Related Experiment Videos
Functional expression of the CXC-chemokine receptor-4/fusin on mouse microglial cells and astrocytes
S Tanabe1, M Heesen, I Yoshizawa
1Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
The mRNA for the seven-transmembrane-spanning G protein-coupled receptor fusin/CXCR-4 is expressed in primary mouse astrocyte cultures and the transformed mouse microglial cell line, N9. Cell surface expression of fusin in these cells was confirmed by staining with a polyclonal anti-fusin Ab. The functional capacity of this chemokine receptor was examined by evaluating the calcium responses following stimulation of glial cells with the CXC-chemokine, stromal-derived cell factor-1alpha (SDF-1alpha). Both astrocytes and microglial cells mobilized calcium following stimulation with chemically synthesized SDF-1alpha. SDF-1alpha- and carbachol-mediated calcium responses of astrocytes were partially inhibited by treatment with pertussis toxin (PTx), suggesting receptor coupling to a combination of G alpha(i) and other G proteins. In contrast, the calcium responses of microglial cells to SDF-1alpha were completely PTx sensitive, while responses to carbachol stimulation were PTx resistant. The ability of SDF-1alpha to induce glial cell migration was also examined. Synthetic SDF-1alpha was a potent chemoattractant for mouse microglial cells at ligand concentrations of 10 to 500 ng/ml; peak responses were noted at 100 ng/ml. In contrast, astrocytes did not migrate toward a gradient of SDF-1alpha. The failure of SDF-1alpha to induce astrocyte migration was specific, as another chemokine, macrophage inflammatory protein-1alpha, triggered astrocyte chemotaxis.
Insights
The G protein-coupled receptor fusin (CXCR-4) is expressed in mouse glial cells. Stromal-derived cell factor-1alpha (SDF-1alpha) induces calcium responses in astrocytes and microglial cells, and chemoattracts microglial cells.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- G protein-coupled receptors (GPCRs) play crucial roles in cellular signaling.
- Fusin (CXCR-4) is a seven-transmembrane-spanning GPCR involved in immune cell trafficking.
- Glial cells, including astrocytes and microglia, are key components of the central nervous system immune system.
Purpose of the Study:
- To investigate the expression and function of fusin (CXCR-4) in primary mouse astrocytes and a mouse microglial cell line (N9).
- To determine the signaling pathways involved in fusin-mediated responses in glial cells.
- To assess the role of fusin in glial cell migration.
Main Methods:
- mRNA expression analysis of fusin in astrocytes and N9 microglial cells.
- Cell surface protein staining using anti-fusin antibody.
- Calcium mobilization assays in response to stromal-derived cell factor-1alpha (SDF-1alpha) and carbachol.
- Pertussis toxin (PTx) sensitivity assays to determine G protein coupling.
- Chemotaxis assays to evaluate glial cell migration towards SDF-1alpha and macrophage inflammatory protein-1alpha.
Main Results:
- Fusin (CXCR-4) mRNA and cell surface protein were detected in both primary mouse astrocytes and N9 microglial cells.
- SDF-1alpha stimulated calcium mobilization in both cell types.
- Astrocyte calcium responses were partially inhibited by PTx, indicating coupling to G alpha(i) and other G proteins.
- Microglial cell calcium responses to SDF-1alpha were completely PTx-sensitive, while carbachol responses were PTx-resistant.
- SDF-1alpha acted as a potent chemoattractant for microglial cells but not for astrocytes.
- Astrocytes migrated towards macrophage inflammatory protein-1alpha, confirming their chemotactic capacity.
Conclusions:
- Fusin (CXCR-4) is functionally expressed on mouse astrocytes and microglial cells, mediating distinct signaling pathways.
- SDF-1alpha acts as a chemoattractant for microglial cells, suggesting a role in neuroinflammation and immune surveillance.
- The differential response of astrocytes and microglia to SDF-1alpha highlights cell-specific functions of chemokine receptors in the CNS.