Corticotropin-releasing hormone exerts direct effects on neuronal progenitor cells: implications for neuroprotection - PubMed (original) (raw)
Corticotropin-releasing hormone exerts direct effects on neuronal progenitor cells: implications for neuroprotection
Y Koutmani et al. Mol Psychiatry. 2013 Mar.
Free PMC article
Abstract
Neurogenesis during embryonic and adult life is tightly regulated by a network of transcriptional, growth and hormonal factors. Emerging evidence indicates that activation of the stress response, via the associated glucocorticoid increase, reduces neurogenesis and contributes to the development of adult diseases.As corticotrophin-releasing hormone (CRH) or factor is the major mediator of adaptive response to stressors, we sought to investigate its involvement in this process. Accordingly, we found that CRH could reverse the damaging effects of glucocorticoid on neural stem/progenitor cells (NS/PCs), while its genetic deficiency results in compromised proliferation and enhanced apoptosis during neurogenesis. Analyses in fetal and adult mouse brain revealed significant expression of CRH receptors in proliferating neuronal progenitors. Furthermore, by using primary cultures of NS/PCs, we characterized the molecular mechanisms and identified CRH receptor-1 as the receptor mediating the neuroprotective effects of CRH. Finally, we demonstrate the expression of CRH receptors in human fetal brain from early gestational age, in areas of active neuronal proliferation. These observations raise the intriguing possibility for CRH-mediated pharmacological applications in diseases characterized by altered neuronal homeostasis, including depression, dementia, neurodegenerative diseases, brain traumas and obesity.
Figures
Figure 1
Altered proliferative and apoptotic properties of neural progenitor cells in the developing brain of _Crh_−null (Crh−/−) mice. (a) 5-bromo-2-deoxyuridine (BrdU) was administered to the mother at gestational age 14.5 for 2 h. Representative images from brain slices (E14.5) of Crh−/− and wild-type (wt) littermates are shown. Scale bar=20 μm. (c) Graph that shows quantification of the BrdU-positive cell count in 180 × 180 μm2 areas. Data are shown as mean±s.e.m. (_n_=20 slices of three animals). (b)Representative images from brain slices (E14.5) of Crh−/− and wt littermates after performance of terminal transferase dUTP nick-end labeling (TUNEL) assay. Scale bar=50 μm. (d) Graph that shows quantification of the TUNEL-positive cell count in 180 × 180 μm2 areas. Data are shown as mean±s.e.m. (_n_=20 slices of four animals) *P<0.05 versus wt mice. vz, svz, LV, lateral ventricle.
Figure 2
Expression of CRH receptors (CRHRs) in embryonal and adult neural progenitor cells in vivo and in vitro. (a) Detection of CRH receptors in neural progenitor cells of embryonic (E14.5) forebrain. Co-expression (yellow) of CRHRs and the neural progenitor marker nestin. (b, e) Detection of CRH receptors in neural progenitor cells of adult forebrain. Co-expression of CRHRs (arrows) and the neural progenitor markers nestin (b) and GFAP (c) was observed in the majority of cells lying in the neurogenic areas of mouse brain. Some cells selectively express CRHRs (black arrowheads) or nestin/GFAP (white arrowheads). Co-expression of CRHRs (green) and the neural progenitor markers Mash1 (red, d–d′) and neuroblast marker DCX (red, e–e′) in several areas is shown. (f) PCR analysis revealed mRNA expression of both CRH-R1 and CRH-R2 in neural stem/progenitor cells (NS/PCs). Adult brain mRNA was used as positive control. (g) Detection of CRH receptors in NS/PCs isolated from E13.5 embryonic forebrain. Co-expression of CRHRs and the neural progenitor marker nestin. While the majority of neural progenitor cells express both CRHRs and nestin (white arrows), some cells are selectively positive only to nestin (yellow arrows). 4′6-diamidino-2-phenylindole (DAPI) was used as counterstain. Scale bar=100 μm (a), 50 μm (b–e), 200 μm (g). Figures in the right panel (a′, g′) or lower panels (b′–e′) are magnifications of the figures shown in the left or upper panel, respectively. aSVZ, anterior SVZ; CP, cortical plate; LV, lateral ventricle; pia, pial surface; RMS, rostral migratory stream.
Figure 3
Effect of CRH in dexamethasone-treated NS/PCs proliferation and apoptosis. (a) Representative figures of 5-bromo-2-deoxyuridine (BrdU)-labeled cells (red) counted 24 h after treatment with or without CRH after pretreatment with dexamethasone. Dexamethasone was added in cell culture 1 h before CRH. 4′6-diamidino-2-phenylindole (DAPI) staining was applied for visualization of total cell abundance. Scale bar=200 μm. (b) Graph that depicts total number of the BrdU-positive cells count in 180 × 180 μm2 areas. Data are shown as mean±s.e.m. (_n_=4). *P<0.01, **P<0.01 versus non-CRH-treated cells. (c) Effect of CRH on dexamethasone-treated NS/PCs apoptosis induced by serum deprivation for 24 h.Representative images of terminal transferase dUTP nick-end labeling (TUNEL)-stained NS/PCs (red) combined with DAPI nuclear staining (blue) after treatment with or without CRH and/or dexamethasone. Scale bar=200 μm. (d) Graph depicts quantification of the TUNEL-positive cells count in 180 × 180 μm2 areas. Data represent the mean±s.e.m. (_n_=4). *P<0.05, **P<0.01.
Figure 4
Effect of CRH in NS/PCs proliferation and apoptosis. (a) Representative figures of 5-bromo-2-deoxyuridine (BrdU)-labeled cells (red) counted 24 h after treatment with or without CRH. CRH receptor antagonists or signaling pathway blockers were added in cell culture 1 h before CRH. Antalarmin used as a specific antagonist for CRH-R1, astressin 2B (A2B) as a specific CRH-R2 antagonist, whereas PD98059 and wortmannin were used as blockers of the MAPK and PI3 K pathway, respectively. 4′6-diamidino-2-phenylindole (DAPI) staining was applied for visualization of total cell abundance. Scale bar=200 μm. (b) Effect of CRH on NS/PCs apoptosis induced by serum deprivation for 24 h. Representative images of terminal transferase dUTP nick-end labeling (TUNEL)-stained NS/PCs (red) combined with DAPI nuclear staining (blue) after treatment with or without CRH and/or specific CRH antagonists and signaling blockers as described for panel. Scale bar=200 μm. (c) Graph that depicts total number of the BrdU-positive cells count in 180 × 180 μm2 areas. Data are shown as mean±s.e.m. (_n_=4). *P<0.01, **P<0.01 versus non-CRH-treated cells. (d) Graph depicts quantification of the TUNEL-positive cells count in 180 × 180 μm2 areas. Data represent the mean±s.e.m. (_n_=4). *P<0.05, **P<0.01.
Figure 5
Expression of CRH receptors (CRHRs) in fetal human brain. (a) Detection of CRH receptors in differentiated neurons of the primordial plexiform layer (PPL) of the thalamus at 13 gestational week (arrows). (b) In the ventricular (VZ) and subventricular (SVZ) neurogenic areas there are many CRH receptor-expressing cells belonging to the neural stem/progenitor cells population as revealed by the Ki67 immunoreactivity in consecutive sections (c). Scale bar=100 μm (a), 100 μm (b, c). Figure b′ is a magnification of figure b. VZ, ventricular zone.
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