Screening methods for identifying Sp35 antagonists
View Patent ↗The invention provides methods of treating diseases, disorders or injuries involving demyelination and dysmyelination, including multiple sclerosis, by the administration of an Sp35 antagonist.
1. A method of identifying an Sp35 antagonist capable of promoting oligodendrocyte-mediated myelination comprising contacting a co-culture of neurons and oligodendrocytes with a compound that binds to Sp35 or to a polynucleotide that encodes Sp35 and measuring myelination, wherein increased myelination compared to a control co-culture indicates the compound is an Sp35 antagonist capable of promoting oligodendrocyte-mediated myelination.
2. The method of claim 1 , wherein the myelination is assessed by measuring myelin basic protein (MBP).
3. The method of claim 2 , wherein the Sp35 antagonist increases MBP at least ten-fold compared to the control co-culture.
4. The method of claim 1 , wherein myelination is assessed by measuring the number of cells expressing MBP.
5. The method of claim 4 , wherein the Sp35 antagonist increases the number of cells expressing MBP at least five-fold compared to the control co-culture.
6. The method of claim 1 , wherein the Sp35 antagonist causes an increase in myelination comparable to the increase in the presence of a soluble Sp35 polypeptide comprising amino acids 34-532 of SEQ ID NO:2.
7. The method of claim 1 , wherein the Sp35 antagonist comprises a nucleic acid molecule which specifically binds to the polynucleotide which encodes Sp35.
8. A method of identifying an Sp35 antagonist capable of promoting oligodendrocyte differentiation comprising contacting a culture of progenitor oligodendrocytes with a compound that binds to Sp35 or to a polynucleotide that encodes Sp35 and quantitating the number of pre-myelinating or mature oligodendrocytes, wherein increased pre-myelinating or mature oligodendrocytes compared to a control culture indicates the compound is an Sp35 antagonist capable of promoting oligodendrocyte differentiation.
9. The method of claim 8 , wherein the number of mature oligodendrocytes is measured by MBP expression.
10. The method of claim 9 , wherein the Sp35 antagonist increases MBP expression at least 3-fold compared to the control culture.
11. The method of claim 8 , wherein the Sp35 antagonist decreases RhoA-GTP.
12. The method of claim 11 , wherein the Sp35 antagonist decreases RhoA-GTP by at least three-fold as compared to the control culture.
13. The method of claim 8 , wherein the Sp35 antagonist increases Fyn protein.
14. The method of claim 13 , wherein the Sp35 antagonist increases Fyn protein at least two-fold as compared to the control culture.
15. The method of claim 8 , wherein the Sp35 antagonist increases Fyn phosphorylation.
16. The method of claim 8 , wherein the Sp35 antagonist causes an increase in the number of pre-myelinating or mature oligodendrocytes comparable to the increase in the presence of a soluble Sp35 polypeptide comprising amino acids 34-532 of SEQ ID NO:2.
17. The method of claim 8 , wherein the Sp35 antagonist comprises a nucleic acid molecule which specifically binds to the polynucleotide which encodes Sp35.
18. A method of identifying an Sp35 antagonist capable of promoting oligodendrocyte survival comprising contacting a culture of mature oligodendrocytes with a compound that binds to Sp35 or to a polynucleotide that encodes Sp35 and measuring oligodendrocyte survival, wherein increased oligodendrocyte survival compared to a control culture indicates the compound is an Sp35 antagonist capable of promoting oligodendrocyte survival.
19. The method of claim 18 , wherein the Sp35 antagonist causes an increase in oligodendrocyte survival comparable to the increase in the presence of a soluble Sp35 polypeptide comprising amino acids 34-532 of SEQ ID NO:2.
20. The method of claim 18 , wherein the Sp35 antagonist comprises a nucleic acid molecule which specifically binds to the polynucleotide which encodes Sp35.