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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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Laminin engagement alters functional hemichannel but not GJIC activity. Functional hemichannel activity was assessed by anionic LY dye uptake (a) of cultures exposed to laminin for 1 DIV (prior to any changes in connexin expression) or 6 DIV (following changes in connexin expression). Spontaneous LY uptake was observed at low levels after 1 and 6 DIV. Open hemichannel activity could be induced by mechanical stimulation with glass microbeads within 1 DIV but not 6 DIV. Dye uptake was inhibited by the hemichannel/chloride channel inhibitor FFA but not the chloride channel blocker DIDS. LY+/RD- cells is expressed as a percentage of the total number of cells per microscopic field ± standard error of the mean (SEM) counted in n = 5 fields per experiment conducted in triplicate experiments. GJIC was assayed using the scrape-loading method (b). Significant LY+/RD- dye transfer was not observed after 1 or 6 DIV when NPCs were cultured in the presence of mitogens (proliferative conditions). As a positive control, we performed the same experiment in a condition known to promote glial cell differentiation and obtained robust GJIC that was significantly inhibited by the gap junction blocker GRA but not the inactive analog GZA (glial differentiation). In GJIC assays, the number of LY+/RD- cells along the scrape line was established in serial photographs taken along the entire length of the scrape (9–14 photos/coverslip) over triplicate cultures. Two coverslips were assessed per culture for a total of n = 54–84 measurements per condition. Data are expressed as the mean number of LY+/RD- cells ± SEM. *p < 0.05, **p < 0.01, ANOVA, post-hoc Tukey test.
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Figure 8: Laminin engagement alters functional hemichannel but not GJIC activity. Functional hemichannel activity was assessed by anionic LY dye uptake (a) of cultures exposed to laminin for 1 DIV (prior to any changes in connexin expression) or 6 DIV (following changes in connexin expression). Spontaneous LY uptake was observed at low levels after 1 and 6 DIV. Open hemichannel activity could be induced by mechanical stimulation with glass microbeads within 1 DIV but not 6 DIV. Dye uptake was inhibited by the hemichannel/chloride channel inhibitor FFA but not the chloride channel blocker DIDS. LY+/RD- cells is expressed as a percentage of the total number of cells per microscopic field ± standard error of the mean (SEM) counted in n = 5 fields per experiment conducted in triplicate experiments. GJIC was assayed using the scrape-loading method (b). Significant LY+/RD- dye transfer was not observed after 1 or 6 DIV when NPCs were cultured in the presence of mitogens (proliferative conditions). As a positive control, we performed the same experiment in a condition known to promote glial cell differentiation and obtained robust GJIC that was significantly inhibited by the gap junction blocker GRA but not the inactive analog GZA (glial differentiation). In GJIC assays, the number of LY+/RD- cells along the scrape line was established in serial photographs taken along the entire length of the scrape (9–14 photos/coverslip) over triplicate cultures. Two coverslips were assessed per culture for a total of n = 54–84 measurements per condition. Data are expressed as the mean number of LY+/RD- cells ± SEM. *p < 0.05, **p < 0.01, ANOVA, post-hoc Tukey test.

Mentions: Transmembrane flux of the low molecular mass fluorescent dye, LY, was used to assess hemichannel activity in neurosphere cultures. RD was used to control for uptake resulting from plasma membrane damage. Hemichannel opening was induced by mechanical stimulation with glass microbeads as previously described [26]. Cultures were expanded in suspension for 8 DIV, then plated and analyzed immediately after adherence (Fig 8a, DIV 1) prior to any detectable change in connexin protein expression (data not shown) or after 6 DIV in contact with laminin (Fig 8a, DIV 6) following the observed changes in connexin expression (Fig 3, 4, 5). Mechanical stimulation elicited a significant increase in dye uptake on DIV 1 (Fig 8a, DIV 1) that was largely inhibited by the dual-specificity chloride channel and connexin/pannexin-channel blocker FFA [27,28] but not the chloride channel inhibitor DIDS (Fig 8a, DIV 1). Hemichannel activity was lost when NPCs were cultured on laminin for 6 DIV (Fig 8a, DIV 6). We cannot, however, rule out that these changes are due to an effect of ECM on pannexin channel formation as we have determined that NPCs cultured in suspension express both pannexin 1 and 2 mRNA (data not shown) yet we have not investigated impact of laminin on this expression at the protein level.


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)

Laminin engagement alters functional hemichannel but not GJIC activity. Functional hemichannel activity was assessed by anionic LY dye uptake (a) of cultures exposed to laminin for 1 DIV (prior to any changes in connexin expression) or 6 DIV (following changes in connexin expression). Spontaneous LY uptake was observed at low levels after 1 and 6 DIV. Open hemichannel activity could be induced by mechanical stimulation with glass microbeads within 1 DIV but not 6 DIV. Dye uptake was inhibited by the hemichannel/chloride channel inhibitor FFA but not the chloride channel blocker DIDS. LY+/RD- cells is expressed as a percentage of the total number of cells per microscopic field ± standard error of the mean (SEM) counted in n = 5 fields per experiment conducted in triplicate experiments. GJIC was assayed using the scrape-loading method (b). Significant LY+/RD- dye transfer was not observed after 1 or 6 DIV when NPCs were cultured in the presence of mitogens (proliferative conditions). As a positive control, we performed the same experiment in a condition known to promote glial cell differentiation and obtained robust GJIC that was significantly inhibited by the gap junction blocker GRA but not the inactive analog GZA (glial differentiation). In GJIC assays, the number of LY+/RD- cells along the scrape line was established in serial photographs taken along the entire length of the scrape (9–14 photos/coverslip) over triplicate cultures. Two coverslips were assessed per culture for a total of n = 54–84 measurements per condition. Data are expressed as the mean number of LY+/RD- cells ± SEM. *p < 0.05, **p < 0.01, ANOVA, post-hoc Tukey test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 8: Laminin engagement alters functional hemichannel but not GJIC activity. Functional hemichannel activity was assessed by anionic LY dye uptake (a) of cultures exposed to laminin for 1 DIV (prior to any changes in connexin expression) or 6 DIV (following changes in connexin expression). Spontaneous LY uptake was observed at low levels after 1 and 6 DIV. Open hemichannel activity could be induced by mechanical stimulation with glass microbeads within 1 DIV but not 6 DIV. Dye uptake was inhibited by the hemichannel/chloride channel inhibitor FFA but not the chloride channel blocker DIDS. LY+/RD- cells is expressed as a percentage of the total number of cells per microscopic field ± standard error of the mean (SEM) counted in n = 5 fields per experiment conducted in triplicate experiments. GJIC was assayed using the scrape-loading method (b). Significant LY+/RD- dye transfer was not observed after 1 or 6 DIV when NPCs were cultured in the presence of mitogens (proliferative conditions). As a positive control, we performed the same experiment in a condition known to promote glial cell differentiation and obtained robust GJIC that was significantly inhibited by the gap junction blocker GRA but not the inactive analog GZA (glial differentiation). In GJIC assays, the number of LY+/RD- cells along the scrape line was established in serial photographs taken along the entire length of the scrape (9–14 photos/coverslip) over triplicate cultures. Two coverslips were assessed per culture for a total of n = 54–84 measurements per condition. Data are expressed as the mean number of LY+/RD- cells ± SEM. *p < 0.05, **p < 0.01, ANOVA, post-hoc Tukey test.
Mentions: Transmembrane flux of the low molecular mass fluorescent dye, LY, was used to assess hemichannel activity in neurosphere cultures. RD was used to control for uptake resulting from plasma membrane damage. Hemichannel opening was induced by mechanical stimulation with glass microbeads as previously described [26]. Cultures were expanded in suspension for 8 DIV, then plated and analyzed immediately after adherence (Fig 8a, DIV 1) prior to any detectable change in connexin protein expression (data not shown) or after 6 DIV in contact with laminin (Fig 8a, DIV 6) following the observed changes in connexin expression (Fig 3, 4, 5). Mechanical stimulation elicited a significant increase in dye uptake on DIV 1 (Fig 8a, DIV 1) that was largely inhibited by the dual-specificity chloride channel and connexin/pannexin-channel blocker FFA [27,28] but not the chloride channel inhibitor DIDS (Fig 8a, DIV 1). Hemichannel activity was lost when NPCs were cultured on laminin for 6 DIV (Fig 8a, DIV 6). We cannot, however, rule out that these changes are due to an effect of ECM on pannexin channel formation as we have determined that NPCs cultured in suspension express both pannexin 1 and 2 mRNA (data not shown) yet we have not investigated impact of laminin on this expression at the protein level.

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