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The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells.

Imbeault S, Gauvin LG, Toeg HD, Pettit A, Sorbara CD, Migahed L, DesRoches R, Menzies AS, Nishii K, Paul DL, Simon AM, Bennett SA - BMC Neurosci (2009)

Bottom Line: Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30.These changes are associated with decreased neurogenesis.Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, Dept. of Biochemistry, Microbiology, and Immunology, University of Ottawa, ON, Canada. even_grable@hotmail.com

ABSTRACT

Background: Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells.

Results: We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication.

Conclusion: Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.

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Cx30 and Cx43 are expressed by Type 1 and Type 2a NPCs and are responsive to laminin. Cx30 and Cx43 was detected in the majority of cells within neurospheres (a, d, arrows) and localized to cells expressing nestin (b, e, insets) and GFAP (c, f, insets). Inset in (a) demonstrates lack of Cx30 immunostaining on -mutant neurospheres confirming antibody specificity. Adherence to laminin did not impact on the frequency of Cx30+ cells (g) although a change in subcellular localization was observed (compare punctate membrane staining (a, arrows) with diffuse cytoplasmic staining (i, arrows)). A decrease in the frequency of Cx43+ cells (arrows) was detected following culture on laminin (j). Cx43 was present at the plasma membrane between cells in direct apposition in the presence (a) or absence (b, small arrows) of laminin. Scale bars, 50 μm, insets in a, b, c, f, 25 μm, inset in e,10 μm. Data represent mean of 5–15 sections (- laminin) or field (+ laminin) counted over n = 5–10 cultures/condition ± SEM (g, j). **p < 0.01 Student's t-test.
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Figure 3: Cx30 and Cx43 are expressed by Type 1 and Type 2a NPCs and are responsive to laminin. Cx30 and Cx43 was detected in the majority of cells within neurospheres (a, d, arrows) and localized to cells expressing nestin (b, e, insets) and GFAP (c, f, insets). Inset in (a) demonstrates lack of Cx30 immunostaining on -mutant neurospheres confirming antibody specificity. Adherence to laminin did not impact on the frequency of Cx30+ cells (g) although a change in subcellular localization was observed (compare punctate membrane staining (a, arrows) with diffuse cytoplasmic staining (i, arrows)). A decrease in the frequency of Cx43+ cells (arrows) was detected following culture on laminin (j). Cx43 was present at the plasma membrane between cells in direct apposition in the presence (a) or absence (b, small arrows) of laminin. Scale bars, 50 μm, insets in a, b, c, f, 25 μm, inset in e,10 μm. Data represent mean of 5–15 sections (- laminin) or field (+ laminin) counted over n = 5–10 cultures/condition ± SEM (g, j). **p < 0.01 Student's t-test.

Mentions: We used immunofluorescence to localize connexins to NPC subtypes grown in the absence of exogenous laminin (Fig 3, 5, 6). For all connexins except Cx26, cultures from -mutant mice served as negative controls to ensure antibody specificity. For Cx26, where deletion is embryonic lethal [23], antibody specificity was determined by Western blot (Fig 4a, inset). Four distinct connexin protein profiles were detected:


The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells.

Imbeault S, Gauvin LG, Toeg HD, Pettit A, Sorbara CD, Migahed L, DesRoches R, Menzies AS, Nishii K, Paul DL, Simon AM, Bennett SA - BMC Neurosci (2009)

Cx30 and Cx43 are expressed by Type 1 and Type 2a NPCs and are responsive to laminin. Cx30 and Cx43 was detected in the majority of cells within neurospheres (a, d, arrows) and localized to cells expressing nestin (b, e, insets) and GFAP (c, f, insets). Inset in (a) demonstrates lack of Cx30 immunostaining on -mutant neurospheres confirming antibody specificity. Adherence to laminin did not impact on the frequency of Cx30+ cells (g) although a change in subcellular localization was observed (compare punctate membrane staining (a, arrows) with diffuse cytoplasmic staining (i, arrows)). A decrease in the frequency of Cx43+ cells (arrows) was detected following culture on laminin (j). Cx43 was present at the plasma membrane between cells in direct apposition in the presence (a) or absence (b, small arrows) of laminin. Scale bars, 50 μm, insets in a, b, c, f, 25 μm, inset in e,10 μm. Data represent mean of 5–15 sections (- laminin) or field (+ laminin) counted over n = 5–10 cultures/condition ± SEM (g, j). **p < 0.01 Student's t-test.
© Copyright Policy - open-access
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Figure 3: Cx30 and Cx43 are expressed by Type 1 and Type 2a NPCs and are responsive to laminin. Cx30 and Cx43 was detected in the majority of cells within neurospheres (a, d, arrows) and localized to cells expressing nestin (b, e, insets) and GFAP (c, f, insets). Inset in (a) demonstrates lack of Cx30 immunostaining on -mutant neurospheres confirming antibody specificity. Adherence to laminin did not impact on the frequency of Cx30+ cells (g) although a change in subcellular localization was observed (compare punctate membrane staining (a, arrows) with diffuse cytoplasmic staining (i, arrows)). A decrease in the frequency of Cx43+ cells (arrows) was detected following culture on laminin (j). Cx43 was present at the plasma membrane between cells in direct apposition in the presence (a) or absence (b, small arrows) of laminin. Scale bars, 50 μm, insets in a, b, c, f, 25 μm, inset in e,10 μm. Data represent mean of 5–15 sections (- laminin) or field (+ laminin) counted over n = 5–10 cultures/condition ± SEM (g, j). **p < 0.01 Student's t-test.
Mentions: We used immunofluorescence to localize connexins to NPC subtypes grown in the absence of exogenous laminin (Fig 3, 5, 6). For all connexins except Cx26, cultures from -mutant mice served as negative controls to ensure antibody specificity. For Cx26, where deletion is embryonic lethal [23], antibody specificity was determined by Western blot (Fig 4a, inset). Four distinct connexin protein profiles were detected:

Bottom Line: Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30.These changes are associated with decreased neurogenesis.Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, Dept. of Biochemistry, Microbiology, and Immunology, University of Ottawa, ON, Canada. even_grable@hotmail.com

ABSTRACT

Background: Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells.

Results: We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication.

Conclusion: Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.

Show MeSH
Related in: MedlinePlus