Chun-Xia Luo

Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95

Stroke is a major public health problem leading to high rates of death and disability in adults. Excessive stimulation of N-methyl-D-aspartate receptors (NMDARs) and the resulting neuronal nitric oxide synthase (nNOS) activation are crucial for neuronal injury after stroke insult. However, directly inhibiting NMDARs or nNOS can cause severe side effects because they have key physiological functions in the CNS. Here we show that cerebral ischemia induces the interaction of nNOS with postsynaptic density protein-95 (PSD-95). Disrupting nNOS–PSD-95 interaction via overexpressing the N-terminal amino acid residues 1–133 of nNOS (nNOS-N1–133) prevented glutamate-induced excitotoxicity and cerebral ischemic damage. Given the mechanism of nNOS–PSD-95 interaction, we developed a series of compounds and discovered a small-molecular inhibitor of the nNOS-PSD-95 interaction, ZL006. This drug blocked the ischemia-induced nNOS–PSD-95 association selectively, had potent neuroprotective activity in vitro and ameliorated focal cerebral ischemic damage in mice and rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Moreover, it readily crossed the blood-brain barrier, did not inhibit NMDAR function, catalytic activity of nNOS or spatial memory, and had no effect on aggressive behaviors. Thus, this new drug may serve as a treatment for stroke, perhaps without major side effects.

Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis

Increasing evidence suggests that depression may be associated with a lack of hippocampal neurogenesis. It is well established that neuronal nitric oxide synthase (nNOS)‐derived NO exerts a negative control on the hippocampal neurogenesis. Using genetic and pharmacological methods, we investigated the roles of nNOS in depression induced by chronic mild stress (CMS) in mice. Hippocampal nNOS over‐expression was first observed 4 days and remained elevated 21 and 56 days after exposure to CMS. The mice exposed to CMS exhibited behavioral changes typical of depression, and impaired neurogenesis in the hippocampus. The CMS‐induced behavioral despair and hippocampal neurogenesis impairment were prevented and reversed in the null mutant mice lacking nNOS gene (nNOS−/−) and in the mice receiving nNOS inhibitor. Disrupting hippocampal neurogenesis blocked the antidepressant effect of nNOS inhibition. Moreover, nNOS−/− mice exhibited antidepressant‐like properties. Our findings suggest that nNOS over‐expression in the hippocampus is essential for chronic stress‐induced depression and inhibiting nNOS signaling in brain may represent a novel approach for the treatment of depressive disorders.

Neuronal Nitric Oxide Synthase Alteration Accounts for the Role of 5-HT1A Receptor in Modulating Anxiety-Related Behaviors

Increasing evidence suggests that 5-HT1A receptor (5-HT1AR) is implicated in anxiety disorders. However, the mechanism underlying the role of 5-HT1AR in these diseases remains unknown. Here, we show that 5-HT1AR-selective agonist 8-OH-DPAT and selective serotonin reuptake inhibitor (SSRI) fluoxetine downregulated hippocampal neuronal nitric oxide synthase (nNOS) expression, whereas 5-HT1AR-selective antagonist NAN-190 upregulated hippocampal nNOS expression. By assessing anxiety-related behaviors using the novelty suppressed feeding, open-field, and elevated plus maze tests, we show that mice lacking nNOS gene [knock-out (KO)] or treated with nNOS-selective inhibitor 7-nitroindazole (7-NI; i.p., 30 mg/kg/d for 28 d; or intrahippocampal microinjection, 16.31 μg/1.0 μl) displayed an anxiolytic-like phenotype, implicating nNOS in anxiety. We also show that, in wild-type (WT) mice, administrations of 8-OH-DPAT (i.p., 0.1 mg/kg/d) or fluoxetine (i.p., 10 mg/kg/d) for 28 d caused anxiolytic-like effects, whereas NAN-190 (i.p., 0.3 mg/kg/d for 28 d) caused anxiogenic-like effects. In KO mice, however, these drugs were ineffective. Moreover, intrahippocampal infusion of 8-OH-DPAT (45.963 μg/100 μl) using 14 d osmotic minipump produced anxiolytic effects. Intrahippocampal microinjection of 7-NI (16.31 μg/1.0 μl) abolished the anxiogenic-like effects of intrahippocampal NAN-190 (4.74 μg/1.0 μl). Additionally, NAN-190 decreased and 8-OH-DPAT increased phosphorylated cAMP response element-binding protein (CREB) levels in WT mice but not in KO mice. Blockade of hippocampal CREB phosphorylation by microinjection of H89 (5.19 μg/1.0 μl), a PKA (protein kinase A) inhibitor, abolished the anxiolytic-like effects of 7-NI (i.p., 30 mg/kg/d for 21 d). These findings indicate that both hippocampal nNOS and CREB activity mediate the anxiolytic effects of 5-HT1AR agonists and SSRIs.

Chronic fluoxetine treatment improves ischemia-induced spatial cognitive deficits through increasing hippocampal neurogenesis after stroke.

Cognitive deficits, including spatial memory impairment, are very common after ischemic stroke. Neurogenesis in the dentate gyrus (DG) contributes to forming spatial memory in the ischemic brain. Fluoxetine, a selective serotonin reuptake inhibitor, can enhance neurogenesis in the hippocampus in physiological situations and some neurological diseases. However, whether it has effects on ischemia‐induced spatial cognitive impairment and hippocampal neurogenesis has not been determined. Here we report that fluoxetine treatment (10 mg kg−1, i.p.) for 4 weeks promoted the survival of newborn cells in the ischemic hippocampus and, consequently, attenuated spatial memory impairment of mice after focal cerebral ischemia. Disrupting hippocampal neurogenesis blocked the beneficial effect of fluoxetine on ischemia‐induced spatial cognitive impairment. These results suggest that chronic fluoxetine treatment benefits spatial cognitive function recovery following ischemic insult, and the improved cognitive function is associated with enhanced newborn cell survival in the hippocampus. Our results raise the possibility that fluoxetine can be used as a drug to treat poststroke spatial cognitive deficits.

Neuronal nitric oxide synthase-derived nitric oxide inhibits neurogenesis in the adult dentate gyrus by down-regulating cyclic AMP response element binding protein phosphorylation.

Neuronal nitric oxide synthase, the major nitric oxide synthase isoform in the mammalian brain, is implicated in some developmental processes, including neuronal survival, precursor proliferation and differentiation. However, reports about the role of neuronal nitric oxide synthase in neurogenesis in the adult dentate gyrus are conflicting. Here we show that 5-bromodeoxyuridine-labeled dividing progenitor cells in the dentate gyrus were significantly increased in mice receiving 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, and in null mutant mice lacking neuronal nitric oxide synthase gene (nNOS-/-) 6 h and 4 weeks after 5-bromodeoxyuridine incorporation. The increase in 5-bromodeoxyuridine positive cells in 7-nitroindazole-treated mice was accompanied by activation of cyclic AMP response element binding protein phosphorylation in the dentate gyrus. Pretreatment with N-methyl-D-aspartate receptor antagonist MK-801 fully abolished the effects of 7-nitroindazole on neurogenesis and cyclic AMP response element binding protein phosphorylation. Furthermore, neuronal nitric oxide synthase inhibition significantly enhanced the survival of newborn cells and the number of 5-bromodeoxyuridine positive/NeuN positive cells in the dentate gyrus. These results indicate that neuronal nitric oxide synthase-derived nitric oxide suppresses neurogenesis in the adult dentate gyrus, in which N-methyl-D-aspartate receptor functions and cyclic AMP response element binding protein phosphorylation may be involved.

Hippocampal Neuronal Nitric Oxide Synthase Mediates the Stress-Related Depressive Behaviors of Glucocorticoids by Downregulating Glucocorticoid Receptor

The molecular mechanisms underlying the behavioral effects of glucocorticoids are poorly understood. We report here that hippocampal neuronal nitric oxide synthase (nNOS) is a crucial mediator. Chronic mild stress and glucocorticoids exposures caused hippocampal nNOS overexpression via activating mineralocorticoid receptor. In turn, hippocampal nNOS-derived nitric oxide (NO) significantly downregulated local glucocorticoid receptor expression through both soluble guanylate cyclase (sGC)/cGMP and peroxynitrite (ONOO−)/extracellular signal-regulated kinase signal pathways, and therefore elevated hypothalamic corticotrophin-releasing factor, a peptide that governs the hypothalamic-pituitary-adrenal axis. More importantly, nNOS deletion or intrahippocampal nNOS inhibition and NO-cGMP signaling blockade (using NO scavenger or sGC inhibitor) prevented the corticosterone-induced behavioral modifications, suggesting that hippocampal nNOS is necessary for the role of glucocorticoids in mediating depressive behaviors. In addition, directly delivering ONOO− donor into hippocampus caused depressive-like behaviors. Our findings reveal a role of hippocampal nNOS in regulating the behavioral effects of glucocorticoids.

Negative regulation of neurogenesis and spatial memory by NR2B-containing NMDA receptors.

Several lines of evidence suggest involvement of NMDA receptors (NMDARs) in the regulation of neurogenesis in adults and the formation of spatial memory. Functional properties of NMDARs are strongly influenced by the type of NR2 subunits incorporated. In adult forebrain regions such as the hippocampus and cortex, only NR2A and NR2B subunits are available to form the receptor complex with NR1 subunit. NR2B is predominant NR2 subunit in any of rat or human neural stem cells (NSCs). Thus, we suppose that NR2B‐containing NMDAR should be critical in regulating adult neurogenesis, and thereby playing a role in the formation of spatial memory. In the cultured NSCs derived from the embryonic brain of rats, NR2B subunit‐specific NMDAR antagonist Ro25‐6981 increased cell proliferation, whereas MK‐801, non‐selective open‐channel blocker of NMDARs, inhibited cell proliferation. Blockade of NR2B‐containing NMDAR stimulated neurogenesis in the adult hippocampus and facilitated the formation of spatial memory. The enhanced spatial memory dropped back to base level when the NR2B antagonist‐induced neurogenesis was neutralized by 3′‐azido‐deoxythymidine, a telomerase inhibitor. In addition, blockade of NR2B inhibited neuronal nitric oxide synthase (nNOS) enzymatic activity. In null mutant mice lacking nNOS gene (nNOS−/−), the effects of NR2B antagonist on neurogenesis disappeared. Moreover, nitric oxide donor DETA/NONOate attenuated and nNOS inhibitor 7‐nitroindazole enhanced the effect of Ro 25‐6981 on NSCs proliferation. Our findings suggest that NR2B‐containing NMDAR subtypes negatively regulate neurogenesis in the adult hippocampus by activating nNOS activity and thereby hinder the formation of spatial memory.

Hippocampal Telomerase Is Involved in the Modulation of Depressive Behaviors

Telomere and telomerase alterations have been reported in mood disorders. However, the role of telomerase in depression remains unclear. Here we show that chronic mild stress (CMS) led to a significant decrease in telomerase reverse transcriptase (TERT) level and telomerase activity in the hippocampus. Treatment with antidepressant fluoxetine reversed the CMS-induced TERT and telomerase activity changes. Inhibiting telomerase by systemic administration (100 mg · kg−1 · d−1, i.p., for 14 d), intrahippocampal microinjection (0.7 μmol, 2 μl), or infusion (using an osmotic minipump, 0.134 μg/μl, 0.25 μl/h) of 3′-azido-deoxythymidine (AZT) resulted in depression-like behaviors and impaired hippocampal neurogenesis in mice. In contrast, overexpressing telomerase by intrahippocampal infusion of recombinant adenovirus vector expressing mouse TERT (Ad-mTERT-GFP) led to neurogenesis upregulation, produced antidepressant-like behaviors, and prevented the CMS-induced behavioral modifications. Disrupting neurogenesis in the dentate gyrus by X-irradiation (15 Gy) of a restricted region of mouse brain containing the hippocampus abolished the antidepressant-like effect of Ad-mTERT-GFP. Additionally, AZT had no effect on DNA polymerase activity and did not cause cell damage in vitro and in vivo. Microinjection of AZT into the subventricular zone of lateral ventricle (0.7 μmol, 2 μl) inhibited local neurogenesis but had no behavioral effect. These results suggest that hippocampal telomerase is involved in the modulation of depression-related behaviors, possibly by regulating adult neurogenesis.

Bidirectional regulation of neurogenesis by neuronal nitric oxide synthase derived from neurons and neural stem cells.

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 N5‐(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−/− 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. STEM CELLS 2010;28:2041–2052

Hippocampal nitric oxide contributes to sex difference in affective behaviors

Mechanisms underlying the female preponderance in affective disorders are poorly understood. Here we show that hippocampal nitric oxide (NO) plays a role in the sex difference of depression-like behaviors in rodents. Female mice had substantially lower NO production in the hippocampus and were significantly more likely to display negative affective behaviors than their male littermates. Eliminating the difference in the basal hippocampal NO level between male and female mice mended the sex gap of affective behaviors. Estradiol exerted a positive control on hippocampal NO production via estrogen receptor-β–mediated neuronal NO synthase expression. Thus, low estrogen in the female hippocampus accounts for lower local NO than in the male hippocampus. Although estrogen has important significance in modulating affective behaviors, it is not estrogen but NO in the hippocampus that mediates the sex difference of affective behaviors directly, because hippocampal NO was necessary for the behavioral effects of estradiol, and NO was an independent factor in modulating behaviors. Stress promoted hippocampal NO production in males because of glucocorticoid release, thus leading to local NO excess. In contrast, stress suppressed NO production in females because of decreased estrogen, thereby resulting in hippocampal NO shortage. Whereas activating cAMP response element binding protein (CREB) rescued the depression-like effects of the intrahippocampal NO donor diethylenetriamine/nitric oxide adduct (DETA/NONOate), inactivating CREB abolished the antidepressant-like effects of the intrahippocampal NO donor DETA/NONOate. Our findings suggest a molecular mechanism underlying the sex difference of affective behaviors.