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De novo production of alpha 2-macroglobulin in cultured astroglia from rat brain
1Department of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan.
Abstract:
Previous studies from our laboratory demonstrated that alpha 2-macroglobulin (alpha 2M) is one of the neurite-promoting factors in the conditioned medium of astroglia. In the present study, we further examined the de novo production of alpha 2M in cultured astroglia by determining the expression of alpha 2M mRNA, and the biosynthesis of [35S]methionine-labeled alpha 2M protein. We analyzed the mRNA of cultured astroglia by differential hybridization using specific probes to alpha 2M and its homologous protein, alpha 1-inhibitor 3 (alpha 1I3), after amplification of reverse-transcribed cDNA with the polymerase chain reaction. The result clearly showed that only alpha 2M mRNA is expressed in cultured astroglia. Northern blotting analysis revealed that alpha 2M mRNA is expressed mainly in the astroglia and is not detected in neurons, microglia and meningeal fibroblasts. Furthermore, the biosynthesis of alpha 2M protein in the astroglia was confirmed by an immunoprecipitation experiment after labeling of each type of cell with [35S]methionine. It was concluded that alpha 2M is produced in the cultured astroglia which is the major source of alpha 2M production among various types of cells in rat brain.
Insights
Alpha 2-macroglobulin (alpha 2M) is produced by cultured astroglia, making them a primary source of this neurite-promoting factor in the rat brain. This study confirms astroglia
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Alpha 2-macroglobulin (alpha 2M) is identified as a neurite-promoting factor in astroglial conditioned medium.
- Previous research established alpha 2M's role in neural development.
Purpose of the Study:
- To investigate the de novo production of alpha 2M in cultured astroglia.
- To determine the expression of alpha 2M mRNA and the biosynthesis of alpha 2M protein in astroglia.
Main Methods:
- Differential hybridization and polymerase chain reaction (PCR) were used to analyze alpha 2M mRNA expression.
- Northern blotting identified the primary cell types expressing alpha 2M mRNA.
- [35S]methionine labeling followed by immunoprecipitation confirmed alpha 2M protein biosynthesis.
Main Results:
- Cultured astroglia exclusively express alpha 2M mRNA, not the homologous alpha 1-inhibitor 3 (alpha 1I3).
- Northern blotting showed alpha 2M mRNA is predominantly in astroglia, absent in neurons, microglia, and meningeal fibroblasts.
- Immunoprecipitation confirmed the biosynthesis of alpha 2M protein in astroglia.
Conclusions:
- Cultured astroglia are confirmed as producers of alpha 2M.
- Astroglia represent the major source of alpha 2M production within the rat brain compared to other cell types.