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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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Functional analysis of Müller glial cells in TrkBGFAP KO mice.(a) Immunoblot analysis of TrkB protein in cultured Müller cells from WT and TrkBGFAP KO mice. (b) Immunoblot analysis of phosphorylated CREB (pCREB) and total CREB in cultured Müller cells from WT and TrkBGFAP KO mice. BDNF (50 ng ml−1)-induced CREB phosphorylation was impaired in TrkBGFAP KO Müller cells. For each determination, the ratio of pCREB/CREB protein level in controls (WT, BDNF 0 min) was normalized to a value of 1.0. Data are shown as the mean+s.e.m. (n=3). (c) Immunohistochemical analysis of pCREB in BDNF (1 μg μl−1)-treated WT and TrkBGFAP KO retinas. Note the absence of pCREB in Müller cells (red arrowhead) of the TrkBGFAP KO retina, despite its persistence in the GCL (arrow). Scale bar, 50 μm. (d) Effect of BDNF (100 ng ml−1 for 48 h) on trophic factor production in cultured Müller cells from WT and TrkBGFAP KO mice. For each determination, the mRNA level in controls (WT, without BDNF) was normalized to a value of 1.0. Data are shown as the mean+s.e.m. (n=6). *P<0.05. NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; CNTF, ciliary neurotrophic factor; GNDF, glial cell line-derived neurotrophic factor.
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f3: Functional analysis of Müller glial cells in TrkBGFAP KO mice.(a) Immunoblot analysis of TrkB protein in cultured Müller cells from WT and TrkBGFAP KO mice. (b) Immunoblot analysis of phosphorylated CREB (pCREB) and total CREB in cultured Müller cells from WT and TrkBGFAP KO mice. BDNF (50 ng ml−1)-induced CREB phosphorylation was impaired in TrkBGFAP KO Müller cells. For each determination, the ratio of pCREB/CREB protein level in controls (WT, BDNF 0 min) was normalized to a value of 1.0. Data are shown as the mean+s.e.m. (n=3). (c) Immunohistochemical analysis of pCREB in BDNF (1 μg μl−1)-treated WT and TrkBGFAP KO retinas. Note the absence of pCREB in Müller cells (red arrowhead) of the TrkBGFAP KO retina, despite its persistence in the GCL (arrow). Scale bar, 50 μm. (d) Effect of BDNF (100 ng ml−1 for 48 h) on trophic factor production in cultured Müller cells from WT and TrkBGFAP KO mice. For each determination, the mRNA level in controls (WT, without BDNF) was normalized to a value of 1.0. Data are shown as the mean+s.e.m. (n=6). *P<0.05. NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; CNTF, ciliary neurotrophic factor; GNDF, glial cell line-derived neurotrophic factor.

Mentions: To further examine the function of TrkB signalling in retinal glia, we next prepared cultured Müller cells from WT and TrkBGFAP KO mice and analysed them by western blot. Consistent with the immunohistochemical results (Fig. 1c), TrkB protein expression in Müller glia was not detectable in TrkBGFAP KO mice (Fig. 3a). We next examined whether exogenous BDNF stimulates TrkB-dependent signal transduction in Müller glia by observing the activation of cyclic AMP response element-binding (CREB) protein. In WT Müller cells, the basal phosphorylated CREB (pCREB) expression level was very low, but BDNF clearly increased the ratio of pCREB to CREB (Fig. 3b). In contrast, the effect of BDNF on CREB activation was only marginal in TrkBGFAP KO Müller cells (Fig. 3b). We also examined the effect of BDNF on CREB phosphorylation in vivo. In WT retinas, intraocular injection of BDNF induced pCREB expression in RGCs and Müller glia (arrow and arrowhead in Fig. 3c). In TrkBGFAP KO mice, BDNF-induced pCREB expression in RGCs was comparable with WT retinas, but expression was barely detectable in Müller glia (Fig. 3c). These results confirmed a lack of TrkB-dependent signal transduction in Müller glia in TrkBGFAP KO mice. We previously demonstrated that treatment of cultured rat Müller cells with BDNF increases the production of several trophic factors that can protect retinal neurons19. Similarly, in mouse Müller cells, BDNF increased mRNA expression levels of BDNF, bFGF, ciliary neurotrophic factor and glial cell line-derived neurotrophic factor (Fig. 3d). However, BDNF failed to stimulate the production of trophic factors in Müller cells prepared from TrkBGFAP KO mice (Fig. 3d). Thus, TrkB elimination in Müller glia impairs the transcription of trophic factors, which may lead to RGC vulnerability during glutamate neurotoxicity.


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)

Functional analysis of Müller glial cells in TrkBGFAP KO mice.(a) Immunoblot analysis of TrkB protein in cultured Müller cells from WT and TrkBGFAP KO mice. (b) Immunoblot analysis of phosphorylated CREB (pCREB) and total CREB in cultured Müller cells from WT and TrkBGFAP KO mice. BDNF (50 ng ml−1)-induced CREB phosphorylation was impaired in TrkBGFAP KO Müller cells. For each determination, the ratio of pCREB/CREB protein level in controls (WT, BDNF 0 min) was normalized to a value of 1.0. Data are shown as the mean+s.e.m. (n=3). (c) Immunohistochemical analysis of pCREB in BDNF (1 μg μl−1)-treated WT and TrkBGFAP KO retinas. Note the absence of pCREB in Müller cells (red arrowhead) of the TrkBGFAP KO retina, despite its persistence in the GCL (arrow). Scale bar, 50 μm. (d) Effect of BDNF (100 ng ml−1 for 48 h) on trophic factor production in cultured Müller cells from WT and TrkBGFAP KO mice. For each determination, the mRNA level in controls (WT, without BDNF) was normalized to a value of 1.0. Data are shown as the mean+s.e.m. (n=6). *P<0.05. NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; CNTF, ciliary neurotrophic factor; GNDF, glial cell line-derived neurotrophic factor.
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f3: Functional analysis of Müller glial cells in TrkBGFAP KO mice.(a) Immunoblot analysis of TrkB protein in cultured Müller cells from WT and TrkBGFAP KO mice. (b) Immunoblot analysis of phosphorylated CREB (pCREB) and total CREB in cultured Müller cells from WT and TrkBGFAP KO mice. BDNF (50 ng ml−1)-induced CREB phosphorylation was impaired in TrkBGFAP KO Müller cells. For each determination, the ratio of pCREB/CREB protein level in controls (WT, BDNF 0 min) was normalized to a value of 1.0. Data are shown as the mean+s.e.m. (n=3). (c) Immunohistochemical analysis of pCREB in BDNF (1 μg μl−1)-treated WT and TrkBGFAP KO retinas. Note the absence of pCREB in Müller cells (red arrowhead) of the TrkBGFAP KO retina, despite its persistence in the GCL (arrow). Scale bar, 50 μm. (d) Effect of BDNF (100 ng ml−1 for 48 h) on trophic factor production in cultured Müller cells from WT and TrkBGFAP KO mice. For each determination, the mRNA level in controls (WT, without BDNF) was normalized to a value of 1.0. Data are shown as the mean+s.e.m. (n=6). *P<0.05. NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; CNTF, ciliary neurotrophic factor; GNDF, glial cell line-derived neurotrophic factor.
Mentions: To further examine the function of TrkB signalling in retinal glia, we next prepared cultured Müller cells from WT and TrkBGFAP KO mice and analysed them by western blot. Consistent with the immunohistochemical results (Fig. 1c), TrkB protein expression in Müller glia was not detectable in TrkBGFAP KO mice (Fig. 3a). We next examined whether exogenous BDNF stimulates TrkB-dependent signal transduction in Müller glia by observing the activation of cyclic AMP response element-binding (CREB) protein. In WT Müller cells, the basal phosphorylated CREB (pCREB) expression level was very low, but BDNF clearly increased the ratio of pCREB to CREB (Fig. 3b). In contrast, the effect of BDNF on CREB activation was only marginal in TrkBGFAP KO Müller cells (Fig. 3b). We also examined the effect of BDNF on CREB phosphorylation in vivo. In WT retinas, intraocular injection of BDNF induced pCREB expression in RGCs and Müller glia (arrow and arrowhead in Fig. 3c). In TrkBGFAP KO mice, BDNF-induced pCREB expression in RGCs was comparable with WT retinas, but expression was barely detectable in Müller glia (Fig. 3c). These results confirmed a lack of TrkB-dependent signal transduction in Müller glia in TrkBGFAP KO mice. We previously demonstrated that treatment of cultured rat Müller cells with BDNF increases the production of several trophic factors that can protect retinal neurons19. Similarly, in mouse Müller cells, BDNF increased mRNA expression levels of BDNF, bFGF, ciliary neurotrophic factor and glial cell line-derived neurotrophic factor (Fig. 3d). However, BDNF failed to stimulate the production of trophic factors in Müller cells prepared from TrkBGFAP KO mice (Fig. 3d). Thus, TrkB elimination in Müller glia impairs the transcription of trophic factors, which may lead to RGC vulnerability during glutamate neurotoxicity.

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