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Role of cyclic nucleotide-gated channels in the modulation of mouse hippocampal neurogenesis.

Podda MV, Piacentini R, Barbati SA, Mastrodonato A, Puzzo D, D'Ascenzo M, Leone L, Grassi C - PLoS ONE (2013)

Bottom Line: Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype.The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade.The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Physiology, Medical School, Università Cattolica, Rome, Italy.

ABSTRACT
Neural stem cells generate neurons in the hippocampal dentate gyrus in mammals, including humans, throughout adulthood. Adult hippocampal neurogenesis has been the focus of many studies due to its relevance in processes such as learning and memory and its documented impairment in some neurodegenerative diseases. However, we are still far from having a complete picture of the mechanism regulating this process. Our study focused on the possible role of cyclic nucleotide-gated (CNG) channels. These voltage-independent channels activated by cyclic nucleotides, first described in retinal and olfactory receptors, have been receiving increasing attention for their involvement in several brain functions. Here we show that the rod-type, CNGA1, and olfactory-type, CNGA2, subunits are expressed in hippocampal neural stem cells in culture and in situ in the hippocampal neurogenic niche of adult mice. Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype. The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade. In addition, patch-clamp recording from neuron-like differentiating neural stem cells revealed cGMP-activated currents attributable to ion flow through CNG channels. The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.

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The cGMP analogue, 8-Br-cGMP, promotes NSC neuronal differentiation through CNG channel activation.(A) Summary bar graph showing an increased number of MAP2+ cells compared to controls following treatment with 8-Br-cGMP (8-Br). This effect was abolished by CNG channel and PKG blockers at D3, D6 and D10. Statistical significance was assessed by ANOVA (F3,76=8.25, P<0.0001, at D3; F3,85=38.5, P<0.0001, at D6; F3,87=32.8, P<0.0001, at D10). The Dunnett’s post-hoc test was used for multiple comparisons: #P<0.05 and # #P<0.001 vs. control; *P<0.05 and **P<0.001 vs. 8-Br-cGMP-treated NSCs. (B–D) Representative images of MAP2+ NSCs at D6 in control differentiation medium (B) and after exposure to 1 mM 8-Br-cGMP (C) or 8-Br-cGMP plus 50 µM LCD (D). Scale bar: 75 µm.
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pone-0073246-g006: The cGMP analogue, 8-Br-cGMP, promotes NSC neuronal differentiation through CNG channel activation.(A) Summary bar graph showing an increased number of MAP2+ cells compared to controls following treatment with 8-Br-cGMP (8-Br). This effect was abolished by CNG channel and PKG blockers at D3, D6 and D10. Statistical significance was assessed by ANOVA (F3,76=8.25, P<0.0001, at D3; F3,85=38.5, P<0.0001, at D6; F3,87=32.8, P<0.0001, at D10). The Dunnett’s post-hoc test was used for multiple comparisons: #P<0.05 and # #P<0.001 vs. control; *P<0.05 and **P<0.001 vs. 8-Br-cGMP-treated NSCs. (B–D) Representative images of MAP2+ NSCs at D6 in control differentiation medium (B) and after exposure to 1 mM 8-Br-cGMP (C) or 8-Br-cGMP plus 50 µM LCD (D). Scale bar: 75 µm.

Mentions: To confirm that CNG channels are the target of cGMP action in hippocampal NSCs we increased the levels of this cyclic nucleotide by applying its membrane permeant analogue, 8-Br-cGMP, and evaluated NSC proliferation and differentiation in the presence and in the absence of blockers of CNG channels and PKG. In our experimental conditions 8-Br-cGMP (1 mM) failed to affect the rate of NSC proliferation as no significant changes occurred in the percentages of BrdU+ cells in 8-Br-cGMP-treated cultures compared to control NSCs (34.5±2.4% [529/1568] vs. 32.6±2.2% in controls). These data confirm that cGMP signaling does not play a role in hippocampal NSC proliferation as suggested by the lack of LCD’s effects we observed in the previous set of experiments. Conversely, the experiments on differentiating NSCs confirmed that cGMP positively modulates NSC differentiation toward neuronal phenotype and its effects are, at least in part, mediated by CNG channels. Indeed, in NSCs that had been exposed to 8-Br-cGMP (1 mM) for three days (D1-D3) the percentage of MAP2+ was increased compared to controls (17.2±1.3% [411/2399] vs. 13.3± 1.4% in control NSCs; P<0.05; Figure 6A). Increased percentages of MAP2+ cells were also observed between 8-Br-cGMP-treated and control cells at D6 (31.3±1.3% [1363/4310] vs. 24.0±1.9%; P<0.001; Figure 6A,C) and D10 (39.1±2.0% [1532/3746] vs. 34.2±1.9%; P<0.05; Figure 6A). At D6 we also found that 8-Br-cGMP treatment reduced the percentage of GFAP+ cells from 72.9±3.5% to 63.3±4.9% (P<0.05) without changing the percentage of undifferentiated nestin+ cells (5.4±1.5% in 8-Br-cGMP-treated cells vs. 5.5±0.9% in controls).


Role of cyclic nucleotide-gated channels in the modulation of mouse hippocampal neurogenesis.

Podda MV, Piacentini R, Barbati SA, Mastrodonato A, Puzzo D, D'Ascenzo M, Leone L, Grassi C - PLoS ONE (2013)

The cGMP analogue, 8-Br-cGMP, promotes NSC neuronal differentiation through CNG channel activation.(A) Summary bar graph showing an increased number of MAP2+ cells compared to controls following treatment with 8-Br-cGMP (8-Br). This effect was abolished by CNG channel and PKG blockers at D3, D6 and D10. Statistical significance was assessed by ANOVA (F3,76=8.25, P<0.0001, at D3; F3,85=38.5, P<0.0001, at D6; F3,87=32.8, P<0.0001, at D10). The Dunnett’s post-hoc test was used for multiple comparisons: #P<0.05 and # #P<0.001 vs. control; *P<0.05 and **P<0.001 vs. 8-Br-cGMP-treated NSCs. (B–D) Representative images of MAP2+ NSCs at D6 in control differentiation medium (B) and after exposure to 1 mM 8-Br-cGMP (C) or 8-Br-cGMP plus 50 µM LCD (D). Scale bar: 75 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750014&req=5

pone-0073246-g006: The cGMP analogue, 8-Br-cGMP, promotes NSC neuronal differentiation through CNG channel activation.(A) Summary bar graph showing an increased number of MAP2+ cells compared to controls following treatment with 8-Br-cGMP (8-Br). This effect was abolished by CNG channel and PKG blockers at D3, D6 and D10. Statistical significance was assessed by ANOVA (F3,76=8.25, P<0.0001, at D3; F3,85=38.5, P<0.0001, at D6; F3,87=32.8, P<0.0001, at D10). The Dunnett’s post-hoc test was used for multiple comparisons: #P<0.05 and # #P<0.001 vs. control; *P<0.05 and **P<0.001 vs. 8-Br-cGMP-treated NSCs. (B–D) Representative images of MAP2+ NSCs at D6 in control differentiation medium (B) and after exposure to 1 mM 8-Br-cGMP (C) or 8-Br-cGMP plus 50 µM LCD (D). Scale bar: 75 µm.
Mentions: To confirm that CNG channels are the target of cGMP action in hippocampal NSCs we increased the levels of this cyclic nucleotide by applying its membrane permeant analogue, 8-Br-cGMP, and evaluated NSC proliferation and differentiation in the presence and in the absence of blockers of CNG channels and PKG. In our experimental conditions 8-Br-cGMP (1 mM) failed to affect the rate of NSC proliferation as no significant changes occurred in the percentages of BrdU+ cells in 8-Br-cGMP-treated cultures compared to control NSCs (34.5±2.4% [529/1568] vs. 32.6±2.2% in controls). These data confirm that cGMP signaling does not play a role in hippocampal NSC proliferation as suggested by the lack of LCD’s effects we observed in the previous set of experiments. Conversely, the experiments on differentiating NSCs confirmed that cGMP positively modulates NSC differentiation toward neuronal phenotype and its effects are, at least in part, mediated by CNG channels. Indeed, in NSCs that had been exposed to 8-Br-cGMP (1 mM) for three days (D1-D3) the percentage of MAP2+ was increased compared to controls (17.2±1.3% [411/2399] vs. 13.3± 1.4% in control NSCs; P<0.05; Figure 6A). Increased percentages of MAP2+ cells were also observed between 8-Br-cGMP-treated and control cells at D6 (31.3±1.3% [1363/4310] vs. 24.0±1.9%; P<0.001; Figure 6A,C) and D10 (39.1±2.0% [1532/3746] vs. 34.2±1.9%; P<0.05; Figure 6A). At D6 we also found that 8-Br-cGMP treatment reduced the percentage of GFAP+ cells from 72.9±3.5% to 63.3±4.9% (P<0.05) without changing the percentage of undifferentiated nestin+ cells (5.4±1.5% in 8-Br-cGMP-treated cells vs. 5.5±0.9% in controls).

Bottom Line: Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype.The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade.The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Physiology, Medical School, Università Cattolica, Rome, Italy.

ABSTRACT
Neural stem cells generate neurons in the hippocampal dentate gyrus in mammals, including humans, throughout adulthood. Adult hippocampal neurogenesis has been the focus of many studies due to its relevance in processes such as learning and memory and its documented impairment in some neurodegenerative diseases. However, we are still far from having a complete picture of the mechanism regulating this process. Our study focused on the possible role of cyclic nucleotide-gated (CNG) channels. These voltage-independent channels activated by cyclic nucleotides, first described in retinal and olfactory receptors, have been receiving increasing attention for their involvement in several brain functions. Here we show that the rod-type, CNGA1, and olfactory-type, CNGA2, subunits are expressed in hippocampal neural stem cells in culture and in situ in the hippocampal neurogenic niche of adult mice. Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype. The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade. In addition, patch-clamp recording from neuron-like differentiating neural stem cells revealed cGMP-activated currents attributable to ion flow through CNG channels. The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.

Show MeSH
Related in: MedlinePlus