nNOS-CAPON blockers produce anxiolytic effects by promoting synaptogenesis in chronic stress-induced animal models of anxiety

Abstract

Background and purpose: Anxiety disorder is a common mental health disorder. However, there are few safe and fast-acting anxiolytic drugs available that can treat anxiety disorder. We previously demonstrated that the interaction of neuronal NOS (nNOS) with its carboxy-terminal PDZ ligand (CAPON) is involved in regulating anxiety-related behaviours. Here, we further investigated the anxiolytic effects of nNOS-CAPON disruptors in chronic stress-induced anxiety in animals.

Experimental approach: Mice were intravenously treated with nNOS-CAPON disruptors, ZLc-002 or Tat-CAPON12C, at the last week of chronic mild stress (CMS) exposure. We also infused corticosterone (CORT) into the hippocampus of mice to model anxiety behaviours and also delivered ZLc-002 or Tat-CAPON12C on the last week of chronic CORT treatment via pre-implanted cannula. Anxiety-related behaviours were examined using elevated plus maze, open field, novelty-suppressed feeding and light-dark (LD) tests. The level of nNOS-CAPON interaction was determined by co-immunoprecipitation (CO-IP) and proximity ligation assay (PLA). The neural mechanisms underlying the behavioural effects of nNOS-CAPON uncoupling in anxiety animal models were assessed by western blot, immunofluorescence and Golgi-Cox staining.

Key results: ZLc-002 and Tat-CAPON12C reversed CMS- or CORT-induced anxiety-related behaviours. ZLc-002 and Tat-CAPON12C increased synaptogenesis along with improved dendritic remodelling in CMS mice or CORT-treated cultured neurons. Meanwhile, blocking nNOS-CAPON interaction significantly activated the cAMP response element-binding protein (CREB)-brain-derived neurotrophic factor (BDNF) pathway, which is associated with synaptic plasticity.

Conclusion and implications: Collectively, these results provide evidence for the anxiolytic effects of nNOS-CAPON uncouplers and their underlying mechanisms in anxiety disorders.

FIGURE 1

ZLc‐002 attenuates chronic mild stress (CMS) ‐induced anxiogenic behaviour. (a) Diagram showing the design of the experiments in (b)–(g). The adult male ICR mice were treated with ZLc‐002 (40 mg·kg⁻¹·day⁻¹) or its vehicle by intravenous administration for 7 consecutive days on 21 days after CMS exposure. (b) The time spent in open arms, and (c) number of entries in the arms in the elevated plus maze (EPM) test. (d) The latency to feed in a novel environment and in the home cage, and (e) food consumption in the home cage in the novelty‐suppressed feeding (NSF) test. (f,g) The locomotor activity of mice in the open field (OF) test. Parameters for the locomotor activities were the number of square crossings (horizontal) and the time standing (vertical). Mean ± SEM, n = 15 mice in each group, ^* P < 0.05, compared with vehicle‐treated group; ^# P < 0.05, compared with vehicle‐treated non‐CMS group; ^$ P < 0.05, compared with vehicle‐treated CMS group, two‐way ANOVA followed by Tukey's post‐test (b–g). NE, novel environment; total arms, open arms + enclosed arms

FIGURE 2

Tat‐CAPON12C reverses chronic mild stress (CMS)‐induced anxiogenic behaviour. (a) Diagram showing the design of the experiments in (b)–(g). The adult male ICR mice were treated with Tat‐CAPON12C (3 μmol·kg⁻¹·day⁻¹) or its control peptide Tat‐CAPON12C/A22D by intravenous administration for 7 consecutive days on 21 days after CMS exposure. (b) The time spent in open arms, and (c) number of entries in the arms in the elevated plus maze (EPM) test. (d) The latency to feed in a novel environment and in the home cage, and (e) food consumption in the home cage in the novelty‐suppressed feeding (NSF) test. (f–g) The locomotor activity of mice in the open field test. Parameters for the locomotor activities were the number of square crossings (horizontal) and the time standing (vertical). Mean ± SEM, Tat‐CAPON12C/A22D or Tat‐CAPON12C groups, n = 15 mice; CMS + Tat‐CAPON12C/A22D or CMS + Tat‐CAPON12C groups, n = 13 mice, *P < 0.05, compared with Tat‐CAPON12C/A22D‐treated group; ^# P < 0.05, compared with Tat‐CAPON12C/A22D‐treated non‐CMS group; ^$ P < 0.05, compared with Tat‐CAPON12C/A22D‐treated CMS group, two‐way ANOVA followed by Bonferroni's post hoc test (b–f). NE, novel environment; total arms, open arms + enclosed arms

FIGURE 3

ZLc‐002 reverses the behavioural effects of glucocorticoids. (a–g) The adult male ICR mice were treated with 10‐μM corticosterone (CORT) alone or in combination with 10‐μM ZLc‐002 by injection cannula. CORT or its vehicle was microinjected into the hippocampus for 28 consecutive days on day 4 after cannula implantation, ZLc‐002 was intrahippocampal infusion on day 21 after CORT treatment for 7 consecutive days and the volume of each drug or its vehicle was 1.0 μl. (a) The time spent in open arms, and (b) number of entries in the arms in the elevated plus maze (EPM) test in adult mice. (c) Time spent in lit compartment of the light–dark box in adult mice. (d) The latency to feed in a novel environment and in the home cage, and (e) food consumption in the home cage in the novelty‐suppressed feeding (NSF) test in adult mice. (f,g) The locomotor activity of mice in the open field test. Parameters for the locomotor activities were the number of square crossings (horizontal) and the time standing (vertical). Mean ± SEM, n = 11 mice in each group, ^* P < 0.05, compared with vehicle‐treated group; ^# P < 0.05, compared with vehicle‐treated and chronic CORT‐infused group, one‐way ANOVA followed by Tukey's post‐test (a–f). NE, novel environment; total arms, open arms + enclosed arms

FIGURE 4

Tat‐CAPON12C reverses the behavioural effects of glucocorticoids. (a–g) The adult male ICR mice were treated with 10‐μM corticosterone (CORT) alone or in combination with 50‐nM Tat‐CAPON12C or its control peptide Tat‐CAPON12C/A22D by injection cannula. Corticosterone (CORT) or its vehicle was microinjected into the hippocampus for 28 consecutive days on day 4 after cannula implantation, Tat‐CAPON12C or Tat‐CAPON12C/A22D was intrahippocampal infusion on day 21 after CORT treatment for 7 consecutive days and the volume of each drug or its vehicle or control peptide was 1.0 μl. (a) The time spent in open arms, and (b) number of entries in the arms in the elevated plus maze (EPM) test in adult mice. (c) Time spent in lit compartment of the light–dark box in adult mice. (d) The latency to feed in a novel environment and in the home cage, and (e) food consumption in the home cage in the novelty‐suppressed feeding (NSF) test in adult mice. (f,g) The locomotor activity of mice in the open field test. Parameters for the locomotor activities were the number of square crossings (horizontal) and the time standing (vertical). Mean ± SEM, n = 11 mice in each group, ^* P < 0.05, compared with Tat‐CAPON12C/A22D‐treated group; ^# P < 0.05, compared with Tat‐CAPON12C/A22D‐treated and chronic CORT‐infused group, one‐way ANOVA followed by Tukey's post‐test (a–f). NE, novel environment; total arms, open arms + enclosed arms

FIGURE 5

nNOS–CAPON blockers rescue the chronic stress‐induced synaptogenesis impairment. (a–d) The adult male ICR mice were treated with ZLc‐002 (40 mg·kg⁻¹·day⁻¹) or Tat‐CAPON12C (3 μmol·kg⁻¹·day⁻¹) or vehicle or control peptide by intravenous administration for 7 consecutive days on 21 days after CMS exposure. (a,b) Dissociating nNOS–CAPON by ZLc‐002 rescues the CMS‐induced spine loss of dentate gyrus (DG) granule cells. Representative images with Golgi‐Cox staining (a) and bar graph (b) showing dendrite spine density of granular cells in the hippocampal DG of mice administered with ZLc‐002 and exposed to CMS (n = 5 mice in each group, 10 neurons per sample). Scale bar, 20 μm. ^* P < 0.05, compared with vehicle‐treated group; ^# P < 0.05, compared with vehicle‐treated non‐CMS group; ^$ P < 0.05, compared with vehicle‐treated CMS group. (c,d) Dissociating nNOS–CAPON by Tat‐CAPON12C rescues the CMS‐induced spine loss of DG granule cells. Representative images with Golgi‐Cox staining (c) and bar graph (d) showing dendrite spine density of granular cells in the hippocampal DG of mice administered with Tat‐CAPON12C and exposed to CMS (n = 5 mice in each group, 10 neurons per sample). Scale bar, 20 μm. ^* P < 0.05, compared with 12C/A22D‐treated group; ^# P < 0.05, compared with 12C/A22D‐treated non‐CMS group; ^$ P < 0.05, compared with 12C/A22D‐treated CMS group. (e–h) Dendrites growth of neurons treated with 10‐μM CORT alone or in combination with 10‐μM ZLc‐002 or vehicle for 72 h. (e) Representative images of GFP⁺ neurons exposure to corticosterone (CORT) with ZLc‐002 or vehicle. Scale bar, 50 μm. (f) Bar graph showing total dendritic length of neurons treated with CORT alone or in combination with ZLc‐002 or vehicle (n = 5 in each group, from five independent experiments, 10 neurons per sample). (g) Bar graph showing total branch number of neurons treated with ZLc‐002 or vehicle after CORT exposure (n = 5 in each group, from five independent experiments, 10 neurons per sample). (h) Sholl analysis of dendritic complexity of neurons treated with ZLc‐002 or vehicle after CORT exposure (n = 5 in each group, from five independent experiments, 10 neurons per sample). (i) Representative images of neurons stained for synapsin/PSD‐95 with or without 10‐μM ZLc‐002 treatment after 10‐μM CORT exposure (upper) and representative images of synapsin‐positive puncta, PSD‐95‐positive puncta and synapsin/PSD‐95 double‐positive puncta along dendrites of neurons selected from boxed areas (lower). These images were selected from boxed areas. Arrows indicate synapsin/PSD‐95 double‐positive puncta. Scale bar, 10 or 20 μm. (j) Bar graph showing densities of synapsin/PSD‐95 double‐positive puncta along dendrites of neurons treated by CORT in combination with ZLc‐002 or vehicle for 72 h (n = 5 in each group, from five independent experiments, 12 neurons per sample). For (f)–(h) and (j), ^* P < 0.05: ZLc‐002 versus vehicle; ^# P < 0.05: CORT + vehicle versus vehicle; ^$ P < 0.05: CORT + ZLc‐002 versus CORT + vehicle. Mean ± SEM, two‐way ANOVA followed by Tukey's post‐test. 12C/A22D, Tat‐CAPON12C/A22D; 12C, Tat‐CAPON12C

FIGURE 6

CREB–BDNF signalling is involved in the effects of ZLc‐002 on synaptogenesis impairment induced by chronic stress. (a,b) The adult male ICR mice were treated with ZLc‐002 (40 mg·kg⁻¹·day⁻¹) or its vehicle by intravenous administration for 7 consecutive days on 21 days after CMS exposure. (a) ZLc‐002 treatment reduced the amounts of nNOS–CAPON complex (presented as the ratio of CAPON to nNOS) in the hippocampus of mice exposed to CMS for 28 days (n = 5 mice in each group). (b) Representative immunoblots (left) and bar graph (right) showing PSD‐95, synapsin, BDNF, p‐CREB and CREB in the hippocampus of mice treated with ZLc‐002 and exposed to CMS (n = 5 mice in each group). (c,d) The cultured neurons treated with 10‐μM corticosterone (CORT) alone or in combination with 10‐μM ZLc‐002 or their vehicle for 72 h. (c) Representative confocal images of in situ proximity ligation assay (PLA) between nNOS and CAPON (red) in primary neurons. Maximum intensity projections of a confocal z‐stack including a whole cell were performed to observe the maximum amount of PLA puncta. The neurons were counter‐stained with β3‐tubulin (green) and DAPI (blue). Scale bar: 20 μm. (d) Representative immunoblots (left) and bar graph (right) showing PSD‐95, synapsin, BDNF and p‐CREB in the cultured hippocampal neurons treated with ZLc‐002 and exposed to CORT (n = 5 in each group, from five independent experiments). For (a) and (b), ^* P < 0.05, compared with vehicle‐treated group; ^# P < 0.05, compared with vehicle‐treated non‐CMS group; ^$ P < 0.05, compared with vehicle‐treated CMS group; for (d), ^* P < 0.05, compared with vehicle‐treated group; ^# P < 0.05, compared with vehicle‐treated group; ^$ P < 0.05, compared with vehicle‐treated CORT‐incubated group. Mean ± SEM, two‐way ANOVA followed by Tukey's post‐test

FIGURE 7

CREB–BDNF signalling is involved in the effects of Tat‐CAPON12C on synaptogenesis impairment induced by chronic stress. (a,b) The adult male ICR mice were treated with Tat‐CAPON12C (3 μmol·kg⁻¹·day⁻¹) or its control peptide Tat‐CAPON12C/A22D by intravenous administration for 7 consecutive days on 21 days after CMS exposure. (a) Tat‐CAPON12C treatment reduced the amounts of nNOS–CAPON complex (presented as the ratio of CAPON to nNOS) in the hippocampus of mice exposed to CMS for 28 days (n = 5 mice in each group). (b) Representative immunoblots (left) and bar graph (right) showing PSD‐95, synapsin, BDNF and p‐CREB in the hippocampus of mice treated with Tat‐CAPON12C and exposed to CMS (n = 5 mice in each group). (c) The cultured neurons treated with 10‐μM corticosterone (CORT) alone or in combination with 50‐nM Tat‐CAPON12C or its control peptide for 72 h. Representative immunoblots (left) and bar graph (right) showing PSD‐95, synapsin, BDNF and p‐CREB in the cultured hippocampal neurons treated with Tat‐CAPON12C and exposed to CORT (n = 5 in each group, from five independent experiments). For (a) and (b), ^* P < 0.05, compared with 12C/A22D‐treated group; ^# P < 0.05, compared with 12C/A22D‐treated non‐CMS group; ^$ P < 0.05, compared with 12C/A22D‐treated CMS group; for (c), ^* P < 0.05, compared with 12C/A22D‐treated group; ^# P < 0.05, compared with 12C/A22D‐treated group; ^$ P < 0.05, compared with 12C/A22D‐treated CORT‐incubated group. Mean ± SEM, two‐way ANOVA followed by Tukey's post‐test. 12C/A22D, Tat‐CAPON12C/A22D; 12C, Tat‐CAPON12C