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Isolation and characterization of defective jimpy oligodendrocytes in culture
A C Feutz1, I Bellomi, B Allinquant
1Laboratoire de Neurobiologie Ontogénique (UPR 417-CNRS), Centre de Neurochimie, Strasbourg, France.
Abstract:
This study characterizes jimpy oligodendrocyte-enriched secondary cultures isolated from 10-12 days in vitro primary glial cell cultures derived from 1-2-day-old jimpy mouse brains. Proliferation of defective oligodendrocytes was carefully investigated with regard to the expression of myelin basic protein and proteolipid protein and their respective mRNAs. Less than 5% of contaminating astrocytes (GFAP+ cells) were usually present. The identity of jimpy oligodendrocytes was confirmed using an antibody directed against a peptide from the wild type proteolipid protein C-terminal sequence for immunocytochemistry and an oligonucleotide complementary to mRNA derived from exon 5 of the proteolipid protein gene for in situ hybridization. Both the antibody and the probe recognize only normal oligondedrocytes while jimpy oligodendrocytes always remain unstained. Proteolipid protein in normal and jimpy oligodendrocytes was detected with antibody recognizing normal and mutated forms. Between 80 and 95% of the cells in normal and jimpy cultures at 2 and 4 days in vitro in secondary cultures express myelin basic protein and proteolipid protein and their respective mRNAs. The percentage of oligodendrocytes (PLP+ or MBP+) in S phase of the cell cycle was 7-10% for both normal and jimpy oligodendrocytes. This contrasts with the in vivo situation where the proliferation rate of oligodendrocytes in jimpy brains is higher than in normal brains. In addition, jimpy oligodendrocytes remain unresponsive to basic fibroblast growth factor treatment while a similar treatment stimulates the proliferation of normal oligodendrocytes.
Insights
Jimpy oligodendrocytes, crucial for myelin production, show normal proliferation and gene expression in vitro, unlike their in vivo counterparts. They also fail to respond to growth factor stimulation.
Area of Science:
- Neuroscience
- Cell Biology
- Genetics
Background:
- Oligodendrocytes are glial cells responsible for myelin sheath formation in the central nervous system.
- The jimpy mouse model exhibits dysmyelination due to mutations affecting oligodendrocyte development and function.
- Understanding oligodendrocyte behavior in vitro is crucial for studying demyelinating diseases.
Purpose of the Study:
- To characterize jimpy oligodendrocyte-enriched secondary cultures.
- To investigate the proliferation and gene expression of defective oligodendrocytes in vitro.
- To compare in vitro findings with the in vivo situation in jimpy mice.
Main Methods:
- Isolation and culture of oligodendrocyte-enriched glial cells from jimpy and normal mouse brains.
- Immunocytochemistry and in situ hybridization to identify and confirm oligodendrocyte identity.
- Assessment of myelin basic protein (MBP) and proteolipid protein (PLP) expression.
- Cell cycle analysis to determine proliferation rates.
- Treatment with basic fibroblast growth factor (bFGF).
Main Results:
- Jimpy and normal oligodendrocytes in secondary cultures expressed MBP and PLP and their mRNAs at similar rates (80-95%).
- The percentage of oligodendrocytes in S phase was comparable (7-10%) in both normal and jimpy cultures in vitro.
- Jimpy oligodendrocytes were unresponsive to bFGF, while normal oligodendrocytes showed stimulated proliferation.
Conclusions:
- In vitro, jimpy oligodendrocytes exhibit normal proliferation and myelin protein gene expression, contrasting with their increased proliferation in vivo.
- The lack of response to bFGF in jimpy oligodendrocytes suggests a defect in growth factor signaling pathways.
- These findings highlight the importance of the in vivo microenvironment in regulating oligodendrocyte behavior in the jimpy model.