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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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Cultured hippocampal NSCs express CNG channels.(A–D) Time course of hippocampal NSC differentiation assessed by immunofluorescence. The vast majority of neurosphere-derived cells grown in the proliferation medium (D0) were positive for nestin (A); during differentiation this immunoreactivity diminished (B) and ultimately disappeared on D10 (C). Cell nuclei (DAPI+) are labeled in blue. The percentages of cells identified as neurons by MAP2 immunoreactivity significantly increased during the 10 days of differentiation (D). Undifferentiated NSCs, characterized by positivity for nestin and lack of MAP2 immunoreactivity, showed both CNGA1 (E,F) and CNGA2 staining (I,J). NSCs differentiating toward neuronal phenotype (MAP2+) exhibited CNGA1 (G,H) and CNGA2 (K,L) labeling. Scale bars: 50 µm.
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pone-0073246-g001: Cultured hippocampal NSCs express CNG channels.(A–D) Time course of hippocampal NSC differentiation assessed by immunofluorescence. The vast majority of neurosphere-derived cells grown in the proliferation medium (D0) were positive for nestin (A); during differentiation this immunoreactivity diminished (B) and ultimately disappeared on D10 (C). Cell nuclei (DAPI+) are labeled in blue. The percentages of cells identified as neurons by MAP2 immunoreactivity significantly increased during the 10 days of differentiation (D). Undifferentiated NSCs, characterized by positivity for nestin and lack of MAP2 immunoreactivity, showed both CNGA1 (E,F) and CNGA2 staining (I,J). NSCs differentiating toward neuronal phenotype (MAP2+) exhibited CNGA1 (G,H) and CNGA2 (K,L) labeling. Scale bars: 50 µm.

Mentions: Adult hippocampal NSC culture were isolated according to previously published protocols [44]. Briefly, brains of newborn (0-1 day old) C57bl/6 mice were microdissected to obtain the hippocampal region upon sagittal sectioning. Tissues were finely minced and digested by accutase (in DPBS, 0.5 mM EDTA; Innovative Cell Tecnologies, Inc., San Diego, CA, USA) at 37°C for 30 min. After centrifugation, cells were carefully dissociated by passaging in fire-polished Pasteur pipettes and resuspended in NeurobsalA medium, supplemented by 2% B27 (Gibco, Grand Island, NY, USA), Glutamax (0.5 mM; Invitrogen, Carlsbad, CA), mouse fibroblast growth factor 2 (FGF2, 10 ng/ml; Invitrogen), epidermal growth factor (EGF, 10 ng/ml; Invitrogen), mouse platelet-derived growth factor bb (PDGFbb, 10 ng/ml; Invitrogen). Cells were seeded onto 25-cm2 T-flask and incubated at 37°C in 5% CO2 atmosphere. During the first week NSCs began to form neurospheres in vitro. At 2-day intervals, the neurospheres were collected, and passaged by a gently enzymatic and mechanical dissociation. These processed NSCs retained the potential to grow infinitely, as demonstrated by the percentage of the cells positive for nestin, a marker for immature neural progenitors (Figure 1A).


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)

Cultured hippocampal NSCs express CNG channels.(A–D) Time course of hippocampal NSC differentiation assessed by immunofluorescence. The vast majority of neurosphere-derived cells grown in the proliferation medium (D0) were positive for nestin (A); during differentiation this immunoreactivity diminished (B) and ultimately disappeared on D10 (C). Cell nuclei (DAPI+) are labeled in blue. The percentages of cells identified as neurons by MAP2 immunoreactivity significantly increased during the 10 days of differentiation (D). Undifferentiated NSCs, characterized by positivity for nestin and lack of MAP2 immunoreactivity, showed both CNGA1 (E,F) and CNGA2 staining (I,J). NSCs differentiating toward neuronal phenotype (MAP2+) exhibited CNGA1 (G,H) and CNGA2 (K,L) labeling. Scale bars: 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750014&req=5

pone-0073246-g001: Cultured hippocampal NSCs express CNG channels.(A–D) Time course of hippocampal NSC differentiation assessed by immunofluorescence. The vast majority of neurosphere-derived cells grown in the proliferation medium (D0) were positive for nestin (A); during differentiation this immunoreactivity diminished (B) and ultimately disappeared on D10 (C). Cell nuclei (DAPI+) are labeled in blue. The percentages of cells identified as neurons by MAP2 immunoreactivity significantly increased during the 10 days of differentiation (D). Undifferentiated NSCs, characterized by positivity for nestin and lack of MAP2 immunoreactivity, showed both CNGA1 (E,F) and CNGA2 staining (I,J). NSCs differentiating toward neuronal phenotype (MAP2+) exhibited CNGA1 (G,H) and CNGA2 (K,L) labeling. Scale bars: 50 µm.
Mentions: Adult hippocampal NSC culture were isolated according to previously published protocols [44]. Briefly, brains of newborn (0-1 day old) C57bl/6 mice were microdissected to obtain the hippocampal region upon sagittal sectioning. Tissues were finely minced and digested by accutase (in DPBS, 0.5 mM EDTA; Innovative Cell Tecnologies, Inc., San Diego, CA, USA) at 37°C for 30 min. After centrifugation, cells were carefully dissociated by passaging in fire-polished Pasteur pipettes and resuspended in NeurobsalA medium, supplemented by 2% B27 (Gibco, Grand Island, NY, USA), Glutamax (0.5 mM; Invitrogen, Carlsbad, CA), mouse fibroblast growth factor 2 (FGF2, 10 ng/ml; Invitrogen), epidermal growth factor (EGF, 10 ng/ml; Invitrogen), mouse platelet-derived growth factor bb (PDGFbb, 10 ng/ml; Invitrogen). Cells were seeded onto 25-cm2 T-flask and incubated at 37°C in 5% CO2 atmosphere. During the first week NSCs began to form neurospheres in vitro. At 2-day intervals, the neurospheres were collected, and passaged by a gently enzymatic and mechanical dissociation. These processed NSCs retained the potential to grow infinitely, as demonstrated by the percentage of the cells positive for nestin, a marker for immature neural progenitors (Figure 1A).

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