BIdirectional Regulation of Neurogenesis by Neuronal Nitric Oxide Synthase Derived from Neurons and Neural Stem Cells

Abstract

It has been demonstrated that neuronal nitric oxide synthase (nNOS) negatively regulates adult neurogenesis. However, the cellular and molecular mechanisms underlying are poorly understood. Here, we show that nNOS from neural stem cells (NSCs) and from neurons play opposite role in regulating neurogenesis. The NSCs treated with nNOS inhibitor N 5 -(1-imino-3-butenyl)-L-ornithine (L-VNIO) or nNOS gene deletion exhibited significantly decreased proliferation and neuronal differentiation, indicating that NSCs-derived nNOS is essential for neurogenesis. The NSCs cocultured with neurons displayed a significantly decreased proliferation, and deleting nNOS gene in neurons or scavenging extracellular nitric oxide (NO) abolished the effects of coculture, suggesting that neurons-derived nNOS, a source of exogenous NO for NSCs, exerts a negative control on neurogenesis. Indeed, the NSCs exposed to NO donor DETA/NONOate displayed decreased proliferation and neuronal differentiation. The bidirectional regulation of neurogenesis by NSCs-and neurons-derived nNOS is probably related to their distinct subcellular localizations, mainly in nuclei for NSCs and in cytoplasm for neurons. Both L-VNIO and DETA/NONOate inhibited telomerase activity and proliferation in wild-type (WT) but not in nNOS 2/2 NSCs, suggesting a nNOS-telomerase signaling in neurogenesis. The NSCs exposed to DETA/NONOate exhibited reduced cAMP response element binding protein (CREB) phosphorylation, nNOS expression, and proliferation. The effects of DETA/NONOate were reversed by forskolin, an activator of CREB signaling. Moreover, disrupting CREB phosphorylation by H-89 or LV-CREB133-GFP simulated the effects of DETA/NONOate, and inhibited telomerase activity. Thus, we conclude that NSCs-derived nNOS stimulates neurogenesis via activating telomerase, whereas neurons-derived nNOS represses neurogenesis by supplying exogenous NO that hinders CREB activation, in turn, reduces nNOS expression in NSCs.

Delayed Administration of Tat-HA-NR2B9c Promotes Recovery After Stroke in Rats

“…by guest on May 7, 2018 http://stroke.ahajournals.org/ Downloaded from NO donor and neuron-derived nNOS inhibit neural stem cell proliferation and neuronal differentiation. 34 dissociating the NMdAR-PSd-95 interaction by Tat-HA-NR2B9c decreases the production of NO. 11 Thus, enhancement of neurogenesis in the ischemic hippocampus by delayed treatment with Tat-HA-NR2B9c may be because of inhibition of NO production.…”

Hippocampal Telomerase Is Involved in the Modulation of Depressive Behaviors

“…Adult NSCs were isolated and cultured as described previously (Luo et al, 2010], with some modifications. In brief, the dentate gyri of 2-monthold female mice (10 mice were used in each primary isolation experiment) were dissected and digested with 0.125% trypsin (Invitrogen) and 250 U/ml DNase I (Sigma-Aldrich) at 37°C for 20 min, and undifferentiated progenitors were enriched by centrifugation with Percoll (GE Healthcare Bio-Sciences).…”

“…Primary hippocampal neurons were isolated from an embryonic day 18 mouse and cultured in neurobasal medium (Invitrogen) containing 2% B27 supplement as reported (Luo et al, 2010). Cultured neurons were identified at 10 d in vitro, and the proportion of ␤-III-tubulin ϩ cells was about 92%.…”

“…Samples from hippocampal tissues of mice were prepared as described in our previous studies (Luo et al, 2010). The samples containing equivalent amounts of protein (10 g) were applied to 8% acrylamide denaturing gels (SDS-PAGE).…”

Intracellular Nitric Oxide Mediates Neuroproliferative Effect of Neuropeptide Y on Postnatal Hippocampal Precursor Cells

“…Exposure to Pharmacological Agonists and Antagonists and BrdU Incorporation-The concentrations of agonists or antagonists were chosen based on published literature and included NPY (1 M; Sigma-Aldrich) (15), the nonselective NOS inhibitor N()-nitro-L-arginine methyl ester (L-NAME) (500 M; Sigma-Aldrich) (23), the inactive enantiomer N()-nitro-D-arginine methyl ester (D-NAME) (500 M; Sigma), the selective Y 1 agonist [F7, P34]NPY (1 M; courtesy of Prof. Dr. A. G. BeckSickinger, Universität Leipzig, Germany) (15), the NOS substrate L-arginine (500 M; Sigma-Aldrich), the inactive enantiomer D-arginine (500 M; Sigma-Aldrich), the cGMP analog 8-Br-cGMP (20 M; Tocris Bioscience) (23) (42), and the NO scavenger carboxy-PTIO (CPTIO, 10 M; Tocris Bioscience) (42,43). Agonists or antagonists were added to cultures for 3 or 5 DIV or for 6 h on the final day before cells were rinsed once in 0.1 M phosphate-buffered saline (PBS; Sigma-Aldrich) and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 min.…”