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Expression of P0 glycoprotein in CNS glia: Effects of overexpression in N20.1 cells
Author(s) -
Studzinski Diane M.,
Benjamins Joyce A.
Publication year - 2005
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/glia.20240
Subject(s) - biology , transfection , gfap stain , microbiology and biotechnology , glial fibrillary acidic protein , western blot , cell culture , oligodendrocyte , myelin basic protein , myelin , immunocytochemistry , gene , immunology , immunohistochemistry , central nervous system , biochemistry , genetics , neuroscience , endocrinology
To examine effects of expression of the PNS myelin P0 glycoprotein in glial cells of CNS lineage, we transfected murine N20.1 glial cells with a rat P0 cDNA. A stably transfected cell line expressing high levels of P0 message showed P0 immunostaining, along with changes in morphology. Polymerase chain reaction (PCR) identified the predicted rat P0 sequence in the transfected N20.1 cells and further revealed low levels of mouse P0 message in the nontransfected cells and in primary mouse astrocytes. This is the first evidence of endogenous expression of message for P0 glycoprotein in CNS glia. Quantitative RT‐PCR confirmed the expression of rat P0 mRNA in the transfected N20.1 cells, at levels about 400 times greater than murine P0 in nontransfected cells. A 27‐kD band was detected in the transfected cells by Western blot with P0 antibody, but not in mock‐transfected or nontransfected N20.1 cells. Immunocytochemistry following permeabilization showed intracellular vesicular localization of P0 in the cytoplasm and perinuclear rings in transfected cells, with a similar pattern but much lower levels in nontransfected cells. Faint surface staining for P0 protein without permeabilization was seen only on the transfected cells. A few transfected cells with membrane sheets stained more intensely for surface P0. Quantitative RT‐PCR was used to determine if P0 overexpression altered expression of other myelin‐related genes compared with glial fibrillary acidic protein (GFAP); the ratios of myelin basic protein (MBP)/GFAP and proteolipid protein (PLP)/GFAP were increased 2‐ to 3‐fold in the P0‐transfected cells. We conclude that P0 overexpression alters N20.1 gene expression and cell morphology, and shifts the cells from astroglial to oligodendroglial phenotype. © 2005 Wiley‐Liss, Inc.