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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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Blockade of CNG channels reduces neuronal differentiation of hippocampal NSCs but does not affect their proliferation.(A,B) Representative images of BrdU-positive cells (red) in NSC cultures grown in (A) normal proliferation medium and (B) in the presence of the CNG channel blocker, LCD (50 µM). (C) Bar graph showing the percentages of cells incorporating the proliferation marker BrdU in control and LCD-exposed NSC cultures. (D–G) Representative images of MAP2+ NSCs at D6 in control differentiative medium (D) and after 3 day-exposure (D1-D3) to 50 µM LCD (E), 1 µM KT5823 (F) and LCD plus KT5823 (G). Cell nuclei (DAPI+) are labeled in blue. Scale bars: 75 µm. (H) Bar graph showing the percentages of NSCs differentiating towards the neuronal phenotype (MAP2+) at D3, D6 and D10 in control conditions and in the culturing conditions described in the graph legend. Error bars show SEM values. Statistical significance was assessed by ANOVA (F3,52=3.80, P<0.05, at D3; F3,74=7.45, P<0.0005, at D6; F3,84=11.6, 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; n.s., not significant P-value.
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pone-0073246-g005: Blockade of CNG channels reduces neuronal differentiation of hippocampal NSCs but does not affect their proliferation.(A,B) Representative images of BrdU-positive cells (red) in NSC cultures grown in (A) normal proliferation medium and (B) in the presence of the CNG channel blocker, LCD (50 µM). (C) Bar graph showing the percentages of cells incorporating the proliferation marker BrdU in control and LCD-exposed NSC cultures. (D–G) Representative images of MAP2+ NSCs at D6 in control differentiative medium (D) and after 3 day-exposure (D1-D3) to 50 µM LCD (E), 1 µM KT5823 (F) and LCD plus KT5823 (G). Cell nuclei (DAPI+) are labeled in blue. Scale bars: 75 µm. (H) Bar graph showing the percentages of NSCs differentiating towards the neuronal phenotype (MAP2+) at D3, D6 and D10 in control conditions and in the culturing conditions described in the graph legend. Error bars show SEM values. Statistical significance was assessed by ANOVA (F3,52=3.80, P<0.05, at D3; F3,74=7.45, P<0.0005, at D6; F3,84=11.6, 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; n.s., not significant P-value.

Mentions: Undifferentiated dividing NSCs (D0) were identified by immunocytochemical detection of BrdU incorporation. Cells were exposed to BrdU (5 µM for 4 h) after 24 h culture in proliferation medium in the absence (control condition) and in the presence of LCD (50 µM). Immunoreactivity for the proliferation marker was observed in 32.6±2.2% of the control cells (529/1568; Figure 5A,C) and it was not significantly different from that observed in LCD-treated cells (29.2±2.4% [506/1608]; Figure 5B,C), thus suggesting that CNG channels, although expressed in undifferentiated NSCs, do not affect their proliferation.


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)

Blockade of CNG channels reduces neuronal differentiation of hippocampal NSCs but does not affect their proliferation.(A,B) Representative images of BrdU-positive cells (red) in NSC cultures grown in (A) normal proliferation medium and (B) in the presence of the CNG channel blocker, LCD (50 µM). (C) Bar graph showing the percentages of cells incorporating the proliferation marker BrdU in control and LCD-exposed NSC cultures. (D–G) Representative images of MAP2+ NSCs at D6 in control differentiative medium (D) and after 3 day-exposure (D1-D3) to 50 µM LCD (E), 1 µM KT5823 (F) and LCD plus KT5823 (G). Cell nuclei (DAPI+) are labeled in blue. Scale bars: 75 µm. (H) Bar graph showing the percentages of NSCs differentiating towards the neuronal phenotype (MAP2+) at D3, D6 and D10 in control conditions and in the culturing conditions described in the graph legend. Error bars show SEM values. Statistical significance was assessed by ANOVA (F3,52=3.80, P<0.05, at D3; F3,74=7.45, P<0.0005, at D6; F3,84=11.6, 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; n.s., not significant P-value.
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pone-0073246-g005: Blockade of CNG channels reduces neuronal differentiation of hippocampal NSCs but does not affect their proliferation.(A,B) Representative images of BrdU-positive cells (red) in NSC cultures grown in (A) normal proliferation medium and (B) in the presence of the CNG channel blocker, LCD (50 µM). (C) Bar graph showing the percentages of cells incorporating the proliferation marker BrdU in control and LCD-exposed NSC cultures. (D–G) Representative images of MAP2+ NSCs at D6 in control differentiative medium (D) and after 3 day-exposure (D1-D3) to 50 µM LCD (E), 1 µM KT5823 (F) and LCD plus KT5823 (G). Cell nuclei (DAPI+) are labeled in blue. Scale bars: 75 µm. (H) Bar graph showing the percentages of NSCs differentiating towards the neuronal phenotype (MAP2+) at D3, D6 and D10 in control conditions and in the culturing conditions described in the graph legend. Error bars show SEM values. Statistical significance was assessed by ANOVA (F3,52=3.80, P<0.05, at D3; F3,74=7.45, P<0.0005, at D6; F3,84=11.6, 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; n.s., not significant P-value.
Mentions: Undifferentiated dividing NSCs (D0) were identified by immunocytochemical detection of BrdU incorporation. Cells were exposed to BrdU (5 µM for 4 h) after 24 h culture in proliferation medium in the absence (control condition) and in the presence of LCD (50 µM). Immunoreactivity for the proliferation marker was observed in 32.6±2.2% of the control cells (529/1568; Figure 5A,C) and it was not significantly different from that observed in LCD-treated cells (29.2±2.4% [506/1608]; Figure 5B,C), thus suggesting that CNG channels, although expressed in undifferentiated NSCs, do not affect their proliferation.

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