CAPON-nNOS Coupling Can Serve As A Target For Developing Novel Anxiolytics

Anxiety is a mental state that is elicited in anticipation of threat or potential threat.Sensations of anxiety are a normal part of human experience,but excessive or inappropriate anxiety can become an illness.As a common human emotional illness,anxiety is accompanied by a characteristic set of behavioural and physiological responses including avoidance,vigilance and arousal,which evolved to protect the individual from danger.Recent epidemiological studies indicated that anxiety disorders affect over 29%of the population at some point in their lifetime.However,the pathophysiology of anxiety disorders and molecular mechanisms underlying anxiety-related behaviors are poorly understood.Benzodiazepines(BZDs)are widely used as clinical anxiolytics.However,BZDs usually produce severe side effects,including anterograde amnesia,impairment of motor coordination,potentiation of ethanol effects and addiction.In the past two decades,another class of drugs,the selective serotonin re-uptake inhibitors(SSRIs),have replaced BZDs as first-line treatment for anxiety,mostly because they lack the addictive properties of BZDs,are now used effectively to treat most anxiety disorders.Unfortunately,however,anxiolytic effects of SSRIs occur only after several weeks of treatment.Thus,there is need for a deeper understanding of anxiety control mechanisms in mammalian brain,and to develop novel anxiolytic agents that have quicker anxiolytic potential and are clinically well tolerated.The hippocampus,one of the components of the limbic system,is structurally and functionally implicated in mood disorders.nNOS,a main nitric oxide synthase in the central nervous system,is expressed in limbic system abundantly.Our study reveals that hippocampal nNOS contributes to stress-and corticosteroid-induced excessive activation of HPA axis.Moreover,we find that hippocampal nNOS accounts for the role of postsynaptic 5-HT1A acceptor in regulating anxiety-related behaviors.CAPON is a cytoplasmic protein whose C terminus binds to the PDZ domain of nNOS(carboxy-terminal PDZ ligand of nNOS).At least two isoforms of CAPON were identified so far.One is the full-length isoform(CAPON-L)encompasses ten exons and encodes a 503 amino acid protein,another is the short form(CAPON-S)contains the last two exons of CAPON and is predicted to produce a 211 amino acids protein.CAPON-L contains two known functional domains,an amino-terminal phosphotyrosine binding domain and a carboxyl-terminal PDZ-binding domain.CAPON-L but not CAPON-S can bind to synapsin,mediats the development,growth and mentainence of dendrites,because CAPON-S lacks the N-terminal phosphotyrosine binding domain.Many studies identified significant linkage disequilibrium between schizophrenia,bipolar disorder and PTSD with the markers within the gene for CAPON.Prior sequencing of the ten exons of CAPON failed to reveal a coding mutation associated with illness.But increased CAPON expression is observed in postmortem samples from individuals with schizophrenia and bipolar disorder,suggesting a possible mechanism of action of CAPON in psychiatric illness.More recently,our studies have shown that nNOS-derived NO contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis.In addition,nNOS plays an important role in the development of synapse and contributes to synaptic damage and subsequent neuronal loss in the pathological states.There are studies showing that CAPON was related to the construction of synapse.CAPON can also serve as a nNOS adaptor protein binding to synapsin,resulting in the localization of nNOS to the presynaptic terminals.Therefore,it is possible that nNOS-CAPON interaction may be implicated in the regulation of anxiety-related behaviors,thereby serves as a target for the treatment of anxiety disorder.To test this possibility,we investigated whether the interaction of nNOS with CAPON in the hippocampus plays an important role in the regulation of anxiety-related behaviors,to explore a target for developing novel anxiolytics,and further analysed possible molecular mechanism underlying the regulation of anxiety by nNOS-CAPON coupling.In part 1,to examine whether nNOS-CAPON coupling contributes to the modulation of anxiety-related behaviors,we generated a lentiviral vector that expresses CAPON-L and GFP,and named it LV-CAPON-L-GFP.We infused LV-CAPON-L-GFP into the hippocampus of mice and assessed anxiety-related behaviors 14 d later.It effectively infected the hippocampus,produced considerable CAPON-L-GFP,and caused the mice to display significantly increased latency to feed in novel environment in the novelty suppressed feeding(NSF)test,and spend less time in open arms in the elevated plus maze(EPM)test and in the lit compartment of the light/dark box,suggesting an anxiogenic-like effect.But this effect disappeared in nNOS gene knock-out(nNOS-/-)mice.To disrupt the interaction of nNOS with CAPON,we generated two lentiviral vectors expressing different CAPON dominants.One is LV-GFP-CAPON-20C that contains 20 amino acids of C-terminal of CAPON.Another is LV-CAPON-125-GFP that contains 125 amino acids of C-terminal of CAPON.We also prepared a small peptide,Tat-CAPON-12C,which contains 12 amino acids of C-terminal of CAPON.We microinjected these lentiviral vectors and peptide into the hippocampus of mice and found that they blocked nNOS-CAPON binding and produced substantial anxiolytic-like effects in NSF test,EPM test and OF test.Moreover,both lentiviral vectors and peptide were ineffective in nNOS-/-mice.Since Stressful life events have a substantial causal association with affective disorders,we therefore investigated the role of nNOS-CAPON coupling in the stress-induced anxiety behaviors.We exposed mice to chronic mild stress(CMS),an unpredictable chronic stress leading to anxiety-like behaviors.CMS significantly enhanced the amount of nNOS-CAPON-L complex in the hippocampus but not in the cortex,probably owing to that up-regulation of nNOS by CMS,in turn,up-regulated CAPON-L in the hippocampus.Next,we infused LV-CAPON-125C-GFP into the hippocampus of mice by microinjection.Four days later,these mice were subjected to CMS for 21 d.LV-CAPON-125C-GFP produced considerable CAPON-125C-GFP,reversed CMS-induced interaction of nNOS with CAPON in the hippocampus and behavioral modifications in the NSF,EPM,OF and light/dark box tests.Taken together,our findings demonstrate that nNOS-CAPON coupling in the hippocampus is critical for the regulation of anxiety-related behaviors,and dissociating this coupling produces significant anxiolytic-like effects,suggesting a target for developing novel anxiolytics.In part 2,to develop small molecules suitable for clinically treating anxiety disorders by disrupting the nNOS-CAPON interaction,we designed and synthesized a series of compounds based on the chemical mechanism for binding the C-terminal of CAPON to nNOS PDZ domain.Among them,ZLc-002 was the best one.ZLc-002 disrupted the interaction of nNOS with CAPON in vitro and in vivo.By systemic administration and intrahippocampal injection of ZLc-002,we found that this drug produced substantial anxiolytic-like effects in NSF test,EPM test and OF test,with a quicker anxiolytic potential(beginning at 24h after treatment)and potent efficacy.To test the metabolic pathway of ZLc-002 in vivo,we treated mice with ZLc-002 and measured concentrations of ZLc-002 and its metabolites in blood and brain.In the systemic circulation,ZLc-002 is rapidly hydrolyzed into ZLc-002-1 and then into ZLc-002-2,but only ZLc-002-1 readily crosses the blood-brain barrier.Thus,ZLc-002 should be a prodrug and ZLc-002-1 may explain the effects of ZLc-002.Indeed,ZLc-002-1 produced significant anxiolytic-like effects and blocked nNOS-CAPON binding.Moreover,we found that there was a correlation between potencies in blocking nNOS-CAPON binding and behavioral effects of ZLc-002 and its analogues.In part 3,we examined the possible mechanism underlying anxiolytic effects of disrupting nNOS-CAPON association.Firstly,it is very unlikely that ZLc-002 acts as a 5-HT reuptake inhibitor or 5-HT1AR agonist,because it had a quicker onset of action,whereas both fluoxetine,a classical SSRI,and 8-OH-DPAT,a selective 5-HT1AR agonist,had no effect until 3 weeks after treatment in mice.In addition,ZLc-002 is unlikely to work via up-regulating GABA function,because it did not affect GABA inhibitory postsynaptic currents.Moreover,ZLc-002 had no sedative effect,and did not cause motor incoordination and myorelaxant.Although nNOS was required for the behavioral effects of disrupting nNOS-CAPON association in the hippocampus,the effects of ZLc-002 could not be explained by altered nNOS,owing to ZLc-002 did not affect nNOS expression.Both ZLc-002 and Tat-CAPON-L-12G did not change PSD-95 expression in the hippocampal neurons.Furthermore,the behavioral effects of dissociating nNOS-CAPON are not due to PSD-95 alteration,because both ZLc-002 and Tat-CAPON-12C did not change PSD-95 expression.To determine whether nNOS-CAPON interaction affects synaptic structure,we treated the cultured hippocampal neurons with Tat-CAPON-12C,ZLc-002,LV-CAPON-125C-GFP or LV-GFP-CAPON-20C to block nNOS-CAPON binding and found that they significantly increased synapsin and spinophilin expressions.In contrast,promoting the interaction of nNOS with CAPON by LV-CAPON-L-GFP in hippocampal neurons caused significant decreases in levels of synapsin and spinophilin.Similar results were acquired in vivo by regulating nNOS-CAPON interaction.And also,blocking nNOS-CAPON association increases the dendrite branching significantly.Thus,synaptogenesis may accounts for the anxiolytic-like effects of blocking nNOS-CAPON association.In summary,our data demonstrate the implication of hippocampal nNOS-CAPON interaction in mediating anxiety-related behaviors,and provide a target for developing novel anxiolytics.We discoverey a new drug,ZLc-002,which selectively blocks nNOS-CAPON interaction and has potent anxiolytic-like effects with a quicker anxiolytic potential and potent efficacy.Remodeling of synaptic structure may accounts for the roles of nNOS-CAPON coupling in regulating anxiety-related behaviors.