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Spatial distribution of myelin basic protein mRNA and polypeptide in quaking oligodendrocytes in culture
1Department of Neurology, University of Connecticut Health Center, Farmington 06032.
Abstract:
In the CNS, myelin is formed from the expansion of oligodendrocyte processes. In order to study myelin assembly in the hypomyelinating mutant mouse quaking (qk), cultures of oligodendrocytes were established from affected and control animals. The cytoarchitecture of the oligodendrocytes was analyzed by performing morphometric measurements after immunostaining with antitubulin. The results indicate that the gross morphology of the processes is similar in control and mutant cells. The localization of the message for the myelin structural component, myelin basic protein (MBP), was examined by in situ hybridization. In control oligodendrocytes, 80% of MBP mRNA is found in the processes. In contrast, only 23% of MBP mRNA is localized to these structures in the mutant; the majority of MBP mRNA remains in the cell body. The mutant cells are capable of distributing mRNAs to the periphery as shown by the presence of tubulin mRNA in their processes. MBP polypeptide was visualized by immunofluorescence and found in the perikaryon, processes and membranous expansions of the control cells. In the mutant, it is largely confined to the perikaryon, reflecting the distribution of the mRNA. These results suggest that the localization of MBP polypeptide is achieved by restricting the distribution of its mRNA, and that MBP assembly into the myelin membrane occurs in the processes. This step appears to be blocked in qk oligodendrocytes in culture.
Insights
In quaking (qk) mutant mice, myelin basic protein mRNA fails to localize to oligodendrocyte processes, blocking myelin assembly. This defect in mRNA transport hinders myelin formation in the central nervous system.
Area of Science:
- Neuroscience
- Cell Biology
- Developmental Biology
Background:
- Myelin, a crucial component of the central nervous system (CNS), is formed by oligodendrocyte processes.
- The quaking (qk) mouse model exhibits hypomyelination, making it valuable for studying myelin assembly defects.
Purpose of the Study:
- To investigate the cellular and molecular mechanisms underlying myelin assembly defects in quaking (qk) mutant oligodendrocytes.
- To determine the role of mRNA localization in myelin basic protein (MBP) incorporation into myelin.
Main Methods:
- Primary oligodendrocyte cultures were established from quaking (qk) mutant and control mice.
- Immunostaining with antitubulin for morphometric analysis of cell processes.
- In situ hybridization to examine myelin basic protein (MBP) mRNA localization.
- Immunofluorescence to visualize MBP polypeptide distribution.
Main Results:
- Oligodendrocyte process morphology was similar between control and mutant cells.
- In control oligodendrocytes, 80% of MBP mRNA was localized to processes, compared to only 23% in mutant cells.
- MBP polypeptide was largely confined to the perikaryon in mutant oligodendrocytes, unlike control cells where it was found throughout processes and extensions.
Conclusions:
- Myelin basic protein (MBP) polypeptide localization to oligodendrocyte processes is regulated by the transport of its mRNA.
- The assembly of MBP into myelin membranes occurs within the cell processes.
- This mRNA localization-dependent assembly step is impaired in quaking (qk) mutant oligodendrocytes, leading to hypomyelination.