Hai-Yin Wu

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 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.

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.

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.

Interaction of nNOS with PSD-95 negatively controls regenerative repair after stroke.

Stroke is a major public health concern. The lack of effective therapies heightens the need for new therapeutic targets. Mammalian brain has the ability to rewire itself to restore lost functionalities. Promoting regenerative repair, including neurogenesis and dendritic remodeling, may offer a new therapeutic strategy for the treatment of stroke. Here, we report that interaction of neuronal nitric oxide synthase (nNOS) with the protein postsynaptic density-95 (PSD-95) negatively controls regenerative repair after stroke in rats. Dissociating nNOS–PSD-95 coupling in neurons promotes neuronal differentiation of neural stem cells (NSCs), facilitates the migration of newborn cells into the injured area, and enhances neurite growth of newborn neurons and dendritic spine formation of mature neurons in the ischemic brain of rats. More importantly, blocking nNOS–PSD-95 binding during the recovery stage improves stroke outcome via the promotion of regenerative repair in rats. Histone deacetylase 2 in NSCs may mediate the role of nNOS–PSD-95 association. Thus, nNOS–PSD-95 can serve as a target for regenerative repair after stroke.

CAPON-nNOS coupling can serve as a target for developing new anxiolytics

Anxiety disorders are highly prevalent psychiatric diseases. There is need for a deeper understanding of anxiety control mechanisms in the mammalian brain and for development of new anxiolytic agents. Here we report that the coupling between neuronal nitric oxide synthase (nNOS) and its carboxy-terminal PDZ ligand (CAPON) can serve as a target for developing new anxiolytic agents. Augmenting nNOS-CAPON interaction in the hippocampus of mice by overexpressing full-length CAPON gave rise to anxiogenic-like behaviors, whereas dissociating CAPON from nNOS by overexpressing CAPON-125C or CAPON-20C (the C-terminal 125 or 20 amino acids of CAPON) or delivering Tat-CAPON-12C (a peptide comprising Tat and the 12 C-terminal amino acids of CAPON) in the hippocampus of mice produced anxiolytic-like effects. Mice subjected to chronic mild stress (CMS) displayed a substantial increase in nNOS-CAPON coupling in the hippocampus and a consequent anxiogenic-like phenotype. Disrupting nNOS-CAPON coupling reversed the CMS-induced anxiogenic-like behaviors. Moreover, small-molecule blockers of nNOS-CAPON binding rapidly produced anxiolytic-like effects. Dexamethasone-induced ras protein 1 (Dexras1)–extracellular signal–regulated kinase (ERK) signaling was involved in the behavioral effects of nNOS-CAPON association. Thus, nNOS-CAPON association contributes to the modulation of anxiety-related behaviors via regulating Dexras1-ERK signaling and can serve as a target for developing potential anxiolytics.

The synergetic effect of edaravone and borneol in the rat model of ischemic stroke.

Free radical production contributes to the early ischemic response and the neuroinflammatory response to injury initiates the second wave of cell death following ischemic stroke. Edaravone is a free radical scavenger, and borneol has shown anti-inflammatory effect. We investigated the synergistic effect of these two drugs in the rat model of transient cerebral ischemia. Edaravone scavenged OH, NO and ONOO─ concentration-dependently, and borneol inhibited ischemia/reperfusion-induced tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) expressions. In the rat model of transient cerebral ischemia and reperfusion, the combination of edaravone and borneol significantly ameliorated ischemic damage with an optimal proportion of 4:1. Emax (% inhibition) of edaravone, borneol and two drugs in combination was 55.7%, 65.8% and 74.3% respectively. ED50 of edaravone and borneol was 7.17 and 0.36 mg/kg respectively. When two drugs in combination, ED50 was 0.484 mg/kg, in which edaravone was 0.387 mg/kg (ineffective dose) and borneol was 0.097 mg/kg (ineffective dose). Combination index (CI)<1 among effects observed in experiments, suggesting a significant synergistic effect. Reduced levels of pro-inflammatory mediators and free radicals were probably associated with the synergistic effect of edaravone and borneol. The combination exhibited a therapeutic time window of 6h in ischemia/reperfusion model, and significantly ameliorated damages in permanent ischemia model. Moreover, two drugs in combination promoted long-term effect, including improved elemental vital signs, sensorimotor functions and spatial cognition. Our results suggest that the combination of edaravone and borneol have a synergistic effect for treating ischemic stroke.

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

Background and Purpose— Previous studies reported that Tat-NR2B9c, a peptide disrupting the N-methyl-D-aspartate receptor–postsynaptic density protein-95 interaction, reduced ischemic damage in the acute phase after stroke. However, its effect in the subacute phase is unknown. The aim of this study is to determine whether disrupting the N-methyl-D-aspartate receptor–postsynaptic density protein-95 interaction in the subacute phase promotes recovery after stroke. Methods— Studies were performed on Sprague-Dawley rats or nNOS−/− mice, and experimental ischemic stroke was induced by middle cerebral artery occlusion. Animals were treated with drugs starting at day 4 after ischemia. Sensorimotor functions and spatial learning and memory ability were assessed after drug treatment. Then, rats were euthanized for morphological observation and biochemical tests. Results— Disrupting the N-methyl-D-aspartate receptor–postsynaptic density protein-95 interaction with Tat-HA-NR2B9c significantly ameliorated the ischemia-induced impairments of spatial memory and sensorimotor functions in rats during subacute stage but did not improve stroke outcome in nNOS−/− mice. Consistent with the functional recovery, Tat-HA-NR2B9c substantially increased neurogenesis in the dentate gyrus and dendritic spine density of mature neurons in the motor cortex of rats, meanwhile, reversed the ischemia-induced formation of S-nitrosylation-cyclin-dependent kinase 5 and increased cyclin-dependent kinase 5 activity in ipsilateral hippocampus. However, directly blocking N-methyl-D-aspartate receptors with MK-801 or Ro 25-6981 did not show the beneficial effects above. Conclusions— Dissociating N-methyl-D-aspartate receptor–postsynaptic density protein-95 coupling by Tat-HA-NR2B9c in the subacute phase after stroke promotes functional recovery, probably because of that it increases neurogenesis and dendritic spine density of mature neurons via regulating cyclin-dependent kinase 5 in the ischemic brain.