Carboxy-terminal PDZ Ligand Of nNOS Research Articles

Hippocampal excitation-inhibition balance underlies the 5-HT2C receptor in modulating depressive behaviours.

The implication of 5-hydroxytryptamine 2C receptor (5-HT2CR) in depression is a topic of debate, and the underlying mechanisms remain largely unclear. We now elucidate hippocampal excitation-inhibition (E/I) balance underlies the regulatory effects of 5-HT2CR in depression. Molecular biological analyses showed that chronic mild stress (CMS) reduced the expression of 5-HT2CR in hippocampus. We revealed that inhibition of 5-HT2CR induced depressive-like behaviors, reduced GABA release and shifted the E/I balance towards excitation in CA3 pyramidal neurons by using behavioral analyses, microdialysis coupled with mass spectrum, and electrophysiological recording. Moreover, 5-HT2CR modulated neuronal nitric oxide synthase (nNOS)-carboxy-terminal PDZ ligand of nNOS (CAPON) interaction through influencing intracellular Ca2+ release, as determined by fiber photometry and coimmunoprecipitation. Notably, disruption of nNOS-CAPON by specific small molecule compound ZLc-002 or AAV-CMV-CAPON-125C-GFP, abolished 5-HT2CR inhibition-induced depressive-like behaviors, as well as the impairment in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly-mediated GABA vesicle release and a consequent E/I imbalance. Importantly, optogenetic inhibition of CA3 GABAergic neurons prevented the effects of AAV-CMV-CAPON-125C-GFP on depressive behaviors in the presence of 5-HT2CR antagonist. Conclusively, our findings disclose the regulatory role of 5-HT2CR in depressive-like behaviors and highlight the hippocampal nNOS-CAPON coupling-triggered E/I imbalance as a pivotal cellular event underpinning the behavioral consequences of 5-HT2CR inhibition.

NNOS and Neurological, Neuropsychiatric Disorders: A 20-Year Story.

In the central nervous system, nitric oxide (NO), a free gas with multitudinous bioactivities, is mainly produced from the oxidation of L-arginine by neuronal nitric oxide synthase (nNOS). In the past 20 years, the studies in our group and other laboratories have suggested a significant involvement of nNOS in a variety of neurological and neuropsychiatric disorders. In particular, the interactions between the PDZ domain of nNOS and its adaptor proteins, including post-synaptic density 95, the carboxy-terminal PDZ ligand of nNOS, and the serotonin transporter, significantly influence the subcellular localization and functions of nNOS in the brain. The nNOS-mediated protein-protein interactions provide new attractive targets and guide the discovery of therapeutic drugs for neurological and neuropsychiatric disorders. Here, we summarize the work on the roles of nNOS and its association with multiple adaptor proteins on neurological and neuropsychiatric disorders.

Requirement of hippocampal DG nNOS-CAPON dissociation for the anxiolytic and antidepressant effects of fluoxetine.

Background: Adult hippocampal neurogenesis and synaptic plasticity are necessary for the behavioral response to the selective serotonin reuptake inhibitor (SSRI) fluoxetine, but the molecular mechanisms underlying these effects are only partially understood.Methods: Anxiety and depressive-like behaviors in mice were developed by chronic mild stress (CMS) or chronic corticosterone (CORT) treatment. Pharmacological and genetic approaches were used to investigate the role of the neuronal nitric oxide synthase (nNOS)-carboxy-terminal PDZ ligand of nNOS (CAPON) interaction in behavioral and neuroplasticity effects of serotoninergic system. Molecular biological and morphological studies were performed to examine the mechanisms underlying the behavioral effects of nNOS-CAPON interaction that modulated by 5-HT1A receptor (5-HT1AR).Results: Fluoxetine prevented chronic stress-induced nNOS-CAPON upregulation and coupling in the dentate gyrus (DG), and promoting nNOS-CAPON association weakened the anxiolytic and antidepressant effects of fluoxetine in stressed mice. The chronic fluoxetine elevated 5-HT and 5HT1AR agonist 8-OH-DPAT decreased the expression and binding of nNOS with CAPON, whereas 5-HT1AR antagonist NAN-190 had the opposite effects. Importantly, augmenting nNOS-CAPON binding neutralized 8-OH-DPAT-upregulated spine density of DG granule cells and well-characterized synaptic-related proteins, including brain-derived neurotrophic factor (BDNF) and phosphorylation of extracellular signal regulated kinase (ERK), cAMP-response element binding protein (CREB), and synapsin in the DG and abolished the anxiolytic and antidepressant-like effects of 8-OH-DPAT. In contrast, dissociation of nNOS from CAPON rescued the effects of NAN-190 on behavior and neuroplasticity.Conclusion: Taken together, our results indicated that fluoxetine modifies mood behaviors and hippocampal neuroplasticity by disrupting the nNOS-CAPON interaction that links postsynaptic 5-HT1AR activation.

NNOS-mediated protein-protein interactions: promising targets for treating neurological and neuropsychiatric disorders.

Neurological and neuropsychiatric disorders are one of the leading causes of disability worldwide and affect the health of billions of people. Nitric oxide (NO), a free gas with multitudinous bioactivities, is mainly produced from the oxidation of L-arginine by neuronal nitric oxide synthase (nNOS) in the brain. Inhibiting nNOS benefits a variety of neurological and neuropsychiatric disorders, including stroke, depression and anxiety disorders, post-traumatic stress disorder, Parkinson's disease, Alzheimer's disease, chronic pain, and drug addiction. Due to critical roles of nNOS in learning and memory and synaptic plasticity, direct inhibition of nNOS may cause severe side effects. Importantly, interactions of several proteins, including post-synaptic density 95 (PSD-95), carboxy-terminal PDZ ligand of nNOS (CAPON) and serotonin transporter (SERT), with the PSD/Disc-large/ZO-1 homologous (PDZ) domain of nNOS have been demonstrated to influence the subcellular distribution and activity of the enzyme in the brain. Therefore, it will be a preferable means to interfere with nNOS-mediated protein-protein interactions (PPIs), which do not lead to undesirable effects. Herein, we summarize the current literatures on nNOS-mediated PPIs involved in neurological and neuropsychiatric disorders, and the discovery of drugs targeting the PPIs, which is expected to provide potential targets for developing novel drugs and new strategy for the treatment of neurological and neuropsychiatric disorders.

Neuroprotective Effect of N-Cyclohexylethyl-[A/G]-[D/E]-X-V Peptides on Ischemic Stroke by Blocking nNOS-CAPON Interaction.

The protein-protein interaction between neuronal nitric oxide syntheses (nNOS) and the carboxy-terminal PDZ ligand of nNOS (CAPON) is a potential target for the treatment of ischemic stroke. Our previous study had identified ZLc-002 as a promising lead compound for inhibiting nNOS-CAPON coupling. To find better neuroprotective agents disrupting the ischemia-induced nNOS-CAPON interaction, a series of N-cyclohexylethyl-[A/G]-[D/E]-X-V peptides based on the carboxy-terminal tetrapeptide of CAPON was designed, synthesized, and evaluated in this study. Herein, we reported an affinity-based fluorescence polarization (FP) method using 5-carboxyfluorescein (5-FAM) labeled CAPON (496-506) peptide as the probe for high-throughput screening of the small-molecule inhibitors of the PDZ domain of nNOS. N-Cyclohexylethyl-ADAV displayed the most potent affinity for the nNOS PDZ domain in the FP and isothermal titration calorimetry (ITC) (ΔH = -1670 ± 151.0 cal/mol) assays. To improve bioavailability, lipophilicity, and membrane permeability, the Asp methylation was employed to get N-cyclohexylethyl-AD(OMe)AV, which possesses good blood-brain barrier (BBB) permeability in vitro parallel artificial membrane permeability assay (PAMPA)-BBB (Pe = 6.07 cm/s) and in vivo assays. In addition, N-cyclohexylethyl-AD(OMe)AV (10 mg/kg body weight, i.v., immediately after reperfusion) substantially reduced infarct size in rats, which was measured 24 h after reperfusion and subjected to 120 min of middle cerebral artery occlusion (MCAO).

Tau binding protein CAPON induces tau aggregation and neurodegeneration

To understand the molecular processes that link Aβ amyloidosis, tauopathy and neurodegeneration, we screened for tau-interacting proteins by immunoprecipitation/LC-MS. We identified the carboxy-terminal PDZ ligand of nNOS (CAPON) as a novel tau-binding protein. CAPON is an adaptor protein of neuronal nitric oxide synthase (nNOS), and activated by the N-methyl-D-aspartate receptor. We observed accumulation of CAPON in the hippocampal pyramidal cell layer in the AppNL-G-F -knock-in (KI) brain. To investigate the effect of CAPON accumulation on Alzheimer’s disease (AD) pathogenesis, CAPON was overexpressed in the brain of AppNL-G-F mice crossbred with MAPT (human tau)-KI mice. This produced significant hippocampal atrophy and caspase3-dependent neuronal cell death in the CAPON-expressing hippocampus, suggesting that CAPON accumulation increases neurodegeneration. CAPON expression also induced significantly higher levels of phosphorylated, oligomerized and insoluble tau. In contrast, CAPON deficiency ameliorated the AD-related pathological phenotypes in tauopathy model. These findings suggest that CAPON could be a druggable AD target.

Hippocampal nuclear factor kappa B accounts for stress-induced anxiety behaviors via enhancing neuronal nitric oxide synthase (nNOS)-carboxy-terminal PDZ ligand of nNOS-Dexras1 coupling.

Anxiety disorders are associated with a high social burden worldwide. Recently, increasing evidence suggests that nuclear factor kappa B (NF-κB) has significant implications for psychiatric diseases, including anxiety and depressive disorders. However, the molecular mechanisms underlying the role of NF-κB in stress-induced anxiety behaviors are poorly understood. In this study, we show that chronic mild stress (CMS) and glucocorticoids dramatically increased the expression of NF-κB subunits p50 and p65, phosphorylation and acetylation of p65, and the level of nuclear p65 invivo and invitro, implicating activation of NF-κB signaling in chronic stress-induced pathological processes. Using the novelty-suppressed feeding (NSF) and elevated-plus maze (EPM) tests, we found that treatment with pyrrolidine dithiocarbamate (PDTC; intra-hippocampal infusion), an inhibitor of NF-κB, rescued the CMS- or glucocorticoid-induced anxiogenic behaviors in mice. Microinjection of PDTC into the hippocampus reversed CMS-induced up-regulation of neuronal nitric oxide synthase (nNOS), carboxy-terminal PDZ ligand of nNOS (CAPON), and dexamethasone-induced ras protein 1 (Dexras1) and dendritic spine loss of dentate gyrus (DG) granule cells. Moreover, over-expression of CAPON by infusing LV-CAPON-L-GFP into the hippocampus induced nNOS-Dexras1 interaction and anxiety-like behaviors, and inhibition of NF-κB by PDTC reduced the LV-CAPON-L-GFP-induced increases in nNOS-Dexras1 complex and anxiogenic-like effects in mice. These findings indicate that hippocampal NF-κB mediates anxiogenic behaviors, probably via regulating the association of nNOS-CAPON-Dexras1, and uncover a novel approach to the treatment of anxiety disorders.

Whole body periodic acceleration is an effective therapy to ameliorate muscular dystrophy in mdx mice.

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca2+ and Na+ overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca2+ and Na+ overload, diminished abnormal sarcolemmal Ca2+ entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway.

Effects of Propofol on Expression of Hippocampal Neuronal Nitric Oxide Synthase and Carboxy-Terminal PDZ Ligand of Neuronal Nitric Oxide Synthase in Stressed Rats Undergoing Electroconvulsive Shock

This study aimed at investigating the effects of propofol on the expression of hippocampal neuronal nitric oxide synthase (nNOS) and carboxy-terminal PDZ ligand of nNOS (CAPON) in stressed rats after electroconvulsive shock (ECS). Sprague-Dawley rats were stressed repeatedly for 28 days to establish a stressed model. Forty stressed rats were randomly divided into 4 groups (n = 10 for each group): the stressed group (unapplied group), propofol group (applied with intraperitoneal injection of propofol 80 mg/kg for 7 days), ECS group (applied with ECS and intraperitoneal injection of normal saline 8 mL/kg), or propofol + ECS group (applied with ECS and intraperitoneal injection of propofol 80 mg/kg). Sucrose preference test, open-field test, and Morris water maze were used to assess behavioral changes. Immunohistochemistry was carried out to measure the expression of nNOS and CAPON in hippocampal CA1, CA3, and DG areas. Rats in the ECS and propofol + ECS groups had higher sucrose preference percentages and open-field scores when compared with the stressed group. Rats in the ECS group exhibited longer escape latency, shorter space exploration time, up-regulated expression of nNOS, down-regulated expression of CAPON, and increased ratio of nNOS/CAPON in hippocampus. Compared with the ECS group, the propofol + ECS group exhibited shorter escape latency, longer space exploration time, down-regulated expression of nNOS, up-regulated expression of CAPON in hippocampus, and lower nNOS/CAPON values. Propofol may alleviate ECS-induced impairment of learning and memory in stressed rats by inhibiting excessive expression of nNOS and enhancing the expression of CAPON to maintain the balance between nNOS and CAPON in hippocampus.

Effects of propofol on nNOS activity and CAPON expression in hippocampal neurons after electroconvulsive therapy in mentally depressed rats

Objective To evaluate the effects of propofol on neuronal nitric oxide synthase (nNOS) activity and carboxy-terminal PDZ ligand of nNOS (CAPON) expression in hippocampal neurons after electroconvulsive therapy (ECT) in mentally depressed rats.Methods Healthy adult male Sprague-Dawley rats,aged 2-3 months,weighing 200-250 g,were used in this study.Mentally depression was induced by exposing the rats to chronic unpredictable mild stress (CUMS).Forty mentally depressed rats were randomly divided into 4 groups (n =10 each):mental depression group (group D),propofol group (group DP),ECT group (group DE),and propofol +ECT group (DPE group).Groups D and DE received intraperitoneal normal saline 8 ml/kg,and in addition group DE received ECT once a day for 7 consecutive days.Groups DP and DPE received intraperitoneal propofol 80 mg/kg,and in addition after the righting reflex disappeared,group DPE received ECT once a day for 7 consecutive days.All rats underwent sucrose preference test and Morris water maze test before CUMS,after CUMS,and after the end of treatment.The rats were sacrificed after completion of Morris water maze test and hippocampi were removed for determination of nNOS and CAPON expression in hippocampal neurons.Results Compared with group D,the sucrose preference percentage was significantly increased in DE and DPE groups,the escape latency was prolonged and space exploration time was shortened,the expression of nNOS in hippocampal DG,CA1 and CA3 regions was up-regulated and the expression of CAPON in hippocampal DG,CA1 and CA3 regions was down-regulated in group DE,and the expression of nNOS and CAPON in hippocampal DG,CA1 and CA3 regions was downregulated in group DP (P ＜ 0.05).Compared with group DE,the escape latency was significantly shortened and space exploration time was prolonged,the expression of nNOS in hippocampal DG,CA1 and CA3 regions was down-regulated and the expression of CAPON in hippocampal DG,CA1 and CA3 regions was up-regulated in group DPE (P ＜ 0.05).Conclusion The mechanism by which propofol reduces the cognitive impairment induced by ECT in mentally depressed rats is related to inhibition of nNOS activity and up-regulation of CAPON expression in hippocampal neurons. Key words: Propofol; Electroconvulsive therapy; Depression; Nitric oxide synthase type Ⅰ ; Hippocampus

Effects of ketamine on nNOS activity and CAPON expression in prefrontal lobe of mentally depressed rats

Objective To investigate the effects of ketamine on neuronal nitric oxide synthase (nNOS) activity and carboxy-terminal PDZ ligand of nNOS (CAPON) expression in the prefrontal lobe of mentally depressed rats.Methods Adult male Sprague-Dawley rats,aged 2.5-3.0 months,weighing 210-260 g,were used in the study.Menial depression was induced by exposing the rats to chronic unpredictable mild stress.Twenty-four animals in which mental depression was successfully induced were randomly divided into 2 groups (n =12 each):mental depression group (group D) and ketamine group (group K).Another 12 rats were chosen and served as control group (group C).Group K received intraperitoneal ketamine 10 mg/kg once a day for 7 consecutive days,while groups C and D received intraperitoneal normal saline 10 ml/kg instead of ketamine.Sucrose preference test and open field test were performed before administration and at 1 day after the end of administration.The total distance,number of rearing and sucrose preference percentage (SPP) were recorded.The rats were sacrificed 1 day after the last test for determination of the expression of nNOS and CAPON protein (using immuno-histochemistry)and mRNA (by RT-PCR) in the prefrontal lobe.Results Compared with group C,the total distance was shortened,the number of rearing and SPP were significantly decreased,the expression of nNOS protein and mRNA was up-regulated and the expression of CAPON protein and mRNA was down-regulated in groups D and K (P ＜ 0.05).Compared with group D,the total distance was prolonged,the number of rearing and SPP were significantly increased,the expression of nNOS and mRNA was down-regulated and the expression of CAPON protein and mRNA was up-regulated in group K (P ＜ 0.05).Conclusion Ketamine can improve the depressive state through promoting the expression of CAPON and inhibiting nNOS activity in the prefrontal lobe of mentally depressed rats. Key words: Ketamine; Depressive disorder; Nitric oxide synthase Type Ⅰ

Research progress on neurobiology of neuronal nitric oxide synthase

Neuronal nitric oxide synthase (nNOS) is mainly expressed in neurons, to some extent in astrocytes and neuronal stem cells. The alternative splicing of nNOS mRNA generates 5 isoforms of nNOS, including nNOS-α, nNOS-β, nNOS-µ, nNOS-γ and nNOS-2. Monomer of nNOS is inactive, and dimer is the active form. Dimerization requires tetrahydrobiopterin (BH4), heme and L-arginine binding. Regulation of nNOS expression relies largely on cAMP response element-binding protein (CREB) activity, and nNOS activity is regulated by heat shock protein 90 (HSP90)/HSP70, calmodulin (CaM), phosphorylation and dephosphorylation at Ser847 and Ser1412, and the protein inhibitor of nNOS (PIN). There are primarily 9 nNOS-interacting proteins, including post-synaptic density protein 95 (PSD95), clathrin assembly lymphoid leukemia (CALM), calcium/calmodulin-dependent protein kinase II alpha (CAMKIIA), Disks large homolog 4 (DLG4), DLG2, 6-phosphofructokinase, muscle type (PFK-M), carboxy-terminal PDZ ligand of nNOS (CAPON) protein, syntrophin and dynein light chain (LC). Among them, PSD95, CAPON and PFK-M are important nNOS adapter proteins in neurons. The interaction of PSD95 with nNOS controls synapse formation and is implicated in N-methyl-D-aspartic acid-induced neuronal death. nNOS-derived NO is implicated in synapse loss-mediated early cognitive/motor deficits in several neuropathological states, and negatively regulates neurogenesis under physiological and pathological conditions.

Increased expression of CAPON (Carboxy‐terminal PDZ ligand of nNOS) within the paraventricular nucleus (PVN) of rats with heart failure (HF).

Activation of NMDA receptor initiates a Ca2+ influx that stimulates the production of NO by neuronal nitric oxide synthase (nNOS). A physical coupling of the NMDA receptor and nNOS occurs by an intermediary adaptor protein PSD95 (post synaptic density 95). CAPON, a protein rich in PVN competes with PSD95 for interaction with nNOS. Previously, we showed a decreased expression of nNOS and enhanced expression of NMDA NR1 within the PVN of rats with HF. To further elucidate the underlying mechanism of upregulation of NMDA NR1 with a concomitant downregulation of nNOS, we measured the levels of CAPON in left coronary ligation‐induced HF model of rats. CAPON expression is augmented by 22% in rats with HF. Previously we showed that Angiotensin II receptor (AT1) blocker, losartan (LOS) treatment in rats with HF normalized the changes in expression of NR1 and nNOS protein in the PVN. Consistent with these data, LOS treatment (10 mg/kg/day in the drinking water for three weeks) in rats with HF normalized protein expression of the CAPON in the PVN. Studies in neuronal NG108‐15 hybrid cell line treated with Ang II (6.25–100 μM) for 24h revealed 30% increase in CAPON expression at highest dose as compared to untreated cell samples. Co‐treatment with LOS (1μM) for 24h significantly, ameliorates the increased CAPON expression to Ang II. Immunostaining revealed colocalization of CAPON and nNOS in NG108 cell line as well as in PVN. These data suggest that perhaps the disparate changes in NR1 (upregulation) and nNOS (downregulation) may be due to an over expression of CAPON and this over expression of CAPON may be mediated by increased Ang II influence in rats with HF.

Spatiotemporal Patterns of Dexamethasone-Induced Ras Protein 1 Expression in the Central Nervous System of Rats with Experimental Autoimmune Encephalomyelitis

Dexamethasone-induced Ras protein 1 (Dexras 1), a brain-enriched member of Ras subfamily of guanosine triphosphatases, as a novel physiologic nitric oxide (NO) effector, anchor neuronal nitric oxide synthase (nNOS) that could form a ternary complex with carboxy-terminal PDZ ligand of nNOS (CAPON) and nNOS, to specific targets to enhance NO signaling. The present study was to explore the expression pattern of Dexras 1 in the development of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Western blot and immunochemistry analysis showed that the gene and protein expression of Dexras 1 in the central nervous system (CNS) of rats increased significantly during the process of EAE compared with control groups (p < 0.01) and maintain a high level in the remission period. The protein expressions of nNOS and CAPON in hippocampus were approximately paralleled Dexras 1. Immunofluorescence revealed that both neurons and glial cells expressed the Dexras 1 in EAE CNS. Importantly, the damaged CNS in EAE-affected rats showed the codistribution between Dexras 1 and caspase 3, indicating the role of Dexras 1 played in the apoptotic process in EAE. Furthermore, colocalizations of Dexras 1 were observed in neurons and glial cells in CNS with nNOS or CAPON, supporting the ternary complex in this model. Thus, these findings suggest the postulation that Dexras 1 might participate into CNS neuronal cell death and demyelination in the whole process of EAE through regulating the NO signaling by binding to nNOS and CAPON.

Inhibition of nitric oxide-induced nuclear localization of CAPON by NMDA receptor antagonist in cultured rat primary astrocytes

Astrocytes play a key role in regulating aspects of inflammation in the central nervous system. It was observed that nNOS had located in the nucleus of cultured cerebral cortical astrocytes of 7 days. In the present study, we found that carboxy-terminal PDZ ligand of nNOS (CAPON) mainly located in the nucleus of astrocytes stimulated with NO donor sodium nitroprusside (SNP) or GSNO or N-methyl-d-aspartate (NMDA) receptor agonist-NMDA. However, originally, it was localized mostly in the cytoplasm of normal astrocytes. Immunocytochemistry showed that nNOS was co-localized with CAPON in the nucleus of astrocytes stimulated with SNP. In addition to the nuclear localization, treatment with SNP increased the mRNA and protein expression of CAPON. When SNP was removed from media, CAPON accumulated in nucleus transported back to cytoplasm. MK801, an inhibitor of NMDA receptor, was able to reverse the nuclear localization of CAPON resulted from SNP, suggesting that there is a functional relationship of NO with NMDA receptor in the regulation of the nuclear localization of CAPON. These findings provide a new insight in the understanding of the physical and pathological significances of CAPON/nNOS/NMDA receptor.

Expression of CAPON after Spinal Cord Injury in Rats

The adaptor protein, carboxy-terminal PDZ ligand of nNOS (CAPON), regulates the distribution of neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI) and produces the key signaling molecule nitric oxide (NO). But little is known about the role of CAPON in the pathological process of SCI. The main objective of the present study was to investigate expression of CAPON and nNOS in a spinal cord contusion model in adult rats. Real time-polymerase chain reaction (PCR) and Western blot analysis revealed that mRNA and protein for CAPON increased at 2 h after SCI and reached the peak at 8 h, gradually recovered to the baseline level at 14 days. The expression of nNOS mRNA and protein was similar to that of CAPON. During the peak expression, CAPON mRNA was found in the ventral horn, mediate zone, dorsal horn, and white matter by in situ hybridization. Immunofluorescence showed that CAPON was colocalized with nNOS in neurons, oligodendrocytes, and some astrocytes of spinal cord tissues within 5 mm from the epicenter. Interaction between CAPON and nNOS was also detected by co-immunoprecipitation. Thus, the transient expression of high levels of CAPON may provide new insight into the secondary response after SCI.

Involvement of CAPON and Nitric Oxide Synthases in Rat Muscle Regeneration After Peripheral Nerve Injury

Carboxy-terminal PDZ ligand of nNOS (CAPON) protein, as an adaptor, binds to nNOS via the PDZ domain helping regulate neuronal nitric oxide synthase (nNOS) activity at post-synaptic sites in neurons (Jaffrey et al., Neuron, 20, 115-124, 1998). Recently, it has been reported that CAPON is present in mouse muscle and may be involved in mouse muscle growth, injury, and repair possibly by regulating the stability, activity, or position of nNOS (Segalat et al., Experimental Cell Research, 302, 170-179, 2005). The present study was to explore the expression patterns and roles of CAPON as well as NOS in rat muscle regeneration after nerve injury. Normal Sprague-Dawley rats were subjected to right sciatic nerve crush injury. Walking track analysis, real time polymerase chain reaction, Western blotting, in situ hybridization, immunocytochemistry, and co-immunoprecipitation techniques were used. It revealed that CAPON mRNA increased, which peaked on days 1 and 28, whereas nNOS mRNA underwent a downregulation in the ipsilateral gastrocnemius muscles after sciatic nerve injury. Their proteins approximately paralleled the mRNA expression. CAPON and nNOS were identified in the activated satellite cells or myotubes and their in vivo interaction was verified. However, eNOS and iNOS proteins suffered an upregulation and were detected in activated satellite cells or myotubes. These data suggest that CAPON and all these three isoforms of NOS might be involved in muscle regeneration after nerve injury. Further study is necessary for a better understanding of the potential functional link between CAPON, NOS, and muscle regeneration, with possible application to therapy for skeletal muscle repair from nerve injury.

CAPON expression in skeletal muscle is regulated by position, repair, NOS activity, and dystrophy

In skeletal muscle, the localization of nNOS is destabilized in the absence of dystrophin, which impacts muscle function and satellite cell activation. In neurons, the adaptor protein, carboxy-terminal PDZ ligand of nNOS (CAPON), regulates the distribution of neuronal nitric oxide synthase (nNOS), which produces the key signaling molecule nitric oxide (NO). While a CAPON-like gene is known to compensate functionally for a dystrophic phenotype in muscle of Caenorhabditis elegans, CAPON expression has not been reported for mammalian muscle. Here, CAPON expression was identified in mouse muscle using Northern and Western blotting and in situ hybridization in combination with immunostaining for laminin. CAPON RNA was expressed in developing normal and dystrophic muscles near fiber junctions with tendons, and levels increased from 1 to 3 weeks. In regenerating normal muscle and also in dystrophic muscles in the mdx mouse, CAPON transcripts were prominent in satellite cells and new myotubes. Expression of CAPON RNA increased in diaphragm muscle of normal and mdx mice after treatment with l-arginine, the NOS substrate. Both CAPON and utrophin protein levels increased in dystrophic quadriceps muscle after treatment with the steroid deflazacort plus l-arginine, known to reduce the dystrophic phenotype. The identification of CAPON transcripts and protein in mammalian muscle and responses to l-arginine suggest CAPON may have a functional role in stabilizing neuronal NOS in skeletal muscle in the cytoskeletal complex associated with dystrophin/utrophin, with possible applications to therapy for human muscular dystrophy.

Changes in mRNA for post-synaptic density-95 (PSD-95) and carboxy-terminal PDZ ligand of neuronal nitric oxide synthase following facial nerve transection

When the axon of motoneurons is transected, the number of synaptic boutons contacting the cell body is decreased, and the recovery of synapses depends on muscle reinnervation. Post-synaptic density-95 (PSD-95) is a protein which is located at the post-synaptic density (PSD) and it plays a pivotal role in regulating synaptic plasticity and synaptogenesis. In addition, PSD-95 binds with neuronal nitric oxide synthase (nNOS), which is competitively inhibited by carboxy-terminal PDZ ligand of nNOS (CAPON) and, thereby, nNOS activity is thought to be regulated by PSD-95 and CAPON. We investigated the changes in mRNA for PSD-95, CAPON and nNOS in the facial motor nucleus of adult rats following axotomy, by in situ hybridization, in combination with the time course of muscle reinnervation, by retrograde tracing and nNOS protein expression, by examining nicotinamide adenine nucleotide phosphate diaphorase (NADPH-d) activity. Signals of mRNA for PSD-95 and CAPON were initially expressed in the facial motoneurons, transiently decreased following axotomy and gradually recovered to the control level. When reinnervation of the axotomized nerve into muscle was observed, mRNA expression of PSD-95 and CAPON started to recover in the facial motoneurons. It was also found that mRNA and protein expression of nNOS started to increase in the axotomized facial motoneurons just prior to the recovery of mRNA expression of PSD-95 and CAPON. These results suggest that PSD-95 and CAPON are involved in synaptogenesis and/or recovery of synaptic function in motoneurons after axotomy.