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Glia- and neuron-specific functions of TrkB signalling during retinal degeneration and regeneration.

Harada C, Guo X, Namekata K, Kimura A, Nakamura K, Tanaka K, Parada LF, Harada T - Nat Commun (2011)

Bottom Line: Furthermore in TrkB(GFAP) knockout mice, BDNF did not prevent photoreceptor degeneration and failed to stimulate Müller glial cell proliferation and expression of neural markers in the degenerating retina.These results demonstrate that BDNF signalling in glia has important roles in neural protection and regeneration, particularly in conversion of Müller glia to photoreceptors.In addition, our genetic models provide a system in which glia- and neuron-specific gene functions can be tested in central nervous system tissues in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan.

ABSTRACT
Glia, the support cells of the central nervous system, have recently attracted considerable attention both as mediators of neural cell survival and as sources of neural regeneration. To further elucidate the role of glial and neural cells in neurodegeneration, we generated TrkB(GFAP) and TrkB(c-kit) knockout mice in which TrkB, a receptor for brain-derived neurotrophic factor (BDNF), is deleted in retinal glia or inner retinal neurons, respectively. Here, we show that the extent of glutamate-induced retinal degeneration was similar in these two mutant mice. Furthermore in TrkB(GFAP) knockout mice, BDNF did not prevent photoreceptor degeneration and failed to stimulate Müller glial cell proliferation and expression of neural markers in the degenerating retina. These results demonstrate that BDNF signalling in glia has important roles in neural protection and regeneration, particularly in conversion of Müller glia to photoreceptors. In addition, our genetic models provide a system in which glia- and neuron-specific gene functions can be tested in central nervous system tissues in vivo.

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Establishment of glia- and neuron-specific TrkB conditional KO mice.(a) Schematic diagram of transgenically targeted fate-mapping strategy. GFAP-Cre and c-kit-Cre mice were crossed with Rosa26-LacZ reporter mice. In Cre reporter offspring, only cells that express Cre excise a loxP-flanked stop signal and activate constitutive LacZ expression. Blue colour depicts LacZ-positive cells. (b) X-gal staining (blue) and immunostaining (red) of β-gal-positive cells. Glutamine synthetase (GS), calretinin, calbindin, protein kinase C (PKC) and TUJ1 were used as cell type-specific markers (green). Overlapping immunoreactivities (yellow) of β-gal and GS, calretinin or TUJ1 indicate that Cre-mediated recombination occurs in Müller glial cells in GFAP-Cre LacZ mice, and RGCs (arrows) and amacrine cells (arrowheads) in c-kit-Cre LacZ mice. (c) Immunohistochemical analysis of TrkB (green) and GS (red) showed loss of TrkB from Müller glial cells (arrowheads) in TrkBGFAP KO mice. (d) Immunohistochemical analysis of TrkB (green) and calretinin (red) showed loss of TrkB from RGCs (arrowheads) and amacrine cells (arrows) in TrkBc-kit KO mice. Scale bar, 100 μm. GCL, ganglion cell layer; INL, inner nuclear layer; RGC, retinal ganglion cell.
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f1: Establishment of glia- and neuron-specific TrkB conditional KO mice.(a) Schematic diagram of transgenically targeted fate-mapping strategy. GFAP-Cre and c-kit-Cre mice were crossed with Rosa26-LacZ reporter mice. In Cre reporter offspring, only cells that express Cre excise a loxP-flanked stop signal and activate constitutive LacZ expression. Blue colour depicts LacZ-positive cells. (b) X-gal staining (blue) and immunostaining (red) of β-gal-positive cells. Glutamine synthetase (GS), calretinin, calbindin, protein kinase C (PKC) and TUJ1 were used as cell type-specific markers (green). Overlapping immunoreactivities (yellow) of β-gal and GS, calretinin or TUJ1 indicate that Cre-mediated recombination occurs in Müller glial cells in GFAP-Cre LacZ mice, and RGCs (arrows) and amacrine cells (arrowheads) in c-kit-Cre LacZ mice. (c) Immunohistochemical analysis of TrkB (green) and GS (red) showed loss of TrkB from Müller glial cells (arrowheads) in TrkBGFAP KO mice. (d) Immunohistochemical analysis of TrkB (green) and calretinin (red) showed loss of TrkB from RGCs (arrowheads) and amacrine cells (arrows) in TrkBc-kit KO mice. Scale bar, 100 μm. GCL, ganglion cell layer; INL, inner nuclear layer; RGC, retinal ganglion cell.

Mentions: Previous studies have shown that the GFAP-Cre transgenic strain, in which Cre expression is regulated by the human GFAP promoter, expresses Cre recombinase not only in mature astrocytes but also in multipotent radial glial cells that exhibit neural stem/progenitor cell properties2021. To examine expression of the GFAP-Cre transgene in the adult retina, we crossed GFAP-Cre mice with a Rosa26-LacZ reporter line2223 in which recombination results in expression of β-galactosidase (β-gal; Fig. 1a,b). To identify the specific β-gal immunopositive (IP) cell type(s), we carried out co-staining with markers of other retinal lineages. β-gal-IP cells were double labelled with glutamine synthetase (GS; a marker of Müller glial cells), but not with calretinin (a marker of RGCs and amacrine cells), calbindin (a marker of horizontal cells) or protein kinase C (PKC; a marker of bipolar cells; Fig. 1b). These results revealed that β-gal expression, which reflects Cre recombinase expression, is restricted to glial cells in GFAP-Cre LacZ mice. On the other hand, in c-kit-Cre LacZ mice24, β-gal-IP cells were double labelled with calretinin and TUJ1 (another RGC marker), but not with GS, calbindin or PKC (Fig. 1b). These results indicate that, in contrast to GFAP-Cre LacZ mice, expression of Cre recombinase is restricted to RGCs and amacrine cells in c-kit-Cre LacZ mice (Fig. 1a).


Glia- and neuron-specific functions of TrkB signalling during retinal degeneration and regeneration.

Harada C, Guo X, Namekata K, Kimura A, Nakamura K, Tanaka K, Parada LF, Harada T - Nat Commun (2011)

Establishment of glia- and neuron-specific TrkB conditional KO mice.(a) Schematic diagram of transgenically targeted fate-mapping strategy. GFAP-Cre and c-kit-Cre mice were crossed with Rosa26-LacZ reporter mice. In Cre reporter offspring, only cells that express Cre excise a loxP-flanked stop signal and activate constitutive LacZ expression. Blue colour depicts LacZ-positive cells. (b) X-gal staining (blue) and immunostaining (red) of β-gal-positive cells. Glutamine synthetase (GS), calretinin, calbindin, protein kinase C (PKC) and TUJ1 were used as cell type-specific markers (green). Overlapping immunoreactivities (yellow) of β-gal and GS, calretinin or TUJ1 indicate that Cre-mediated recombination occurs in Müller glial cells in GFAP-Cre LacZ mice, and RGCs (arrows) and amacrine cells (arrowheads) in c-kit-Cre LacZ mice. (c) Immunohistochemical analysis of TrkB (green) and GS (red) showed loss of TrkB from Müller glial cells (arrowheads) in TrkBGFAP KO mice. (d) Immunohistochemical analysis of TrkB (green) and calretinin (red) showed loss of TrkB from RGCs (arrowheads) and amacrine cells (arrows) in TrkBc-kit KO mice. Scale bar, 100 μm. GCL, ganglion cell layer; INL, inner nuclear layer; RGC, retinal ganglion cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105320&req=5

f1: Establishment of glia- and neuron-specific TrkB conditional KO mice.(a) Schematic diagram of transgenically targeted fate-mapping strategy. GFAP-Cre and c-kit-Cre mice were crossed with Rosa26-LacZ reporter mice. In Cre reporter offspring, only cells that express Cre excise a loxP-flanked stop signal and activate constitutive LacZ expression. Blue colour depicts LacZ-positive cells. (b) X-gal staining (blue) and immunostaining (red) of β-gal-positive cells. Glutamine synthetase (GS), calretinin, calbindin, protein kinase C (PKC) and TUJ1 were used as cell type-specific markers (green). Overlapping immunoreactivities (yellow) of β-gal and GS, calretinin or TUJ1 indicate that Cre-mediated recombination occurs in Müller glial cells in GFAP-Cre LacZ mice, and RGCs (arrows) and amacrine cells (arrowheads) in c-kit-Cre LacZ mice. (c) Immunohistochemical analysis of TrkB (green) and GS (red) showed loss of TrkB from Müller glial cells (arrowheads) in TrkBGFAP KO mice. (d) Immunohistochemical analysis of TrkB (green) and calretinin (red) showed loss of TrkB from RGCs (arrowheads) and amacrine cells (arrows) in TrkBc-kit KO mice. Scale bar, 100 μm. GCL, ganglion cell layer; INL, inner nuclear layer; RGC, retinal ganglion cell.
Mentions: Previous studies have shown that the GFAP-Cre transgenic strain, in which Cre expression is regulated by the human GFAP promoter, expresses Cre recombinase not only in mature astrocytes but also in multipotent radial glial cells that exhibit neural stem/progenitor cell properties2021. To examine expression of the GFAP-Cre transgene in the adult retina, we crossed GFAP-Cre mice with a Rosa26-LacZ reporter line2223 in which recombination results in expression of β-galactosidase (β-gal; Fig. 1a,b). To identify the specific β-gal immunopositive (IP) cell type(s), we carried out co-staining with markers of other retinal lineages. β-gal-IP cells were double labelled with glutamine synthetase (GS; a marker of Müller glial cells), but not with calretinin (a marker of RGCs and amacrine cells), calbindin (a marker of horizontal cells) or protein kinase C (PKC; a marker of bipolar cells; Fig. 1b). These results revealed that β-gal expression, which reflects Cre recombinase expression, is restricted to glial cells in GFAP-Cre LacZ mice. On the other hand, in c-kit-Cre LacZ mice24, β-gal-IP cells were double labelled with calretinin and TUJ1 (another RGC marker), but not with GS, calbindin or PKC (Fig. 1b). These results indicate that, in contrast to GFAP-Cre LacZ mice, expression of Cre recombinase is restricted to RGCs and amacrine cells in c-kit-Cre LacZ mice (Fig. 1a).

Bottom Line: Furthermore in TrkB(GFAP) knockout mice, BDNF did not prevent photoreceptor degeneration and failed to stimulate Müller glial cell proliferation and expression of neural markers in the degenerating retina.These results demonstrate that BDNF signalling in glia has important roles in neural protection and regeneration, particularly in conversion of Müller glia to photoreceptors.In addition, our genetic models provide a system in which glia- and neuron-specific gene functions can be tested in central nervous system tissues in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan.

ABSTRACT
Glia, the support cells of the central nervous system, have recently attracted considerable attention both as mediators of neural cell survival and as sources of neural regeneration. To further elucidate the role of glial and neural cells in neurodegeneration, we generated TrkB(GFAP) and TrkB(c-kit) knockout mice in which TrkB, a receptor for brain-derived neurotrophic factor (BDNF), is deleted in retinal glia or inner retinal neurons, respectively. Here, we show that the extent of glutamate-induced retinal degeneration was similar in these two mutant mice. Furthermore in TrkB(GFAP) knockout mice, BDNF did not prevent photoreceptor degeneration and failed to stimulate Müller glial cell proliferation and expression of neural markers in the degenerating retina. These results demonstrate that BDNF signalling in glia has important roles in neural protection and regeneration, particularly in conversion of Müller glia to photoreceptors. In addition, our genetic models provide a system in which glia- and neuron-specific gene functions can be tested in central nervous system tissues in vivo.

Show MeSH
Related in: MedlinePlus