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-N(1-133)) 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.

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.

Using differential display, we cloned a gene with reduced expression in short-term explants of head and neck squamous cell carcinoma (HNSCC) tumors compared to cultured normal oral epithelial cells. The differentially expressed gene was identical to the recently cloned CXC chemokine BRAK, which is ubiquitously expressed in normal tissue extracts but is absent from many tumor cell lines in vitro. To define the cell populations expressing BRAK in vivo, in situ mRNA hybridization was performed on normal and cancerous tissues from six different histological sites. The predominant normal cell type constitutively expressing BRAK in vivo was squamous epithelium. Expression in tumors was heterogeneous, with the majority of HNSCCs and some cervical squamous cell carcinomas (SCCs) showing loss of BRAK mRNA. Although absent in unstimulated peripheral blood mononuclear cells, high levels of BRAK were consistently found in infiltrating inflammatory cells (with lymphocyte morphology) in nearly all cancers examined. Furthermore, BRAK expression was demonstrated in B cells and monocytes, after stimulation of peripheral blood mononuclear cells with lipopolysaccharide. This study demonstrates for the first time up-regulation of BRAK mRNA by inflammatory cells in the tumor microenvironment and lost expression from certain cancers in vivo. The data suggest that BRAK may have a role in host-tumor interactions.

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 Ϫ )/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 Ϫ donor into hippocampus caused depressive-like behaviors. Our findings reveal a role of hippocampal nNOS in regulating the behavioral effects of glucocorticoids.

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