ⁇ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an ' organism, wherein the number of oligodendrocyte progenitor cells that express PTPR ⁇ within the bodily sample is known; (b) measuring the number of oligodendrocyte progenitor cells that express PTPR ⁇ , after contact with the compound or agent.
  • the number oligodendrocyte progenitor cells that express PTPR ⁇ , in the bodily sample after contact with the compound or agent is greater than the number prior to contact, it is indicative that the compound or agent may modulate activity of the PTPR ⁇ .
  • the present invention extends to a method for determining whether a compound or agent modulates activity of an PTPR ⁇ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the extent of myelination in the sample is known; and (b) determining the extent of myelination in the bodily sample after contact with the compound or agent.
  • the present invention further extends to a method for determining whether a compound or agent modulates activity of an PTPR ⁇ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the extent of myelination in the sample is known; and (b) determining the extent of myelination in the bodily sample after contact with the compound or agent.
  • the present invention also extends to a method for determining whether a compound or agent modulates activity of an PTPR ⁇ comprising the steps of:
  • the present invention further extends to a method for determining whether a compound or agent modulates activity of an PTPR ⁇ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the PTPR ⁇ activity or activity of a relevant member of the PTPR ⁇ pathway in the sample is known; and (b) determining the activity of PTPR ⁇ or the activity of the relevant member of the PTPR ⁇ pathway in the bodily sample after contact with the compound or agent.
  • PTPR ⁇ activity or activity of the activity of the relevant member of the PTPR ⁇ pathway in the bodily sample after contact with the compound or agent is greater than that observed prior to contact, it is indicative that the compound or agent may modulate PTPR ⁇ .
  • the sample can be taken from a variety of organisms, e.g. ovine, bovine, feline, rodent, equine, primate, human, rodent, etc.
  • the sample is white matter taken from the brain of the organism, e.g. a human.
  • FIG. 1 Schmatical view of the PTPR ⁇ pathway.
  • FIG: 2 Amino Acid sequence of human PTPR ⁇ (SEQ ID NO: 1).
  • FIG. 3 is a histogram of TAQMAN results showing that the level of RPT ⁇ transcription is higher in human fetal ventricular zone (vz), a region where progenitor cells are enhanced, than the cortex (ex), where mature cells reside, and is enriched in human oligodendrocyte progenitor cells.
  • FIG. 4 graphical presentation of TAQMAN results that show human PTPR ⁇ mRNA is not detected in adult human peripheral organs.
  • the present invention is based upon the novel discovery that PTPR ⁇ is expressed on the surface of progenitor cells within the human central nervous system. Hence, modulating the activity of PTPR ⁇ or the PTPR ⁇ pathway may cause the differentiation of these cells into oligodendrocytes, which then, through enhanced remyelination, may remediate the demyelination that occurs in a demeylinating disease or disorder.
  • the present invention extends to a method for determining whether a compound or agent modulates activity of an PTPR ⁇ , or its pathway, comprising the steps of contacting the compound or agent with the PTPR ⁇ , and then determining whether the compound or agent binds to the PTPR ⁇ ,.
  • Modulation occurs when, as a result of the compound's or agent's binding to PTPR ⁇ , increased numbers of oligodendrocyte progenitor cells observed, increased numbers of oligodendrocytes is observed, an increase in the extent of oligodendrocyte differentiation or amount of myelin produced is observed, or a combination thereof.
  • pleitrophin a growth and differentiation factor and a natural ligand for PTPR ⁇ , binds to and functionally inactivates the constitutive intracellular phosphatase activity of PTPR ⁇ , resulting in an increase in tyrosine phosphorylation of a molecule called ⁇ -catenin.
  • phosphorylated ⁇ -catenin stimulates a transient and limited number of cell divisions that lead to the initiation of differentiating pathways that result in the formation of mature oligodendrocytes.
  • FIG. 1 A schematical view of the PTPR ⁇ pathway is set forth in FIG. 1
  • the terms "compound” or “agent” refer to any composition presently known or subsequently discovered.
  • compounds or agents having applications herein include organic compounds (e.g., man made, naturally occurring and/or optically active), peptides (man made, naturally occurring, and or optically active, i.e., either D or L amino acids), carbohydrates, nucleic acid molecules, etc.
  • enzyme refers to a biomolecule, such as a protein or RNA, that catalyzes a specific chemical reaction. It does not affect the equilibrium of the catalyzed reaction. Rather, the enzyme enhances the rate of reaction by lowering the energy of activation.
  • relevant pathway member refers to genes known or yet to be identified that are downstream from PTPR ⁇ and that ultimate lead to the function consequences of the activated PTPR ⁇ receptor, such as proliferation, migration or differentiation.
  • the PTPR ⁇ can be detectably labeled, the compound or agent can be detectably labeled, or even both can be detectably labeled.
  • Suitable labels include enzymes (e.g.
  • fluorophores e.g., fluorescene isothiocyanate (F1TC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially Eu 3+ , to name a few fluorophores
  • F1TC fluorescene isothiocyanate
  • PE phycoerythrin
  • TR Texas red
  • rhodamine free or chelated lanthanide series salts, especially Eu 3+ , to name a few fluorophores
  • chromophores e.g., radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (e.g., biotin), and che iluminescent agents.
  • the same or different labels may be used for the receptor and control marker.
  • radioactive label such as the isotopes 3 H, 14 C, 32 P, 35 S, 3 ⁇ Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, I31 I, and 186 Re
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • Direct labels are one example of labels which can be used according to the present invention.
  • a direct label has been defined as an entity, which in its natural state, is readily visible, either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e.g. U. V. light to promote fluorescence.
  • colored labels include metallic sol particles, for example, gold sol particles such as those described by Leuvering (U.S. Patent 4,313,734); dye sole particles such as described by Gribnau et al. (U.S. Patent 4,373,932) and May et al.
  • direct labels include a radionucleotide, a fluorescent moiety or a luminescent moiety.
  • indirect labels comprising enzymes can also be used according to the present invention.
  • enzyme linked immunoassays are well known in the art, for example, alkaline phosphatase and horseradish peroxidase, lysozyme, glucose-6- phosphate dehydrogenase, lactate dehydrogenase, urease, these and others have been discussed in detail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70. 419-439, 1980 and in U.S. Patent 4,857,453.
  • labels for use in the invention include magnetic beads or magnetic resonance imaging labels.
  • PTPR ⁇ may be immobilized on a substrate.
  • substrates include but certainly not limited to polyvinylidene difluoride (PVDF) and nitrocellulose, to name only a few.
  • PVDF polyvinylidene difluoride
  • nitrocellulose to name only a few.
  • the compound or agent may be immobilized on such a substrate.
  • high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries" are then screened with a method of the present invention to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • Combinatorial Chemical Libraries are a particular means to assist in the generation of new chemical compound leads.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, one commentator has observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (Gallop et al., J. Med. Chem., 37(9): 1233-1251 (1994)).
  • combinatorial chemical libraries include, but are not limited to peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88).
  • Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but certainly are not limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec.
  • Patent 5,593,853 small organic molecule libraries
  • small organic molecule libraries see, e.g., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33, isoprenoids U.S. Patent 5,569,588, thiazolidinones and metathiazanones U.S. Patent 5,549,974, pyrrolidines U.S. Patents 5,525,735 and 5,519,134, morpholino compounds U.S. Patent 5,506,337, benzodiazepines 5,288,514, and the like).
  • High throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
  • oligodendrocytes the myelin forming cells in the CNS
  • axons are damaged, resulting in severely impaired neuronal function and physical deficits.
  • Newly formed oligodendrocyte progenitor cells are present in and around damaged tissue, suggesting the possibility of self-repair.
  • only limited repair is observed.
  • the resulting data were analyzed by focusing on a number of key pathways implicated in neuronal and glial development. Genes were grouped and visualized as expression maps that profiled correlations between pathway-related genes that were co- regulated with defined clusters of marker genes.
  • Primary rat oligodendrocyte progenitor cells are obtained from the neocortex of newborn (postnatal days 2-3) rats or mice and are enriched, after removal of microglia, by mechanical separation from the astrocytic monolayer using a modification of the technique originally described by McCarthy and de Vellis (1980, J. Cell Biol. 85:890- 902).
  • the meninges are removed and the brain tissue is mechanically dissociated.
  • Cells are plated onto T75 flasks and fed with DMEM/F12 + 10% FBS.
  • Oligodendrocytes that grow on the astrocyte bed layer are collected by "gentle shaking" fourteen days after the original prep date.
  • the suspension is centrifuged and the cell pellet is resuspended in serum free media (SFM; DMEM combined with 25 ⁇ g/ml transferring, 30 nM triiodothyronine, 20 nM hydrocortisone, 20 nM progesterone, 10 nM biotin, lx trace elements, 30 nM selenium, 1 ⁇ g/ml putrescine, 0.1% BSA, 5 U/ml PenStrep, 10 ⁇ g/ml insulin) supplemented with the following growth factors: Platelet derived growth factor-AA (PDGF) and fibroblast growth factor-2 (FGF).
  • PDGF Platelet derived growth factor-AA
  • FGF fibroblast growth factor-2
  • the trypsin is neutralized and the cell suspension is centrifuged at 900g for 5 min.
  • the enriched rat Central Glia-4 (CG4) progenitor cell line may be used, which is maintained in base media (DMEM, with 2 mM glutamine, lmM sodium pyruvate, biotin (40 nM), insulin (1 ⁇ M) and Nl) supplemented with 30% conditioned media from the B-104 neuroblastoma cell line.
  • base media DMEM, with 2 mM glutamine, lmM sodium pyruvate, biotin (40 nM), insulin (1 ⁇ M) and Nl
  • base media fetal calf serum
  • insulin 500 nM
  • A2B5 and MBP immunoreactivity is used to confirm >95% enrichment in immature and mature cultures, respectively.
  • Human neurospheres (collected from E19.5 - E22 human embryo cortex) are cultured for 2 weeks in progenitor media: DMEM/F12 containing 100 ⁇ g/ml transferring, 30 nM triiodothyronine, 20 nM hydrocortisone, 20 nM progesterone, 10 nM biotin, lx trace elements, 30 nM selenium, 60 uM putrescine, 0.1% BSA, 5 U/ml PenStrep, 25 ⁇ g/ml insulin) supplemented with PDGF and FGF.
  • progenitor media DMEM/F12 containing 100 ⁇ g/ml transferring, 30 nM triiodothyronine, 20 nM hydrocortisone, 20 nM progesterone, 10 nM biotin, lx trace elements, 30 nM selenium, 60 uM putrescine, 0.1% BSA, 5 U/ml PenStre
  • the neurospheres are then dissociated with 20 U/ml papain at 37°C for 30-50 min.
  • Proliferating cultures a. The next day, the old medium is replaced with new medium containing the compound or agents and mitogens. b. Compound or agent dose response evaluations are performed at 6 different concentrations (10 ⁇ M, 1 ⁇ M, 100 nM, lOnM, 1 nM, and 0.1 nM). c. Triplicates wells are run for each compound or agent concentration.
  • oligodendrocyte-specific antibodies are used to assess ability of compounds or agents to accelerate/promote oligodendrocyte differentiation . . . (for example, 04, 01, or myelin basic protein immunoreactivity changes over time between compound or agent treated and untreated cultures).
  • Cells are plated onto poly-D-lysine treated 4-well chamber slides at 5xl0 3 to 20xl0 3 cells/well and grown as described above. Sequential staining is performed on oligodendrocyte populations with increasing degrees of cellular differentiation, as determined by days in vitro without PDGF and FGF.
  • oligodendrocyte stage specific cell surface marker expression including A2B5, 04, and 01.
  • oligodendrocyte stage specific marker expression including myelin basic protein, MBP.
  • Non-specific binding sites are blocked with 5-10% goat serum in antibody dilution buffer (0.1% Triton-X 100 and 1% IgG-free bovine serum albumin; also used to dilute antibodies) for 15 min at room temperature. 7. Primary antibodies are added and cells are incubated overnight with gentle rocking at 4° C.
  • Cell nuclei are stained with 4,6-dia idino-2-phenylindole (DAPI) for 15min at room temperature.
  • DAPI 4,6-dia idino-2-phenylindole
  • the following conditions are compared over time and at different compound or agent doses: mitogen-treated, serum free media (SFM) treated, SFM-IGF1 treated, mitogen and compound or agent treated, SFM and compound or agent treated.
  • SFM serum free media
  • BrdU immunostaining is used to confirm that compounds or agents do not promote cell proliferation.
  • Oligodendrocyte progenitor cells are labeled with 10 ⁇ M BrdU for 20 hr and then fixed with either 70% ethanol or 4% paraformaldehyde.
  • the cells are incubated successively with biotinylated mouse anti-BrdU and Streptavidin- Peroxidase, with three intervening washes with PBS.
  • Fluorescent microscopy is used to quantify the extent of oligodendrocyte differentiation after compound or agent exposure.
  • the immunofluorescence images are obtained using a Zeiss AxioVision digital imaging system, with a Zeiss AxioCam HRc cooled CCD camera connected to the same microscope. All microscopic imaging parameters are set for acquiring images for the analysis of cellular immunofluorescence intensity. Cellular autofluorescence should be undetectable under the imaging conditions.
  • Manual Cell Counting Four fields are randomly selected for each experimental condition and 500-600 cells are counted in each field. The percentage of MBP or 04 immunopositive cells (mature process bearing cells with or without myelin sheets) versus DAPI positive cells (total cell number) cells are compared in the control and compound or agent treated groups.
  • ELISA is used to evaluate compound or agent induced PTPR ⁇ pathway activation and the extent of oligodendrocyte maturation (changes in protein levels).
  • the plates are incubated, shaking at 500-700 rpm on orbital microplate shaker, for 2 hr at room temperature. 6. 100 ⁇ l of the MBP Antibody-Biotin conjugate is added to each well. 7. The plates are incubated, shaking at 500-700 rpm on orbital microplate shaker, forl hr at room temperature. 8. The wells are washed 5 times with Wash Solution and blot dried by inverting the plate on absorbent material. 9. The streptavidin-enzyme conjugate concentrate is diluted 1:50 with MBP Elisa Assay buffer (must be diluted immediately prior to use in the assay). 10. 100 ⁇ l streptavidin-enzyme conjugate solution are added to each well. 11.
  • the plates are incubated, shaking at 500-700rpm on orbital microplate shaker, for 30 min at room temperature. 12. Wash well 5 times with the Wash Solution. Blot dry by inverting the plate on absorbent material. 13. Add 100 ⁇ l of TMB Chromogen Solution to each well. 14. Incubate the well, shaking at 500-700 RPM on orbital microplate shaker for 10-20 min at room temperature. Avoid exposure to direct sunlight. 15. 100 ⁇ l of the Stopping Solution is added to each well. 16. Read the absorbance of the solution in the wells within 30 min, using a microplate reader set to 450 nM.
  • RNA is extracted from cultured oligodendrocytes using TRIZOL reagent.
  • RNA is treated with RNase-free DNase, repurified, and then converted to cDNA template using a reverse transcription (RT) reaction (Clontech Advantage RT for PCR Kit).
  • RT reverse transcription
  • PTPR ⁇ pathway member transcript expression is quantified using Sybr Green PCR Master Mix.
  • Quantitative PCR is carried out using real-time TAQMAN technology (Gibson, et al., 1996) with a model 7700 Sequence Detector System (Applied Biosystems, Foster City, CA).
  • the bone is removed and rats are injected with 2 ⁇ l ethidium bromide, lysolecithin, or SESf-l into the right caudal cerebellar peduncle, DV -7.1 mm, over a 2 min period by means of a Hamilton ⁇ l syringe and needle. Alternatively injections are made into the spinal cord, corpus callosum, or cortex. 7. The needle is left in position for the subsequent 2 min.
  • Each rat receives an i.m. injection of 0.003mg buprenorphine into a hind leg.
  • the rat is placed in a warming cupboard until it regains consciousness. At which time it is returned to its home cage. Do not allow more than 2 rats per cage, as they will pull each other's suture out.
  • A2B5 and CNP2:GFP-def ⁇ ned parenchymal progenitor cells resides in adult human subcortical white matter. These cells can act as oligodendrocyte progenitors, and give rise to myelinogenic oligodendrocytes upon transplantation. However, when removed from the tissue environment they behave as multipotential neurogenic progenitor cells.
  • U133 Affymetrix microarrays to analyze the transcriptional profile of A2B5+ white matter progenitor cells (WMPCs) sorted from surgically-resected human white matter. The profile?
  • the isolated WMPCs which comprised ⁇ 3% of viably dissociated white matter cells, expressed high levels of PDGF ⁇ R, GD3 s nthase and NG2, consistent with an oligodendrocyte progenitor phenotype. But they also differentially expressed high levels of MASH1, FHL1 and HES, suggesting a more primitive neurogenic progenitor.
  • Several cognate pairs of ligands and receptors were noted in the A2B5 + population, suggesting prominent autocrine regulation.
  • PTPRZ1 a receptor tyrosine phosphatase
  • RTPzeta receptor tyrosine phosphatase
  • CSPG2-5 chondrotin sulphate proteoglycans
  • NrCAM and LI cell adhesion molecules
  • These apparent autocrine ligand-receptor pairs may provide the means by which parenchymal progenitors are maintained as such, in a multipotential and mitoticalfy-competent state. As such, they may provide targets by which to perturb cell fate choices by adult human neural progenitor cells.
  • a population of nominally glial progenitor cells resides in the parenchyma of the adult human subcortical white matter. These cells may be defined by A2B5-irnmunoreactivity, and by their expression of fluorescent reporters placed under the control of the CNP2 promoter.
  • the cells typically act as oligodendrocyte progenitors, giving rise to myelinogenic oligodendrocytes upon transplantation.
  • the cells were collected by centrifugation at 200x g in an EEC Centra-4B centrifuge, resuspended in DMEM/F-12/N1 with DNase I (10 U/ml), and incubated for 30 min at 37°C. The samples were again spun, and their pellets recovered in 2 ml of DMEM/F-12/N1. They were then dissociated by sequentially triturating for 20, 10, and 5 times, respectively, through three glass Pasteur pipettes fire polished to decreasing bore diameters. The cells were passed through a 40 ⁇ m mesh into DMEM/F-12/N1, with 10% plasma-derived fetal bovine serum (PD-FBS; Cocalico Biologicals, Reamstown, PA) to stop the enzymatic dissociation.
  • PD-FBS plasma-derived fetal bovine serum
  • the cells were then suspended in DMEM F12 N1 and incubated in A2B5-antibody containing supernatant (clone 105; American Type Culture Collection, Manassas, VA) for 30-45 min at 4°C on a shaker.
  • the cells were washed 3x with PBS ⁇ ntaining 0.5% bovine serum albumin and 2 mM EDTA, then incubated with 1:4 diluted microbead-tagged rat anti-mouse IgM antibody (MACS, Miltenyi Biotech) for 30 min at 4°C on a shaker.
  • A2B5-antibody containing supernatant clone 105; American Type Culture Collection, Manassas, VA
  • the cells were washed 3x with PBS ⁇ ntaining 0.5% bovine serum albumin and 2 mM EDTA, then incubated with 1:4 diluted microbead-tagged rat anti-mouse IgM antibody (MACS
  • the A2B5 + cells were washed, resuspended and separated using positive selection columns, type MS+/RS+ or LS+/VS+ (MACS, Miltenyi Biotech). The total number of viable cells was determined using calcein (Molecular Probes).
  • the probe match software on the NetAffx website was used to query the U95 probe data fwww.affymetrix.com/analvsis/probe match.prma). In general, probe sets that did not match greater than 12 out of 16 probe pairs were excluded. In addition, only the best probe set was used unless multiple probe sets were 16/16 perfectly matched. In the case of genes which did not provide at least one qualifying probe set, the search was extended by using the NetAffx & Ensembl generated annotations.
  • Real-time PCR Primers and probes were either designed using Primer express (Applied Biosystems) or obtained as Assays-on-demand directly from Applied Biosystems. For each sample, four separate reverse transcription reactions of 25ng total RNA were performed as per manufacturer's protocol and the resulting cDNA diluted to lOOpg/ul. Four separate real-time PCR reactions with 500pg / reaction, in addition 2 no-RT control reactions were performed to check for RNA- independent product amplification. For taqman real-time PCR, 900nM forward and reverse primers, and 250nM FAM-labelled MGB probes. For SYBR Green real-time PCR, 300nM forward and reverse primers were used.
  • the human 18S RNA was used as an endogenous control (ABI).
  • WMPCs expressed oligodendrocyte progenitor marker genes
  • WMPCs were enriched by magnetic-activated cell sorting (MACS) using the A2B5 marker. From four epileptic temporal lobe resections cases, male ages 30-54, we obtained between 5 x 10 5 and 1 x 10 6 A2B5 + cells, that comprised between 3-5% of the total white matter dissociate.
  • the expression of individual genes in the isolated WMPC was normalized against the unsorted white matter dissociate from which it was derived, and the mean expression ratio calculated from the three samples.
  • the marker used to isolate WMPCs the A2B5 antibody, primarily recognizes gangliosides, GT3 and its O-acetylated derivative.
  • GD3 synthase the enzyme that catalyzes the transfer of sialic acid from CMP-sialic acid to GM3 that produces gangliosides GD3 and GT3, is significantly enriched within the A2B5-isolated WMPC population.
  • qPCR real-time RT-PCR analysis
  • CSPG4 PDGF ⁇ R and NG2
  • the resulting list of approximately 250 probe sets ( ⁇ 5% of total) was then pruned by removing those probe sets which were; 1. ambiguously annotated mapping to multiple genes, 2. un-annotated novel transcripts (not yet annotated to an NCBI LocusLink identifiers, www.ncbi.nih.gov/LocusLink).
  • the remaining probe sets were annotated to 208 distinct genes. For each gene, additional probe sets were identified and we found that a number of genes had conflicting microarray expression data that could not be explained by the presence of specific alternatively spliced transcripts.
  • GAP43 is so called growth-associated protein that is expressed in growth cones during development but has previously been considered by some groups as a useful marker of rodent A2B5+ oligodendrocyte progenitors.
  • a few genes whose expression has previously been described in neural cell types other than oligodendrocyte progenitors were also significantly enriched in the A2B5+ defined WMPC pool.
  • GAD67 encodes one of two forms of glutamate decarboxylase (GAD) and has been commonly used as a marker of GABAergic intemeurons. Although no ⁇ lll-tubulin or MAP2 positive neurons were found by immunostaining immediately post-sort, we found that the mRNA for GAD67 was enriched >8 fold in the A2B5-enriched WMPC profile. Doublecortin (OCX), which is expressed on migrating immature neurons during development, was also found approximately 8 fold enriched in the WMPC. Jjitriguingly, two transcription factors expressed by neural progenitors and stem cells respectively, MASHl (ASCL1) and HES1 were found to be enriched in the WMPC pool. MASHl expression was 12- fold greater by microarray and over 18-fold by qPCR in the A2B5+ vs unsorted cell populations. HES1 was found to be 5 fold higher by microarray and over 12 fold higher by qPCR.
  • GAD67 glutamate decarboxy
  • WMPCs express high levels of several receptor tyrosine kinases and phosphatases We also find enriched expression of other G-protein coupled receptors in the adult human WMPC population.
  • the cannabinoid receptor (CNR1) which is expressed by rodent oligodendrocyte lineage cells, is significantly enriched in the A2B5-sorted WMPC profile and is expressed over 12 fold higher by qPCR.
  • the novel GPR19 which is abundantly expressed in the human and rodent brain, is significantly enriched in both Affymetrix probe sets.
  • Receptor tyrosine phosphatase zeta (PTPRZ1) was the single most significantly enriched gene from our microarray analysis and was over 15 fold enriched in WMPCs versus the unsorted cells by qPCR analysis (p ⁇ 0.01).
  • the high level expression of PTPRZl, which contains both a CPSG-like extracellular domain and intracellular protease domain, by A2B5-expressing WMPCs was remarkable in light of the developmental expression of this molecule by radial cells and neural progenitors of the fetal ventricular zone.
  • this receptor phosphatase has been shown to be expressed by the rat CG4 OPC line in culture; and RPTP ⁇ knock-out mice exhibited impaired recovery to MOG-induced EAE relative to wild-type mice. Jjitriguingly, the only known ligand of RPTP ⁇ , pleiofrophin (PTN), was also found to be expressed significantly higher in the WMPC-enriched profile by both microarray and qPCR analysis (p ⁇ 0.02).
  • WMPCs express a distinct set of cell-cell adhesion molecules
  • NrCAm may be of especial intesrt here since it has been identified as a heterophilic ligand for the RTPzeta ectodomain.
  • the related CAM-family member LI has similarly been identified as an RTP ⁇ binding partner, and it too is differentially expressed by adult WMPCs, notwithstanding the high levels of LI expressed by neuronal axons in the white matter (whose mRNAs would nonetheless be largely cortical in distribution).
  • WMPCs differentially express the CSPGs
  • Four chondroitin-sulphate proteoglycans (CSPG) were represented in the WMPC- enriched genes; versican (CSPG2), neurocan (CSPG3), NG2 (CSPG4) and neuroglycan C (CSPG5) were all significantly enriched in human A2B5-sorted WMPCs between 5-20 fold (Table 2).
  • CSPG2 chondroitin-sulphate proteoglycans
  • CSPG5 neuroglycan C
  • WMPC s may actively suppress local wnt signals
  • a secreted WNT antagonist, SFRP3 (FRZB) is highly enriched in the A2B5+ WMPC profile over 15 fold greater than unsorted cells.
  • SFRP3 binds WNTs x,y and z and blocks WNT ligand activation of frizzled receptors which can drive neural progenitor expansion in rodent neurospheres.
  • RA - RARRES2 (as indicator of activation)
  • ALDH1A3 (as potential for RA synthesis).
  • BMP - mab-21-like 1 & BAMBI (as indicators of activation)
  • increased synthesis of BMP2/7 (as activators).
  • FGF - FGFR3 (receptor responsible for neural progenitor expansion) & sprouty 2 (SPRY2) as potential indicator of activation and negative feedback.
  • the WMPC-enriched expression profile indicates the expression of a number of proteins involved in sterol biosynthesis and metabolism
  • oligodendrocytes are the major lipid-producing cell in the CNS
  • these genes included 3-hydroxy-3- methylglutaryl-coenyzme A reductase (HMGCR), which is the rate-limiting enzyme in cholesterol biosynthesis, and the low density lipoprotein receptor (LDLR), responsible for cellular uptake of LDL increasing the availability of intracellular cholesterol.
  • HMGCR 3-hydroxy-3- methylglutaryl-coenyzme A reductase
  • LDLR low density lipoprotein receptor
  • INSIGl 2.1 fold enriched
  • each symbol For each known human gene we approve a gene name and symbol (short-form abbreviation). All approved symbols are stored in Genew. the Human Gene Nomenclature Database. Each symbol is unique and we ensure that each gene is only given one approved gene symbol. It is necessary to provide a unique symbol for each gene so that we and others can talk about them, it also facilitates electronic data retrieval from publications. In preference each symbol maintains parallel construction in different members of a gene family and can also be used in other species, especially the mouse.
  • the finished draft of the human genome sequence will soon be public and with it will come the expectation of a usable system for quickly identifying genes of interest.
  • the HGNC is providing one of the keys to unlocking the secrets of the human genomic sequence by ensuring that there are unique gene symbols linked to curated data.
  • HGNC HUGO Gene Nomenclature Committee
  • Locuslink (URL: http:/ www.ncbi.nlm.nih.gov/LocusLink/) should be given as an alternative free source of the same data.
  • L 1 J A method of modulating production of neurons and/or oligodendrocytes from neural progenitor cells of post-natal or adult human white matter, said method comprising: administering an agonist or antagonist of one or more molecules set forth in Tables 1, 2, 3, 4, 5, and/or 6 to the neural progenitor cells under conditions effective to modulate production of neurons and/or oligodendrocytes.
  • ⁇ _2.J The method of (1 ⁇ wherein the neural progenitor cells are oligodendrocyte progenitor cells. f3. ⁇ ) The method of jl/, wherein the one or more molecules is set forth in
  • Table 1 The method of
  • Jl V The method of Jl Oj, wherein the one or more molecules is adenylate cyclase 8.
  • l 2 The method of (fL wherein the one or more molecules is set forth in Table 3.
  • l 3j The method of jj- , wherein the one or more molecules is selected from the group consisting of pleiotrophin, platelet-derived growth factor, NEL-like 1, neuralin 1, BMP2 (a Dpp homologue), OP-1, chromoganin B (se ⁇ retogranin 1), or secretogranin II (cljTomogramn C).
  • the method of J wherein the neural progenitor cells are from a post-natal human.
  • J27J The method of Q, wherein the neural progenitor cells are from an adult human.
  • j28J The method of 111 wherein the one or more molecules modulate oligodendrocyte progenitor mobilization, division, proliferation, differentiation, and/or self- maintenance.
  • J47J. The method of J46 wherein the one or molecules is a receptor tyrosine kinase.
  • the method of - J46J wherein the one or more molecules is platelet- derived growth factor receptor, cannabinoid receptor 1, G protein-coupled receptor 19, activated p21cdc42Hs kinase, or transmembrane 4 superfamily member 2.
  • ⁇ 9j. The method of , wherein the one or more molecules is GABA A receptor, glycine receptor beta, glutamate receptor ionotrophic AMPA 2, or glutamate receptor iontropic kainate 1.

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Abstract

Provided herein is a novel and useful methods for determining whether a compound or agent may modulate the activity of a Protein Tyrosine Phosphatase Receptor (PTPRZETA).

Description

METHOD FOR ASSAYING COMPOUNDS THAT MODULATE THE ACTIVrTY OF PROTEIN TYROSINE PHOSPHATASE RECEPTOR ZETA (PTPRO
FIELD OF THE INVENTION The present invention relates to a method for determining whether a compound or agent modulates the activity of PTPRζ. Such compounds may readily have applications in treating and/or preventing demylinating diseases or disorders.
BACKGROUND OP THE INVENTION Signals sent from Central Nervous System (CNS) propagate to the periphery of the body via neurons. An action potential passes from one neuron to another, and thus the signal is transmitted. In order to quickly and efficiently transmit these signals, glial cells, such as Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system, synthesize myelin, which wraps around axons to form the myelin sheath. The myelin sheath performs the same function as insulation around an electrical wire, i.e. insulation for the nerve cell. Thus, the main functions of the myelin sheath are to allow for fast saltatory nerve conduction through the clustering of sodium channels at the nodes of Ranvier, to maintain the nodal regions, and for trophic support to neuronal axons.
In demyelinating diseases or disorders, the myelin sheath surrounding neuronal axons is damaged or destroyed. Consequently, the action potential passing through demyelinated axons is not quickly or efficiently transmitted. Rather, the action potential leaks from the axon, and becomes attenuated. For example, the demyelinating that occurs in Multiple Sclerosis (MS) involves immune-mediated destruction of the myelin sheath surrounding neuronal axons in the brain and spinal cord. As a result, peripheral sensations, such as touch, vision, and balance, can be detrimentally affected. These affects are not necessarily chronic. • Rather, they can occur in "attacks" periodically. For some patients, the attacks are mild, while in others, the attacks can be severe and debilitating, resulting in sensory and/or motor deficits and paralysis. Other demeylinating diseases or disorders include Charcot-Marie- Tooth disease, Pelizaeus-Merzbacher disease, encephalomyelitis, neuromyelitis optica, adrenoleukodystrophy, Guillian-Barre syndrome, and instances in which myelin forming glial cells are damaged, including spinal cord injury, neuropathies, and other types of nerve injuries. Although newly formed oligodendrocyte progenitor cells are present in the CNS and around MS lesions and up to 70% of lesions show some evidence of remyelination, this appears to be a transient and incomplete process since only limited long term repair of the myelin sheath has been observed. Traditional methods for treating the disease are principally concerned with controlling the number of attacks by suppressing the immune system, and/or ameliorating the effects or duration of an attack with the administration of corticosteroids. Unfortunately, these treatments merely ameliorate the progression of the disease, and neither reverse the progression of the disease, nor restore the nervous system or its condition prior to the onset of the disease.
Accordingly, what is needed is a method for identifying compounds or agents that can promote or enhance progenitor cells that reside within regions of demyelination to differentiate into oligodendrocytes, and remyelinate previously demyelinated axons. This type of therapeutic approach may reverse the disease course and foster functional improvement, possibly in combination with therapies that minimize immune responses and allow for aggressive repair strategies to work successfully.
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.
SUMMARY OF THE INVENTION There is provided, in accordance with a present invention, a method for evaluating compounds or agents for their ability to modulate the activity of a Protein Tyrosine Phosphatase Receptor zeta (PTPRζ), or the PTPRζ pathway. PTPRζ is also known as PTPζ, RPTPβ, HPTPζ, and PTB18. PTPRζ is expressed on the surface of oligodendrocyte progenitor cells. In the In the PTPRζ pathway, pleitrophin, a growth and differentiation factor and a natural ligand for PTPRζ, binds to and functionally inactivates the constitutive intracellular phosphatase activity of PTPRζ, resulting in an increase in tyrosine phosphorylation of a molecule called β-catenin. In immature oligodendrocytes, phosphorylated β-catenin stimulates a transient and limited number of cell divisions that lead to the initiation of differentiating pathways that result in the formation of mature oligodendrocytes. [Deuel, F et al. Pleiotrophin, A Cytokine with Diverse Functions and a . Novel Signaling Pathway. Archives of Biochemistry and Biophysics, 397(2): 162-171 (2002); Meng, K. et al. Pleiotrophin Signals Increased Tyrosine Phosphorylation of β-Catenin through Inactivation of the Instrinsic Catalytic Activity of the Receptor-type Protein Tyrosine Phosphatase β/ζ PNAS 97(6): 2603-2608 (2000)].
It has been discovered that, substantial numbers of PTPRζ are expressed on the surface of progenitor cells of post-natal and adult human white matter. [P.D. Canoll et al. , J. Neurosci. Res. 44:199 (1996); Ranjan, M. and Hudson, L.D., Mol. Cell. Neurosci. 7:404 (1996). Moreover, shown herein is PTBRζ mRNA expression in human CNS germinal zone and glial progenitor cells (see FIG. 3, infra). Hence, modulating the activity of PTPRζ or the PTPRζ pathway may cause the differentiation of these immature cells into oligodendrocytes, which then may remediate the damage that occurs in a demyelinating disease or disorder through enhanced remyelination.
Accordingly, the present invention extends to a method for determining whether a compound or agent modulates activity of an PTPRζ, or its pathway, comprising the steps of contacting the compound or agent with the PTPRζ, and then determining whether the compound or agent has interacted with or bound to PTPRζ. In such a method, the PTPRζ may optionally be immobilized on a substrate, or alternatively, the compound or agent may be immobilized on a substrate. Numerous substøtes have applications in a method of the present invention. Particular examples include polyvinylidene difluoride and nitrocellulose, to name only a few.
Moreover, in a method of the present invention, the compound can be detectably labeled, the PTPRζ, can be detectably labeled, both PTPRζ and the compound can be detectably labeled, or a relevant pathway member of the PTPRζ can be detectably labeled. Numerous detectable labels have applications in a method of the present invention and are discussed infra. Particular examples include, but are not limited to radioactive isotopes, chemicals that fluoresce, chemicals that phosphoresce, biotin/avidin, etc. Particular examples are described infra.
Furthermore, PTPRζ from a variety of sources, e.g. equine, bovine, canine, feline, human, primate, ovine, rodent, avian, etc. have applications in a method of the present invention. In a particular embodiment, the PTPRζ is human, and comprises the amino acid sequence of FIG. 2 (SEQ ID NO:l). Naturally, the present invention extends to a method for determining whether a compound or agent modulates activity of an PTPRζ, or its pathway, wherein one compound or agent at a time is evaluated. Alternatively, a method of the present invention can be perfoπned in a high throughput fashion.
In another embodiment, the present invention extends to a method for determining whether a compound or agent modulates activity of a PTPRζ or its pathway. In such a method, the compound or agent is contacted with a sample of brain white matter containing progenitor cells. This sample is then evaluated for a change in the number of oligodendrocytes, the extent of oligodendrocyte differentiation, the amount of myelin produced, and/or the extent of PTPRζ activity or relevant pathway activity. Such results would be indicative that the compound or agent modulated the activity of the PTPRζ.
In a method of the present invention, progenitor cells that express the PTPRζ, can be either in vivo or ex vivo. Moreover, should the progenitor cell be in vivo, the step of contacting the compound or agent with the white matter or post-natal progenitor cell that expresses the PTPRζ comprises administering the compound or agent to the organism.
In another embodiment, the present invention extends to a method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the number of oligodendrocytes that express PTPRζ within the bodily sample is known; (b) measuring the number of oligodendrocytes that express PTPRζ, after contact with the compound or agent. If the number oligodendrocytes that express PTPRζ, in the bodily sample after contact with the compound or agent is greater than the number prior to contact, it is indicative that the compound or agent may modulate activity of the PTPRζ.
The present invention further extends to a method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an ' organism, wherein the number of oligodendrocyte progenitor cells that express PTPRζ within the bodily sample is known; (b) measuring the number of oligodendrocyte progenitor cells that express PTPRζ, after contact with the compound or agent.
If the number oligodendrocyte progenitor cells that express PTPRζ, in the bodily sample after contact with the compound or agent is greater than the number prior to contact, it is indicative that the compound or agent may modulate activity of the PTPRζ.
Furthermore, the present invention extends to a method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the extent of myelination in the sample is known; and (b) determining the extent of myelination in the bodily sample after contact with the compound or agent.
If the extent of myelination in the sample after contact with the compound or agent is greater than observed prior to contact, it is indicative that the compound or agent may modulate PTPRζ.
The present invention further extends to a method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the extent of myelination in the sample is known; and (b) determining the extent of myelination in the bodily sample after contact with the compound or agent.
If the extent of myelination in the sample after contact with the compound or agent is greater than observed prior to contact, it is indicative that the compound or agent may modulate PTPRζ.
The present invention also extends to a method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of:
(a) contacting the compound or agent with a bodily sample taken from an organism, wherein the extent of oligodendrocyte differentiation in the sample is known; and
(b) measuring the extent of oligodendrocyte differentiation in the bodily sample after contact with the compound or agent. If the extent of oligodendrocyte differentiation in the bodily sample is greater after contact with the compound or agent, it is indicative that the compound or agent may modulate the activity of PTPRζ..
The present invention further extends to a method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the PTPRζ activity or activity of a relevant member of the PTPRζ pathway in the sample is known; and (b) determining the activity of PTPRζ or the activity of the relevant member of the PTPRζ pathway in the bodily sample after contact with the compound or agent.
If the PTPRζ activity or activity of the activity of the relevant member of the PTPRζ pathway in the bodily sample after contact with the compound or agent is greater than that observed prior to contact, it is indicative that the compound or agent may modulate PTPRζ.
In a method of the present invention, the sample can be taken from a variety of organisms, e.g. ovine, bovine, feline, rodent, equine, primate, human, rodent, etc. In a particular embodiment, the sample is white matter taken from the brain of the organism, e.g. a human.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1: Schmatical view of the PTPRζ pathway. FIG: 2: Amino Acid sequence of human PTPRζ (SEQ ID NO: 1).
FIG. 3 is a histogram of TAQMAN results showing that the level of RPTζ transcription is higher in human fetal ventricular zone (vz), a region where progenitor cells are enhanced, than the cortex (ex), where mature cells reside, and is enriched in human oligodendrocyte progenitor cells.
FIG. 4: graphical presentation of TAQMAN results that show human PTPRζ mRNA is not detected in adult human peripheral organs. DETAILED DESCRIPTION OF THE INVENTION The present invention is based upon the novel discovery that PTPRζ is expressed on the surface of progenitor cells within the human central nervous system. Hence, modulating the activity of PTPRζ or the PTPRζ pathway may cause the differentiation of these cells into oligodendrocytes, which then, through enhanced remyelination, may remediate the demyelination that occurs in a demeylinating disease or disorder. Thus broadly, the present invention extends to a method for determining whether a compound or agent modulates activity of an PTPRζ, or its pathway, comprising the steps of contacting the compound or agent with the PTPRζ, and then determining whether the compound or agent binds to the PTPRζ,. Modulation occurs when, as a result of the compound's or agent's binding to PTPRζ, increased numbers of oligodendrocyte progenitor cells observed, increased numbers of oligodendrocytes is observed, an increase in the extent of oligodendrocyte differentiation or amount of myelin produced is observed, or a combination thereof.
As explained above, pleitrophin, a growth and differentiation factor and a natural ligand for PTPRζ, binds to and functionally inactivates the constitutive intracellular phosphatase activity of PTPRζ, resulting in an increase in tyrosine phosphorylation of a molecule called β-catenin. In immature oligodendrocytes, phosphorylated β-catenin stimulates a transient and limited number of cell divisions that lead to the initiation of differentiating pathways that result in the formation of mature oligodendrocytes. [Deuel, F et al. Pleiotrophin, A Cytokine with Diverse Functions and a Novel Signaling Pathway. Archives of Biochemistry and Biophysics, 397(2): 162-171 (2002); Meng, K. et al. Pleiotrophin Signals Increased Tyrosine Phospliorylation ofβ-Catenin through Inactivation of the Instrinsic Catalytic Activity of the Receptor-type Protein Tyrosine Phosphatase β/ζ PNAS 97(6):2603-2608 (2000)]. A schematical view of the PTPRζ pathway is set forth in FIG. 1
Numerous terms and phrases used throughout the instant Specification and appended Claims are defined below. Accordingly:
As used herein, the terms "compound" or "agent" refer to any composition presently known or subsequently discovered. Examples of compounds or agents having applications herein include organic compounds (e.g., man made, naturally occurring and/or optically active), peptides (man made, naturally occurring, and or optically active, i.e., either D or L amino acids), carbohydrates, nucleic acid molecules, etc. As used herein, the term "enzyme" refers to a biomolecule, such as a protein or RNA, that catalyzes a specific chemical reaction. It does not affect the equilibrium of the catalyzed reaction. Rather, the enzyme enhances the rate of reaction by lowering the energy of activation.
As used herein, the phrase "relevant pathway member" with respect to the PTPRζ pathway refers to genes known or yet to be identified that are downstream from PTPRζ and that ultimate lead to the function consequences of the activated PTPRζ receptor, such as proliferation, migration or differentiation.
Detectable labels As explained above, in a method of the present invention, the PTPRζ can be detectably labeled, the compound or agent can be detectably labeled, or even both can be detectably labeled. Suitable labels include enzymes (e.g. alkaline phosphatase and horseradish peroxidase) , fluorophores (e.g., fluorescene isothiocyanate (F1TC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially Eu3+, to name a few fluorophores), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (e.g., biotin), and che iluminescent agents. When a control marker is employed, the same or different labels may be used for the receptor and control marker.
In the instance where a radioactive label, such as the isotopes 3H, 14C, 32P, 35S, 3δCl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, I31I, and 186Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
Direct labels are one example of labels which can be used according to the present invention. A direct label has been defined as an entity, which in its natural state, is readily visible, either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e.g. U. V. light to promote fluorescence. Among examples of colored labels, which can be used according to the present invention, include metallic sol particles, for example, gold sol particles such as those described by Leuvering (U.S. Patent 4,313,734); dye sole particles such as described by Gribnau et al. (U.S. Patent 4,373,932) and May et al. (WO 88/08534); dyed latex such as described by May, supra, Snyder (EP-A 0 280559 and 0 281 327); or dyes encapsulated in liposomes as described by Campbell et al. (U.S. Patent 4,703,017). Other direct labels include a radionucleotide, a fluorescent moiety or a luminescent moiety. In addition to these direct labelling devices, indirect labels comprising enzymes can also be used according to the present invention. Various types of enzyme linked immunoassays are well known in the art, for example, alkaline phosphatase and horseradish peroxidase, lysozyme, glucose-6- phosphate dehydrogenase, lactate dehydrogenase, urease, these and others have been discussed in detail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70. 419-439, 1980 and in U.S. Patent 4,857,453.
Other labels for use in the invention include magnetic beads or magnetic resonance imaging labels. Immobilized PTPRC
In a method of the present invention, PTPRζ, may be immobilized on a substrate. Numerous substrates have applications in such a method, including but certainly not limited to polyvinylidene difluoride (PVDF) and nitrocellulose, to name only a few. Alternatively, the compound or agent may be immobilized on such a substrate.
Search of Libraries for Compounds or Agents that Modulate the Activity of PTPRζ Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. However, the current trend is to shorten the time scale for all aspects of drug discovery. Because of the ability to test large numbers quickly and efficiently, high throughput screening (HTS) methods are replacing conventional lead compound identification methods.
In a particular embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries" are then screened with a method of the present invention to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics. Combinatorial Chemical Libraries Combinatorial chemical libraries are a particular means to assist in the generation of new chemical compound leads. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, one commentator has observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (Gallop et al., J. Med. Chem., 37(9): 1233-1251 (1994)).
Preparation of combinatorial chemical libraries is well known to those of ordinary skill in the art. Such combinatorial chemical libraries include, but are not limited to peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention. Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but certainly are not limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), random biooligomers (PCT Publication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, (1993) Proc. Nat. Acad. Sci. USA 90: 69096913), vinylogous polypeptides (Hagihara et al. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimetics with a Beta D Glucose scaffolding (Hirschmann et al.,
(1992) /. Amer. Chem. Soc. 114: 92179218), analogous organic syntheses of small compound libraries (Chen et al. (1994) /. Amer. Chem. Soc. 116: 2661), oligocarbamates (Cho, et al.,
(1993) Science 261:1303), and/or peptidyl phosphonates (Campbell et al., (1994) /. Org. Chem. 59: 658). See, generally, Gordon et al, (1994) I. Med. Chem. 37:1385, nucleic acid libraries, peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083) antibody libraries (see, e.g., Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996) Science, 21 A: 1520-1522, and U.S. Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33, isoprenoids U.S. Patent 5,569,588, thiazolidinones and metathiazanones U.S. Patent 5,549,974, pyrrolidines U.S. Patents 5,525,735 and 5,519,134, morpholino compounds U.S. Patent 5,506,337, benzodiazepines 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). A number of well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate JJ, Zymark Corporation, Hopkinton, Mass.; Orca, HewlettPackard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, Ru; Tripos, Inc., St. Louis, MO; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD, etc.). High throughput assays of chemical libraries A method of the present invention is readily amenable to high throughput screening. High throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like. The present invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following Examples are presented in order to more fully illustrate the particular embodiments of the present invention. They should in no way be construed, however, as limiting the broad scope of the invention.
EXAMPLE I In Multiple Sclerosis, oligodendrocytes (the myelin forming cells in the CNS) are destroyed and axons are damaged, resulting in severely impaired neuronal function and physical deficits. Newly formed oligodendrocyte progenitor cells are present in and around damaged tissue, suggesting the possibility of self-repair. However though, only limited repair is observed.
In an effort to identify proteins that regulate cell fate decisions in human progenitor cells, Affymetrix U95A-E chips were used to profile the second trimester forebrain. Both the rostrolateral forebrain VZ and adjacent cortical mantle were sampled at biweekly intervals from 12-23 weeks of gestation (n=2-3 samples/time-point/region), and their transcript profiles compared. These samples generated expression profiles for -70,000 gene transcripts, for at least triplicate samples at each of 6 time-points spanning second trimester development. In addition, cDNA libraries were established from 18 and 23 week VZ and cortex, as were subtracted and normalized libraries at each time point; each library was sequenced to yield 5- 18,000 ESTs per library. The resulting data were analyzed by focusing on a number of key pathways implicated in neuronal and glial development. Genes were grouped and visualized as expression maps that profiled correlations between pathway-related genes that were co- regulated with defined clusters of marker genes.
Gestational week 23 tissue expressed high levels of PDGFRα, Oligl/2, SoxlO, NG2, CNP JJ, consistent with their oligodendrocyte progenitor cell phenotype. The transcript profile of gestational week 23 tissue and sorted progenitor cells (A2B5+/PSA-NCAM-) was then compared with that of earlier gestational time points. Bioinformatic and qPCR analyses confirmed that one of the most highly enriched genes during the period when glial cells were forming was PTPRζ, a receptor tyrosine phosphatase. This analysis allowed causal relationships to be inferred between the appearance and disappearance PTPRζ pathway- related transcripts with the serial generation in ontogeny of glial progenitor cells. Furthermore, surprisingly and unexpectedly, an upregulation of the PTPRζ was observed in human adult oligodendrocyte progenitor cells and in human MS lesions (FIG. 3). However, the presence of PTPRζ mRNA transcripts is not detected in peripheral organs (FIG. 4).
EXAMPLE π Biological Assavs Used to Screen PTPR Compound or Agent Efficacy in Oligodendrocvtes: Determine Whether Compounds or Agents Modulate PTPRC Pathway Activity or Accelerates/Promotes Oligodendrocyte Progenitor Cell Differentiation
RAT/MICE Oligodendrocyte Cultures Preparation of cells:
1. Primary rat oligodendrocyte progenitor cells are obtained from the neocortex of newborn (postnatal days 2-3) rats or mice and are enriched, after removal of microglia, by mechanical separation from the astrocytic monolayer using a modification of the technique originally described by McCarthy and de Vellis (1980, J. Cell Biol. 85:890- 902).
2. The meninges are removed and the brain tissue is mechanically dissociated. Cells are plated onto T75 flasks and fed with DMEM/F12 + 10% FBS.
3. Oligodendrocytes that grow on the astrocyte bed layer are collected by "gentle shaking" fourteen days after the original prep date. The suspension is centrifuged and the cell pellet is resuspended in serum free media (SFM; DMEM combined with 25 μg/ml transferring, 30 nM triiodothyronine, 20 nM hydrocortisone, 20 nM progesterone, 10 nM biotin, lx trace elements, 30 nM selenium, 1 μg/ml putrescine, 0.1% BSA, 5 U/ml PenStrep, 10 μg/ml insulin) supplemented with the following growth factors: Platelet derived growth factor-AA (PDGF) and fibroblast growth factor-2 (FGF).
4. Cells are plated onto PDL-coated dishes and incubated at 37°C with 6-7% C02.
5. Media components are replaced every 48 hrs to keep the cells in a progenitor state.
Progenitor cell passaging and expansion for screening assays
1. When the cultures reach confluence, they are rinsed with PBS; trypsin is added and the cultures are then incubated for -2-3 min at 37°C.
2. The trypsin is neutralized and the cell suspension is centrifuged at 900g for 5 min.
3. Cell pellets are resuspended in SFM + the mitogens, PDGF/FGF. 4. Cultures are fed with fresh growth factors every 48 hrs to enrich for the rapidly dividing progenitor cells.
5. Cells are passaged no more than 4-5 times prior to experimental assays.
6. All experiments involving oligodendrocyte progenitor cells are done using cells that are continuously maintained under these conditions. Greater than 95% of all cells are A2B5 immunopositive and express 2' 3' -cyclic nucleotide 3' -phosphodiesterase II mRNA.
7. To generate mature oligodendrocytes, 24 hours after plating progenitor cells are switched to SFM supplemented with or without IGF-I, and are grown under these conditions for 7 days prior to experimental assays.
8. Alternatively, the enriched rat Central Glia-4 (CG4) progenitor cell line may be used, which is maintained in base media (DMEM, with 2 mM glutamine, lmM sodium pyruvate, biotin (40 nM), insulin (1 μM) and Nl) supplemented with 30% conditioned media from the B-104 neuroblastoma cell line. To induce differentiation, CG4 cells are switched to base media with 1% fetal calf serum (removed after 2 days) and insulin (500 nM). A2B5 and MBP immunoreactivity is used to confirm >95% enrichment in immature and mature cultures, respectively.
HUMAN Oligodendrocyte cultures Preparation of cells:
1. Human neurospheres (collected from E19.5 - E22 human embryo cortex) are cultured for 2 weeks in progenitor media: DMEM/F12 containing 100 μg/ml transferring, 30 nM triiodothyronine, 20 nM hydrocortisone, 20 nM progesterone, 10 nM biotin, lx trace elements, 30 nM selenium, 60 uM putrescine, 0.1% BSA, 5 U/ml PenStrep, 25 μg/ml insulin) supplemented with PDGF and FGF.
2. The neurospheres are then dissociated with 20 U/ml papain at 37°C for 30-50 min.
3. Cells are plated onto PDL coated dishes at density of 50,000-100,000 cell/well in progenitor media containing PDGF/FGF and incubated at 37°C with 5-6% C02.
4. Media and growth factors are replenished every 48 hrs.
Compound or Agent Treatment
1. 20,000 - 25,000 cells are plated into each well of 24-well poly-D-lysine-coated plates and cultured in the presence of mitogen (lOng/ml).
2. Proliferating cultures: a. The next day, the old medium is replaced with new medium containing the compound or agents and mitogens. b. Compound or agent dose response evaluations are performed at 6 different concentrations (10 μM, 1 μM, 100 nM, lOnM, 1 nM, and 0.1 nM). c. Triplicates wells are run for each compound or agent concentration.
3. Differentiating cultures: a. The next day, the medium is replaced with new medium and compounds or agents (without mitogen). b. Compound or agent dose response evaluations are performed at 6 concentrations (10 μM, 1 μM, 100 nM, 10 nM, 1 nM, and 0.1 nM). c. Triplicates wells are run for each compound or agent concentration.
4. Compound or agent treated cultures are maintained for 7 days.
5. After 7 days exposure to compounds or agents, immunostaining procedures are performed.
Oligodendrocyte Specific Immunostaining
Following compound or agent exposure, oligodendrocyte-specific antibodies are used to assess ability of compounds or agents to accelerate/promote oligodendrocyte differentiation . . . (for example, 04, 01, or myelin basic protein immunoreactivity changes over time between compound or agent treated and untreated cultures).
1. Cells are plated onto poly-D-lysine treated 4-well chamber slides at 5xl03 to 20xl03 cells/well and grown as described above. Sequential staining is performed on oligodendrocyte populations with increasing degrees of cellular differentiation, as determined by days in vitro without PDGF and FGF.
2. Live staining for 30 min at 37°C is used to detect oligodendrocyte stage specific cell surface marker expression (including A2B5, 04, and 01).
3. Subsequently, cells are fixed with 4% paraformaldehyde, 10 min, room temperature.
4. Additional immunostaining procedures are used to detect intracellular oligodendrocyte stage specific marker expression (including myelin basic protein, MBP).
5. Cells are rinsed and permeabilized with 0.1% Triton/0.01 % NaAz diluted in IX PBS for 10 min, room temperature.
6. Non-specific binding sites are blocked with 5-10% goat serum in antibody dilution buffer (0.1% Triton-X 100 and 1% IgG-free bovine serum albumin; also used to dilute antibodies) for 15 min at room temperature. 7. Primary antibodies are added and cells are incubated overnight with gentle rocking at 4° C.
8. The next day, cells are rinsed with PBS and incubated with appropriate secondary antibodies for 45 min at room temperature.
9. Cell nuclei are stained with 4,6-dia idino-2-phenylindole (DAPI) for 15min at room temperature.
10. Cells are rinsed several times with PBS and evaluated using fluorescent microscopy.
11. The following conditions are compared over time and at different compound or agent doses: mitogen-treated, serum free media (SFM) treated, SFM-IGF1 treated, mitogen and compound or agent treated, SFM and compound or agent treated.
Bromodeoxyuridine (BrdU) immunostaining
BrdU immunostaining is used to confirm that compounds or agents do not promote cell proliferation.
1. Oligodendrocyte progenitor cells are labeled with 10 μM BrdU for 20 hr and then fixed with either 70% ethanol or 4% paraformaldehyde.
2. The cells are incubated successively with biotinylated mouse anti-BrdU and Streptavidin- Peroxidase, with three intervening washes with PBS.
3. Colormetric visualization of the BrdU immunoreactivity is developed with DAB and total cell numbers are assessed using the counter-stain hematoxylin.
4. Two independent observers count BrdU immunopositive cells.
Image analysis
Fluorescent microscopy is used to quantify the extent of oligodendrocyte differentiation after compound or agent exposure.
1. Randomly select at least 6 fields/well to assess the number of differentiating oligodendrocytes among the total population (~1-I5xl03 cells are counted in each well).
2. Automated Cell Counting: The immunofluorescence images are obtained using a Zeiss AxioVision digital imaging system, with a Zeiss AxioCam HRc cooled CCD camera connected to the same microscope. All microscopic imaging parameters are set for acquiring images for the analysis of cellular immunofluorescence intensity. Cellular autofluorescence should be undetectable under the imaging conditions.
3. Manual Cell Counting: Four fields are randomly selected for each experimental condition and 500-600 cells are counted in each field. The percentage of MBP or 04 immunopositive cells (mature process bearing cells with or without myelin sheets) versus DAPI positive cells (total cell number) cells are compared in the control and compound or agent treated groups.
ELISA
ELISA is used to evaluate compound or agent induced PTPRζ pathway activation and the extent of oligodendrocyte maturation (changes in protein levels).
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1. Plates are washed with PBS, and then keep on ice. Add 200 μl ice cold lysis buffer (TRIS 50 mM, pH7.4, MgCl22mM, EDTA lmM, β-mercaptoethanol 5mM, Nonidet P-40 1%, Protease inhibitor cocktail (Roche): (1 tablet/50 ml) to each well. 2. Cells are lysed and the plates are spun at 2000 rpm at 4°C for 5 min. The supernatant is ready to use. 3. Pipet 50 μl of standard, controls and samples into the wells. 4. 50 μl of MBP assay buffer is added to each well. 5. The plates are incubated, shaking at 500-700 rpm on orbital microplate shaker, for 2 hr at room temperature. 6. 100 μl of the MBP Antibody-Biotin conjugate is added to each well. 7. The plates are incubated, shaking at 500-700 rpm on orbital microplate shaker, forl hr at room temperature. 8. The wells are washed 5 times with Wash Solution and blot dried by inverting the plate on absorbent material. 9. The streptavidin-enzyme conjugate concentrate is diluted 1:50 with MBP Elisa Assay buffer (must be diluted immediately prior to use in the assay). 10. 100 μl streptavidin-enzyme conjugate solution are added to each well. 11. The plates are incubated, shaking at 500-700rpm on orbital microplate shaker, for 30 min at room temperature. 12. Wash well 5 times with the Wash Solution. Blot dry by inverting the plate on absorbent material. 13. Add 100 μl of TMB Chromogen Solution to each well. 14. Incubate the well, shaking at 500-700 RPM on orbital microplate shaker for 10-20 min at room temperature. Avoid exposure to direct sunlight. 15. 100 μl of the Stopping Solution is added to each well. 16. Read the absorbance of the solution in the wells within 30 min, using a microplate reader set to 450 nM.
Quantitative Polvmerase Chain Reaction (PCR) To evaluate compound or agent induced PTPR ζ pathway activation and the extent of oligodendrocyte maturation (changes in mRNA levels). 1. Total RNA is extracted from cultured oligodendrocytes using TRIZOL reagent. 2. Subsequently, mRNA is treated with RNase-free DNase, repurified, and then converted to cDNA template using a reverse transcription (RT) reaction (Clontech Advantage RT for PCR Kit).
3. PTPRζ pathway member transcript expression is quantified using Sybr Green PCR Master Mix.
4. The 18S ribosomal RNA primer/probe mix (186 bp product), suspended in TAQMAN 2X PCR Master Mix is used as an internal control.
5. Quantitative PCR is carried out using real-time TAQMAN technology (Gibson, et al., 1996) with a model 7700 Sequence Detector System (Applied Biosystems, Foster City, CA).
6. The results are analyzed using Sequence Detection Systems software version 1.91.
In Vivo Proof of Concept Models Focal Lesions: used to assess whether compounds or agents protect myelin integrity and/or accelerate and/or enhance the rate of remyelination. 1. Rats 7 weeks of age are given free access to food and water and acclimatized for a minimum of 4 days before use in experiments. 2. Prior to surgery each animal is weighed. The rat is then anaesthetized with ketamine (100 mg/ l) in combination with xylazine (20 mg/ml) in a ratio of 1.8 : 1. The rats are injected with 0.15ml/180g body weight i.p. of the anesthetic solution prior to the surgical procedure. The animal is prepared for surgery using aseptic conditions in accordance with the IACUC guidelines. All surgical instruments will be autoclaved. The hair is clipped between the ears and this region will then be scrubbed with Betadine, flushed with sterile saline and finally wiped with a pre-packaged sterile alcohol swab. 3. For the surgical procedure, the rat is placed on its ventral surface in a small animal stereotaxic instrument designed to hold the head steady. The incisor bar is always set at - 3.9 mm, since this has been shown to achieve a flat-skull position for SD rats. 4. An incision is made in the previously shaven skin overlying the skull between the ears. 5. A small area of bone (0.75mm in diameter) is drilled at the following coordinates AP - 1.8, ML -3.1 from lambda. 6. The bone is removed and rats are injected with 2μl ethidium bromide, lysolecithin, or SESf-l into the right caudal cerebellar peduncle, DV -7.1 mm, over a 2 min period by means of a Hamilton μl syringe and needle. Alternatively injections are made into the spinal cord, corpus callosum, or cortex. 7. The needle is left in position for the subsequent 2 min.
8. After withdrawal of the needle the incision is sutured.
9. Each rat receives an i.m. injection of 0.003mg buprenorphine into a hind leg.
10. The rat is placed in a warming cupboard until it regains consciousness. At which time it is returned to its home cage. Do not allow more than 2 rats per cage, as they will pull each other's suture out.
11. Similar procedures are also done using mice.
EXAMPLE πi
Transcriptional Identification of Acutely Isolated Progenitor Cells of the Adult Human Subcortical White Matter
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We have previously shown that a population of A2B5 and CNP2:GFP-defϊned parenchymal progenitor cells resides in adult human subcortical white matter. These cells can act as oligodendrocyte progenitors, and give rise to myelinogenic oligodendrocytes upon transplantation. However, when removed from the tissue environment they behave as multipotential neurogenic progenitor cells. To identify proteins that regulate the homeostasis and cell fate decisions of this adult progenitor population, we used U133 Affymetrix microarrays to analyze the transcriptional profile of A2B5+ white matter progenitor cells (WMPCs) sorted from surgically-resected human white matter. The profile? of each sorted cell population was then normalized against that of the dissociated white matter from which it was derived. The isolated WMPCs, which comprised <3% of viably dissociated white matter cells, expressed high levels of PDGFαR, GD3 s nthase and NG2, consistent with an oligodendrocyte progenitor phenotype. But they also differentially expressed high levels of MASH1, FHL1 and HES, suggesting a more primitive neurogenic progenitor. Several cognate pairs of ligands and receptors were noted in the A2B5+ population, suggesting prominent autocrine regulation. The most significantly enriched gene, PTPRZ1 (RTPzeta), a receptor tyrosine phosphatase, was found along with its ligand, pleiotrophin, at 5-10 fold greater expression than in unsorted white matter cells. In addition, the cells expressed a large repertoire of chondrotin sulphate proteoglycans (CSPG2-5) and cell adhesion molecules including NrCAM and LI, that have been reported to interact heterophilically with RTPzeta. These apparent autocrine ligand-receptor pairs may provide the means by which parenchymal progenitors are maintained as such, in a multipotential and mitoticalfy-competent state. As such, they may provide targets by which to perturb cell fate choices by adult human neural progenitor cells.
Introduction A population of nominally glial progenitor cells resides in the parenchyma of the adult human subcortical white matter. These cells may be defined by A2B5-irnmunoreactivity, and by their expression of fluorescent reporters placed under the control of the CNP2 promoter. The cells typically act as oligodendrocyte progenitors, giving rise to myelinogenic oligodendrocytes upon transplantation. However, we previously noted that when removed from the tissue environment they behave as multipotential and neurogenic progenitor cells. This observation suggested that the local tissue environment regulates both the self-renewal and phenotype of parenchymal glial progenitors, such that the latter actually represent a pool of multipotential progenitors whose fate is tonically restricted by their local tissue environment. As a result, the environmental cues presented to these these cells, and their responsiveness to these signals, may directly deteπriine their not only their mitotic turnover, but also their undifferentiated self- renewal and post-mitotic lineage choice. Yet no studies to date have specifically examined the environment of the adult human white matter from the standpoint of steady-state cues and cell- specific responsiveness by resident progenitor cells. In order to identify genes that regulate both the turnover and fate decisions of the adult progenitor cell population in vivo, we therefore used U133 Affymetrix microarrays to analyze the transcriptional profile of A2B5+ white matter progenitor cells (WMPCs) sorted from the adult human white matter, in samples derived from surgically-resected adult human temporal lobe. The profile of each sorted cell population was then normalized against that of the unsorted white matter dissociate from which it was derived, to identify WMPC-enriched transcripts that were otherwise underrepresented in the white matter. By this strategy, we identified several novel ligands and receptors that appear to uniquely characterize the interaction of oligodendrocyte progenitor cells with the ambient white matter in which they reside.
MATERIALS AND METHODS Adult human subcortical white matter was obtained from temporal lobe tissue removed from 4 patients at surgery formedication-rfractory epilepsy. The tissues were prepared and white matter progeniotr cells freshly isolated. Briefly, samples were minced into PIPES solution (in mM: 120 NaCl, 5 KC1, 25 glucose, and 20 PIPES), then digested in papain-PIPES (11.4 U/ml papain; Worthington, Freehold, NJ) and DNase I (10 U/ml; Sigma, St. Louis, MO), on a shaker for 1.5 hr at 37°C. The cells were collected by centrifugation at 200x g in an EEC Centra-4B centrifuge, resuspended in DMEM/F-12/N1 with DNase I (10 U/ml), and incubated for 30 min at 37°C. The samples were again spun, and their pellets recovered in 2 ml of DMEM/F-12/N1. They were then dissociated by sequentially triturating for 20, 10, and 5 times, respectively, through three glass Pasteur pipettes fire polished to decreasing bore diameters. The cells were passed through a 40 μm mesh into DMEM/F-12/N1, with 10% plasma-derived fetal bovine serum (PD-FBS; Cocalico Biologicals, Reamstown, PA) to stop the enzymatic dissociation. The cells were then suspended in DMEM F12 N1 and incubated in A2B5-antibody containing supernatant (clone 105; American Type Culture Collection, Manassas, VA) for 30-45 min at 4°C on a shaker. The cells were washed 3x with PBS ∞ntaining 0.5% bovine serum albumin and 2 mM EDTA, then incubated with 1:4 diluted microbead-tagged rat anti-mouse IgM antibody (MACS, Miltenyi Biotech) for 30 min at 4°C on a shaker. The A2B5+ cells were washed, resuspended and separated using positive selection columns, type MS+/RS+ or LS+/VS+ (MACS, Miltenyi Biotech). The total number of viable cells was determined using calcein (Molecular Probes).
Affymetrix GeneChip protocol RNA was then extracted with Trizol (Invitrogen) and then purified using RNeasy (Qiagen) according to manufacturers specifications. lOOng of total RNA was amplified using the Affymetrix' small sample protocol, as per the manufacturers' recommendations. 15μg of cRNA was used on each U95A GeneChip. Although this method of amplification results in a greater 3' bias, 95% of the concordant probe sets using the standard protocol (x) were identical and the remaining 5% did not show signifciant bias toward absent (lOOng)/ present (lOμg).
Analysis of GeneChip expression data Data analysis. Image files were processed using MAS5.0 to produce CHP files. Images were masked to remove streaks/smears present. No scaling of data was performed during analysis. The CHP files were then imported into GeneSpring (5.0, Silicon Genetics) and the data normalized using the default settings for Affymetrix data sets. The normalized data was then processed using either GeneSpring tools or exported into an in-house Microsoft Access database. Selection of probe sets. Qualifying probe sets for each gene on the Affymetrix Human U95 chip set (Santa Clara, CA) were identified using the reference sequences from NCBI LocusLink (www.ncbi.nih.gov/locuslink') and Ensembl (www.ensembl.org/human). The probe match software on the NetAffx website was used to query the U95 probe data fwww.affymetrix.com/analvsis/probe match.prma). In general, probe sets that did not match greater than 12 out of 16 probe pairs were excluded. In addition, only the best probe set was used unless multiple probe sets were 16/16 perfectly matched. In the case of genes which did not provide at least one qualifying probe set, the search was extended by using the NetAffx & Ensembl generated annotations. These annotations were manually confirmed using by BLAST seach against the UniGene human EST database, requiring a perfect hit to at least one read within the same cluster as the gene, and Ensembl BLAST, to exclude intron containing and sense orientation probe sets which was not uncommon amongst NetAffx annotation. Annotation and data analysis was performed within the MS Access database.
Real-time PCR Primers and probes were either designed using Primer express (Applied Biosystems) or obtained as Assays-on-demand directly from Applied Biosystems. For each sample, four separate reverse transcription reactions of 25ng total RNA were performed as per manufacturer's protocol and the resulting cDNA diluted to lOOpg/ul. Four separate real-time PCR reactions with 500pg / reaction, in addition 2 no-RT control reactions were performed to check for RNA- independent product amplification. For taqman real-time PCR, 900nM forward and reverse primers, and 250nM FAM-labelled MGB probes. For SYBR Green real-time PCR, 300nM forward and reverse primers were used. The human 18S RNA was used as an endogenous control (ABI). The relative abundance of transcript expression was calculated following normalization of the Ct value to the matched unsorted white matter dissociate control, and the final expression ratio then normalized to the endogenous control. Significance was tested with two-way one-sample t-test against a ratio of 1 (n=3).
RESULTS Adult human WMPCs expressed oligodendrocyte progenitor marker genes Adult human subcortical white matter progenitor cells (WMPCs) were enriched by magnetic-activated cell sorting (MACS) using the A2B5 marker. From four epileptic temporal lobe resections cases, male ages 30-54, we obtained between 5 x 105 and 1 x 106 A2B5+ cells, that comprised between 3-5% of the total white matter dissociate. To identify those genes whose expression distinguishes the A2B5+ WMPC population from the other glial subtypes present in normal human adult white matter, we performed microarray analysis on RNA extracted from WMPCs immediately after sorting by A2B5-based MACs (n=3) and RNA from the matched unsorted white matter dissociate. Due to the limiting amounts of RNA obtained, starting from lOOng total RNA, we performed two rounds of RNA amplification prior to hybridization to Affymetrix HG-U95Av2 GeneChips according to Affymetrix's small sample protocol. Following microarray-wide normalization, the expression of individual genes in the isolated WMPC was normalized against the unsorted white matter dissociate from which it was derived, and the mean expression ratio calculated from the three samples. To analyze the microarray data, we first examined the expression of several known marker genes expressed by glial cells in adult white matter. The marker used to isolate WMPCs, the A2B5 antibody, primarily recognizes gangliosides, GT3 and its O-acetylated derivative. We found that the expression of GD3 synthase (SIAT8A), the enzyme that catalyzes the transfer of sialic acid from CMP-sialic acid to GM3 that produces gangliosides GD3 and GT3, is significantly enriched within the A2B5-isolated WMPC population. This observation was confirmed with real-time RT-PCR analysis (qPCR) of GD3 synthase mRNA levels following normalization to 18S ribosomal RNA (one sample t-test, Ho = 1, p<0.01). Furthermore, we found strong expression of PDGFαR and NG2 (CSPG4) in the WMPCs by microarray analysis and confirmed by real-time RT-PCR. These two genes are canonical in vivo markers of oligodendrocyte progenitors. Intriguingly, we also found evidence of expression of a novel truncated 1.5kb transcriptional variant of PDGFζR that is significantly enriched in WMPCs (5.16 ± 0.98). This variant does not contain the extracellular ligand-binding domain and has only previously been shown to be expressed by an undifferentiated teratocarcinoma cell line. The oligodendrocyte progenitor-expressed bHLH transcription factor Olig2, expressed throughout the oligodendrocyte lineage, was detected in the WMPC profile although it was not significantly enriched compared to the unsorted white matter dissociate; this was not surprising, since the latter was likely to contain other Olig2+ oligodendrocyte lineage cells. Markers of more mature oligodendrocyte lineage cells, the myelin protein genes, myelin basic protein (MBP) and proteolipid protein (PLPl), were not highly expressed by WMPCs. Furthermore, markers of other glial type cells present in adult white matter, namely astrocyte, microglial and endothelial cell markers were not significantly enriched in WMPCs.
Adult WMPCs are transcriptionally distinct from the local white matter environment We next identified a set of genes whose expression was enriched in the A2B5-sorted WMPC-enriched population compared to the unsorted white matter dissociate. Microarray data was analyzed using Genespring (Silicon Genetics). We first removed those probe sets whose data was deemed unreliable, by removing probe sets which were 'absent' in all three A2B5-sorted profiles. This list was then used to generate a list of Affymetrix probe sets whose A2B5+: unsorted expression ratio was significantly different from 1 (a one-sample t-test using cross-gene error model variances, with a Benjamini and Hochberg False Discovery Rate (FDR) of 20% post-hoc test). The resulting list of approximately 250 probe sets (<5% of total) was then pruned by removing those probe sets which were; 1. ambiguously annotated mapping to multiple genes, 2. un-annotated novel transcripts (not yet annotated to an NCBI LocusLink identifiers, www.ncbi.nih.gov/LocusLink). The remaining probe sets were annotated to 208 distinct genes. For each gene, additional probe sets were identified and we found that a number of genes had conflicting microarray expression data that could not be explained by the presence of specific alternatively spliced transcripts. As described above, a number of genes whose expression has previously been described in oligodendrocyte progenitors were found differentially enriched in the A2B5+ progenitor pool. Furthermore we found that expression of GAP43 was significantly enriched four fold in the WMPC-enriched profile. GAP43 is so called growth-associated protein that is expressed in growth cones during development but has previously been considered by some groups as a useful marker of rodent A2B5+ oligodendrocyte progenitors. However, a few genes whose expression has previously been described in neural cell types other than oligodendrocyte progenitors were also significantly enriched in the A2B5+ defined WMPC pool. GAD67 encodes one of two forms of glutamate decarboxylase (GAD) and has been commonly used as a marker of GABAergic intemeurons. Although no βlll-tubulin or MAP2 positive neurons were found by immunostaining immediately post-sort, we found that the mRNA for GAD67 was enriched >8 fold in the A2B5-enriched WMPC profile. Doublecortin (OCX), which is expressed on migrating immature neurons during development, was also found approximately 8 fold enriched in the WMPC. Jjitriguingly, two transcription factors expressed by neural progenitors and stem cells respectively, MASHl (ASCL1) and HES1 were found to be enriched in the WMPC pool. MASHl expression was 12- fold greater by microarray and over 18-fold by qPCR in the A2B5+ vs unsorted cell populations. HES1 was found to be 5 fold higher by microarray and over 12 fold higher by qPCR.
WMPCs express high levels of several receptor tyrosine kinases and phosphatases We also find enriched expression of other G-protein coupled receptors in the adult human WMPC population. The cannabinoid receptor (CNR1), which is expressed by rodent oligodendrocyte lineage cells, is significantly enriched in the A2B5-sorted WMPC profile and is expressed over 12 fold higher by qPCR. In addition, the novel GPR19, which is abundantly expressed in the human and rodent brain, is significantly enriched in both Affymetrix probe sets. Receptor tyrosine phosphatase zeta (PTPRZ1) was the single most significantly enriched gene from our microarray analysis and was over 15 fold enriched in WMPCs versus the unsorted cells by qPCR analysis (p<0.01). The high level expression of PTPRZl, which contains both a CPSG-like extracellular domain and intracellular protease domain, by A2B5-expressing WMPCs was remarkable in light of the developmental expression of this molecule by radial cells and neural progenitors of the fetal ventricular zone. More recently, this receptor phosphatase has been shown to be expressed by the rat CG4 OPC line in culture; and RPTPζ knock-out mice exhibited impaired recovery to MOG-induced EAE relative to wild-type mice. Jjitriguingly, the only known ligand of RPTPζ, pleiofrophin (PTN), was also found to be expressed significantly higher in the WMPC-enriched profile by both microarray and qPCR analysis (p<0.02). In addition to RPTPζ, PTN has been shown to bind the syndecan family of transmembrane heparin-sulphate proteoglycans and interestingly we find that one member, syndecan-3 (SDC3) was significantly enriched in the WMPC mRNA, 8 fold higher by qPCR (p<0.05). We also found that the PDZ-containing protein CASK, which has shown to bind the cytoplasmic domain of syndecans, was significantly enriched in WMPCs (2 fold higher than unsorted white matter).
WMPCs express a distinct set of cell-cell adhesion molecules We next examined the frequency of functionally related transcripts to determine relevant functional categories of genes. Over represented functional categories in the A2B5-sorted WMPC cell profile were determined by comparison with the entire population of genes on the HG-U95Av2 microarray. Using the EASE software tool (NIAJD) to examine the Gene Ontology (GO) biological process annotation (www.GeneOntology.org) of WMPC-enriched genes and identify statistically relevant classes of genes (p < 0.05, EASE score/adjusted Fisher exact test. Cell adhesion molecules and extracellular matrix proteins were significantly over represented in the WMPC-enriched gene list. These genes included members of the proteoglycan, immunoglobulin-like, and cadherin genes families. NrCAm may be of especial intesrt here since it has been identified as a heterophilic ligand for the RTPzeta ectodomain. Moreover, the related CAM-family member LI has similarly been identified as an RTPζ binding partner, and it too is differentially expressed by adult WMPCs, notwithstanding the high levels of LI expressed by neuronal axons in the white matter (whose mRNAs would nonetheless be largely cortical in distribution).
WMPCs differentially express the CSPGs Four chondroitin-sulphate proteoglycans (CSPG) were represented in the WMPC- enriched genes; versican (CSPG2), neurocan (CSPG3), NG2 (CSPG4) and neuroglycan C (CSPG5) were all significantly enriched in human A2B5-sorted WMPCs between 5-20 fold (Table 2). In addition to NG2, the rodent A2B5-posϊtive oligodendrocyte has been shown to express versican and neurocan. Neuroglycan C, a relatively recently cloned member of the aggrecan family, exhibits brain specific expression but the cellular localization in glia has yet to be addressed.
WMPC s may actively suppress local wnt signals A secreted WNT antagonist, SFRP3 (FRZB) is highly enriched in the A2B5+ WMPC profile over 15 fold greater than unsorted cells. SFRP3 binds WNTs x,y and z and blocks WNT ligand activation of frizzled receptors which can drive neural progenitor expansion in rodent neurospheres.
Expression of specific members of WNT, BMP, FGF and other signaling pathways within the WMPC Other than the WNT signaling pathway, we also find evidence for potential activation of the retinoic acid, BMP, and FGF pathways. RA - RARRES2 (as indicator of activation) & ALDH1A3 (as potential for RA synthesis). BMP - mab-21-like 1 & BAMBI (as indicators of activation) & increased synthesis of BMP2/7 (as activators). FGF - FGFR3 (receptor responsible for neural progenitor expansion) & sprouty 2 (SPRY2) as potential indicator of activation and negative feedback.
The WMPC-enriched expression profile indicates the expression of a number of proteins involved in sterol biosynthesis and metabolism Although oligodendrocytes are the major lipid-producing cell in the CNS, we found that a relatively large number of genes that are involved in sterol biosynthesis and metabolism are enriched in the A2B5+ WMPC profile. Specifically, these genes included 3-hydroxy-3- methylglutaryl-coenyzme A reductase (HMGCR), which is the rate-limiting enzyme in cholesterol biosynthesis, and the low density lipoprotein receptor (LDLR), responsible for cellular uptake of LDL increasing the availability of intracellular cholesterol. Furthermore we found significantly increased expression of INSIGl (2.1 fold enriched), an intracellular protein that is thought to control both cholesterol homeostasis and pre-adipocyte differentiation and maturation.
Discussion In this study, we used a genomic analysis to define the differences in gene expression between adult human WMPC and the white matter environment from which they derive. This permitted us to identify ligand-receptor relationships that modulate the turnover and fate of these cells. Specifically, by comparing the expressed RNA profiles of adult human WMPCs to those of the parental white matter tissue from which each progenitor sample has been extracted, we identified differentially expressed genes in the tissue that appeared to complement others selectively expressed by the progenitor pool. By this means, we identified several hitherto unpredicted ligand-receptor interactions and their in cis modifiers. These data suggest the importance of the RTPz/PTN/syndecan system as a potential targets for both genetic and pharmacological modulation of progenitor cell turnover and induction.
i NftotJAu-i LEF? /ZufaKJ
Cell adhesion and extracellular matrix
Figure imgf000033_0001
TABLE. 2
Figure imgf000034_0001
o
Ligands
Figure imgf000035_0001
soluble
Figure imgf000035_0002
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
HUGO homepage http://www.gene.ucl.ac.uk/nomenclature/
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For each known human gene we approve a gene name and symbol (short-form abbreviation). All approved symbols are stored in Genew. the Human Gene Nomenclature Database. Each symbol is unique and we ensure that each gene is only given one approved gene symbol. It is necessary to provide a unique symbol for each gene so that we and others can talk about them, it also facilitates electronic data retrieval from publications. In preference each symbol maintains parallel construction in different members of a gene family and can also be used in other species, especially the mouse.
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Thus the present invention extends various embodiments described below. Each bracketed number refers to an embodiment of the present invention.
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L 1 • J A method of modulating production of neurons and/or oligodendrocytes from neural progenitor cells of post-natal or adult human white matter, said method comprising: administering an agonist or antagonist of one or more molecules set forth in Tables 1, 2, 3, 4, 5, and/or 6 to the neural progenitor cells under conditions effective to modulate production of neurons and/or oligodendrocytes.
{_2.J The method of (1 ^wherein the neural progenitor cells are oligodendrocyte progenitor cells. f3.~) The method of jl/, wherein the one or more molecules is set forth in
Table 1. 4 The method of |3J, wherein the one or more molecules is a receptor tyrosine phosphatase. jj . The method of Q, wherein the one or more molecules is RPTP-zeta. ( The method of 1 wherein the one or more molecules is a chondrotin sulfate proteoglycan.
( The method of [όj wherein the one or more molecules is phosphocan, versican, neurocan, NG2, or neuroglycan C/NGC. g. The method of 3J, wherein the one or more molecules is a syndecan. 9J. - The method of {8] wherein the one or more molecules is syndecan-3. jl 7 The method of βj, wherein the one or more molecules is set forth in
Table 2. Jl V. The method of Jl Oj, wherein the one or more molecules is adenylate cyclase 8. l 2 The method of (fL wherein the one or more molecules is set forth in Table 3. l 3j. The method of jj- , wherein the one or more molecules is selected from the group consisting of pleiotrophin, platelet-derived growth factor, NEL-like 1, neuralin 1, BMP2 (a Dpp homologue), OP-1, chromoganin B (seαretogranin 1), or secretogranin II (cljTomogramn C).
4} The method of" - f 3j, wherein one or more molecules is pleiofrophin. μ5j The method o^ μ3j, wherein the one or more molecules is NEL-like
jl 6J. The method of J13J, wherein the one or more molecules is neuralin 1. l 7 The method of jlj, wherein the one or more molecules is set forth in
Table 4. jl 8J. The method of (17J, wherein the one or molecules is a receptor tyrosine kinase.
'[I | The method of [17}, wherein the one or more molecules is platelet- derived growth factor receptor, cannabinoid receptor 1, G protein-coupled receptor 19, activated p21cdc42Hs kinase, or transmembrane 4 superfarnily member 2. |2θj. The method of- Jl % wherein the one or more molecules is GAB A A receptor, glycine receptor beta, glutamate receptor ionotrophic AMPA 2, or glutamate receptor iontropic kainate 1. j2 . The method of la, wherein the one or more molecules is set forth in Table 5.
122] The method c fl, wherein the one or more molecules is set forth in Table 6.
(23j. The method of J22, wherein one or more molecules is zinc finger DAZ interacting protein 1.
[24] The method of l], wherein said administering is carried out in vivo.
The method of πi wherein said administering is carried out in vitro.
The method of J ), wherein the neural progenitor cells are from a post-natal human.
J27J. The method of Q, wherein the neural progenitor cells are from an adult human. j28J The method of 111 wherein the one or more molecules modulate oligodendrocyte progenitor mobilization, division, proliferation, differentiation, and/or self- maintenance.
129]. The method o - ffl,-wherein the one or more molecules modulate oligodendrocyte maturation, differentiation, myelin production, and/or axonal myelination. ]3(j. A method of treating a subject for a condition modulated by underproduction or overproduction of neurons and/or oligodendrocytes from neural progenitor cells of post-natal or adult human white matter, said method comprising: administering to the subject an agonist or antagonist of one or more molecules molecules set forth in Tables 1, 2, 3, 4, 5, and/or 6 under conditions effective to treat the condition modulated by underproduction or overproduction of neurons and/or oligodendrocytes.
[3 J. The method of J3QJ, wherein the neural progenitor cells are oligodendrocyte progenitor cells.
|3^. The method of J30L wherein the one or more molecules is set forth in
Table 1.
J33J. The method of |32j> wherein the one or more molecules is a receptor tyrosine phosphatase. The method of B3J, wherein the one or more molecules is RPTP-zeta.
Figure imgf000045_0001
The method of 32j wherein the one or more molecules is a chondrotin sulfate proteoglycan.
^g. The method of (3 , wherein the one or more molecules is phosphocan, versican, neurocan, NG2, or neuroglycan C/NGC. The method of J3|, wherein the one or more molecules is a syndecan.
^38j. The method of g wherein the one or more molecules is syndecan-
3. J39J. The method of |3(j, wherein the one or more molecules is set forth in Table 2.
^Oj. The method of |3SJ, wherein the one or more molecules is adenylate cyclase 8.
141). The method of wherein the one or more molecules is set forth in
Figure imgf000046_0001
Table 3.
(42 The method of [ fij, wherein the one or more molecules is selected from the group consisting of pleiotrophin, platelet-derived growth factor, NEL-like 1, neuralin 1, BMP2 (a Dpp homologue), OP-1, chromoganin B (secretogranin 1), or secretogrardn II (clrromografiin C).
[43 The method of |42j, wherein one or more molecules is pleiofrophin. '
/44J. The method of "> . (ΪJ2J, wherein the one or more molecules is NEL-like 1. 43. The method of (4^, wherein the one or more molecules is neuralin 1. The method of j30 wherein the one or more molecules is set forth in Table 4.
J47J. ' The method of J46 wherein the one or molecules is a receptor tyrosine kinase. The method of - J46J wherein the one or more molecules is platelet- derived growth factor receptor, cannabinoid receptor 1, G protein-coupled receptor 19, activated p21cdc42Hs kinase, or transmembrane 4 superfamily member 2. β9j. The method of , wherein the one or more molecules is GABA A receptor, glycine receptor beta, glutamate receptor ionotrophic AMPA 2, or glutamate receptor iontropic kainate 1.
J50j. The method of gOJ wherein the one or more molecules is set forth in
Table 5.
J5 lj. The method of |3y, wherein the one or more molecules is set forth in
Table 6.
J52|. The method of p lj, wherein one or more molecules is zinc finger
DAZ interacting protein 1.
(f>^. The method of \30j, wherein the neural progenitor cells are from a post-natal human. p4 The method of (30J, wherein the neural progenitor cells are from an adult human.
(55} The method of 1 (3 (J, wherein the condition is modulated by oligodendrocyte progenitor mobilization, division, proliferation, differentiation, and/or self- maintenance.
J56j. The method of H^, wherein the condition is modulated by oligodendrocyte maturation, differentiation, myelin production, and or axonal myelination. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:
1. A method for determining whether a compound or agent modulates activity of a protein tyrosine phosphatase receptor (PTPRζ), comprising the steps:
(a) contacting the compound or agent with the PTPRζ; and
(b) determining whether the compound or agent binds to the PTPRζ.
2. The method of Claim 1, wherein the agent or compound is immobilized on a substrate.
3. The method of Claim 2, wherein the substrate comprises polyvinylidene difluoride or nitrocellulose.
4. The method of Claim 1, wherein the PTPRζ is immobilized on a substrate.
5. The method of Claim 4, wherein the substrate comprises polyvinylidene difluoride or nitrocellulose.
6. The method of any of Claims 1-5, wherein the compound or agent is detectably labeled.
7. The method of any of Claims 1-6, wherein the PTPRζ is detectably labeled.
8. The method of any of Claims 1-7, wherein the PTPRζ comprises the amino acid sequence of SEQ ID NO: 1.
9. The method of any of Claims 1-8, wherein the method is performed in a high throughput manner.
10. A method for detemήning whether a compound or agent modulates activity of a protein tyrosine phosphatase receptor (PTPRζ), comprising the steps of:
(a) contacting the compound or agent with a CNS derived progenitor cell that expresses PTPRζ; and then
(b) determining whether the progenitor cell differentiates into an oligodendrocyte, wherein such differentiation indicates the compound or agent may modulate the activity of the PTPRζ.
11. The method of Claim 10, wherein the CNS derived progenitor cell is a post-natal progenitor cell, or an adult human white matter progenitor cell.
12. The method of Claims 10, wherein the progenitor cell that expresses the PTPRζ is located within an organism, and the contacting step comprises administering the compound or agent to the organism.
13. A method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the number of oligodendrocytes that express PTPRζ within the bodily sample is known; (b) measuring the number of oligodendrocytes that express PTPRζ, after contact with the compound or agent, wherein an increase the number oligodendrocytes that express PTPRζ, in the bodily sample after contact with the compound is indicative that the compound or agent may modulate activity of the PTPRζ.
14. A method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the number of oligodendrocyte progenitor cells that express PTPRζ within the bodily sample is known; (b) measuring the number of oligodendrocyte progenitor cells that express PTPRζ, after contact with the compound or agent, wherein an increase in number oligodendrocyte progenitor cells that express PTPRζ, in the bodily sample after contact with the compound or agent indicates that the compound or agent may modulate activity of the PTPRζ.
15. A method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the extent of myelination in the sample is known; and (b) determining the extent of myelination in the bodily sample after contact with the compound or agent, wherein an increase in the extent of myelination in the sample after contact with the compound or agent indicates that the compound or agent may modulate PTPRζ.
16. A method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of:
(a) contacting the compound or agent with a bodily sample taken from an organism, wherein the extent of oligodendrocyte differentiation in the sample is known; and
(b) measuring the extent of oligodendrocyte differentiation in the bodily sample after contact with the compound or agent, wherein an increase in the extent of oligodendrocyte differentiation in the bodily sample after contact with the compound or agent indicates that the compound or agent may modulate the activity of PTPRζ..
17. A method for determining whether a compound or agent modulates activity of an PTPRζ comprising the steps of: (a) contacting the compound or agent with a bodily sample taken from an organism, wherein the PTPRζ activity or activity of a relevant member of the PTPRζ pathway in the sample is known; and (b) determining the activity of PTPRζ or the activity of the relevant member of the PTPRζ pathway in the bodily sample after contact with the compound or agent, wherein an increase in PTPRζ activity or activity of the relevant member of the PTPRζ pathway in the bodily sample after contact with the compound or agent indicates that the compound or agent may modulate PTPRζ.
18. The method of any of Claims 12-17, wherein the bodily sample is white matter taken from the brain of the organism.
19. The method of any of Claims 12-18, wherein the organism is selected from the group consisting of an equine, a bovine, a canine, a feline, a human, a primate, an ovine, and a rodent.
PCT/US2004/037514 2003-11-10 2004-11-09 Method for assaying compounds that modulate the activity of protein tyrosine phosphatase receptor zeta (ptprzeta) WO2005047530A2 (en)

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