Reversing anxiety by targeting a stress-responsive signaling pathway

Significance

Persistent stress plays a significant role in the development of various neurodevelopmental and neuropsychiatric disorders. Our study unravels a signaling pathway at inhibitory synapses in the brain, which is highly sensitive to chronic stress. Crucially, we have demonstrated the possibility of reversing high anxiety phenotypes in chronically stressed mice by pharmacologically manipulating this signaling pathway, which holds promise for developing therapeutic strategies against anxiety and depressive disorders.

Abstract

Current treatments of anxiety and depressive disorders are plagued by considerable side effects and limited efficacies, underscoring the need for additional molecular targets that can be leveraged to improve medications. Here, we have identified a molecular cascade triggered by chronic stress that exacerbates anxiety- and depressive-like behaviors. Specifically, chronic stress enhances Src kinase activity and tyrosine phosphorylation of calmodulin, which diminishes MyosinVa (MyoVa) interaction with Neuroligin2 (NL2), resulting in decreased inhibitory transmission and heightened anxiety-like behaviors. Importantly, pharmacological inhibition of Src reinstates inhibitory synaptic deficits and effectively reverses heightened anxiety-like behaviors in chronically stressed mice, a process requiring the MyoVa–NL2 interaction. These data demonstrate the reversibility of anxiety- and depressive-like phenotypes at both molecular and behavioral levels and uncover a therapeutic target for anxiety and depressive disorders.

Chronic stress is a leading factor contributing to various neurodevelopmental and neuropsychiatric disorders, including posttraumatic stress disorder, anxiety, and depression (1, 2). Anxiety and depressive disorders stand out as the most prevalent mental health disorders on a global scale, impacting approximately 33% of the world’s population (3–5). The emergence of the recent COVID-19 pandemic has further exacerbated this already significant public health concern (6, 7). Existing medications for anxiety and depressive disorders targeting GABA_A receptors (GABA_ARs) and serotonin transporters could effectively alleviate anxiety and are often well tolerated, with the flexibility for individualized treatment to improve overall quality of life (8, 9). However, they are also known for significant side effects, addiction risk, delayed therapeutic effects, and limited effectiveness (8, 10, 11). Notably, these drugs primarily modulate crucial receptors and transporters that are critically involved in various biological processes, potentially affecting signaling pathways unrelated to anxiety and depression management and leading to diverse side effects. To optimize drug efficacy while minimizing adverse effects, identifying specific signaling pathways directly involved in anxiety and depressive behaviors may provide an alternative approach to design therapeutic options for anxiety and depression (10, 12–14).

Accumulating evidence has demonstrated that chronic stress can induce marked alterations at chemical synapses by dysregulating a variety of signaling pathways, and it has been recognized that synaptic deficits predominantly underlie the development of cognitive and emotional deficits induced by chronic stress (12, 15, 16). However, the core signaling pathways at synapses translating chronic stress into anxiety and depressive behaviors remain enigmatic. More importantly, it remains to be investigated whether synaptic and behavioral defects induced by chronic stress can be rescued by manipulating these molecular pathways. Answers to these questions will help pave the way to understanding etiologies of stress-related brain disorders.

Results

Identification and Characterization of the Chronic Stress-Sensitive Signaling Pathway.

We have employed a chronic restraint stress (RS) model for 14 consecutive days in ~8-wk-old mice (17) (Fig. 1A) and found that these RS mice showed anxiety- and depressive-like behaviors as observed in the elevated plus maze (EPM) (Fig. 1B), marble bury test (MBT) (Fig. 1C), and forced swim test (FST) (Fig. 1D). To study the effect of chronic stress on the synapses in the brain, we performed biochemical experiments and found that RS did not significantly change the total expression of a number of excitatory and inhibitory synaptic proteins (Fig. 1E). However, expression of several inhibitory synaptic proteins, including Neuroligin2 (NL2), gephyrin, vesicular GABA transporter (vGAT), GABA_AR α1 (Gabra1) and GABA_AR α2 (Gabra2) subunits, were markedly decreased in RS hippocampal synaptosomal fractions (Fig. 1F and SI Appendix, Fig. S1E). Consistently, the frequency, but not the amplitude, of miniature inhibitory postsynaptic currents (mIPSC) was significantly reduced in CA1 neurons of acute hippocampal slices prepared from RS mice (Fig. 1G), indicating that chronic stress reduced inhibitory transmission through down-regulating GABAergic synaptic proteins. We also employed another chronic stress model, prolonged maternal separation stress (MS), to examine whether different chronic stress models would induce similar effects on synapses (SI Appendix, Fig. S1A). Similar to the RS model, we found that in MS mice, NL2, gephyrin, vGAT, and Gabra2 subunit synaptic expression and inhibitory transmission were strongly reduced (SI Appendix, Fig. S1 B–D). Together, these data show that two different models of chronic stress similarly reduce GABAergic synaptic protein expression and inhibitory transmission.

Fig. 1.

Chronic stress (RS) increases anxiety and strongly suppresses GABAergic transmission. (A) Schematic representation of the RS protocol in mice. (B) RS mice spent significantly less time and made fewer entries in the open arm of the EPM than the Control (n = 12 for both Control and RS; Student's t test). (C) RS mice buried a significantly high number of marbles with strongly reduced latency to bury the first marble in the MBT compared to the Control (n = 12 for both Control and RS; Student's t test). (D) RS mice showed significantly higher immobility and lower mobility in the last 4 min of the FST than Control (n = 12 for both Control and RS; Student's t test). (E) RS did not change the total expression of indicated excitatory and inhibitory synaptic proteins in the hippocampal lysates of the RS mice compared to the Control (n = 3; two-way ANOVA test). (F) Expression of indicated inhibitory but not the excitatory synaptic proteins was significantly reduced in the synaptosomal fractions of the RS mice as compared to the Control (n = 3; two-way ANOVA test). (G) mIPSC frequency, but not amplitude, was significantly reduced in CA1 pyramidal neurons in acute hippocampal slices prepared from RS mice as compared to the Control (n = 14 for both Control and RS; Student's t test). For cumulative distributions, the Kolmogorov–Smirnov test was used. (Scale bar, 20 pA and 1 s.) Error bars indicate SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

We next investigated the molecular mechanisms underlying the reduction of NL2 synaptic expression in chronically stressed animals, as NL2 is a critical GABAergic synaptic cell adhesion molecule and strongly implicated in anxiety (18–25). To this end, we first performed a proteomic screen by co-immunoprecipitation (co-IP) of NL2 from wild-type (WT) mouse hippocampal lysates followed by mass spectrometry analysis and identified a number of proteins (26), including Src kinase, calmodulin (CaM), and MyosinVa (MyoVa), associated with NL2 (Fig. 2 A and B and SI Appendix, Fig. S2A). We focused on these proteins because of their relatively high abundance in the NL2 proteome and their potential roles in stress and anxiety (27–29). We next examined how RS and MS modulated NL2 interaction with Src, CaM, or MyoVa, by performing the co-IP assay in RS and MS mouse hippocampal lysates (Fig. 2C and SI Appendix, Fig. S2B), and found that Src association with NL2 was significantly increased; in contrast, CaM and MyoVa associations with NL2 were strongly decreased (Fig. 2D and SI Appendix, Fig. S2C), showing that chronic stress alters the NL2 proteome. Additionally, active Src expression and tyrosine-phosphorylation levels of CaM, a substrate of Src (30, 31), were significantly elevated in hippocampal lysates of RS and MS mice (Fig. 2E and SI Appendix, Fig. S2D), suggesting a common signaling pathway involving Src, CaM, MyoVa, and NL2 in response to two different chronic stressors.

Fig. 2.

Chronic stress disrupts the NL2 proteome in the mouse hippocampus. (A) Representative proteins that were associated with NL2 in mass spectroscopy analysis. NL2 was not detected in IgG Control samples, and the ratio of 100 is the default number for maximum fold change allowed in Proteome Discoverer software. (B) Indicated proteins were coimmunoprecipitated with NL2 from the hippocampal lysates of 7- to 9-wk-old WT mice. IP, coimmunoprecipitation; IB, immunoblot; IgG, immunoglobulin G. (n = 3). (C) Schematic representation of the RS protocol in mice. (D) In the hippocampal lysates of the RS mice, coimmunoprecipitation with NL2 revealed a notable increase in the association between NL2 and Src, along with a marked reduction in the interactions between NL2 and MyoVa or CaM, in comparison to the Control. (n = 3; two-way ANOVA test). (E) Active Src and tyrosine phosphorylated CaM (pCaM) but not the total Src or total CaM expressions were significantly increased in the hippocampal lysates of RS mice as compared to the Control (n = 3; two-way ANOVA test). (F) Surface but not total NL2 expression was significantly decreased in hippocampal neurons expressing CA Src (constitutively active Src); however, WT Src or KD Src (kinase-dead Src) expression has no significant effect on the surface and total NL2 expression compared to the Control [Control, n = 14; GFP, n = 15; WT Src, n = 14; CA Src, n = 16; KD Src, n = 18; one-way ANOVA test (Scale bar, 5 μm.)] (G) mIPSC frequency but not amplitude was significantly reduced in hippocampal neurons expressing CA Src but not in cells expressing either WT Src or KD Src constructs as compared to the Control (Control, n = 16; GFP, n = 14; WT Src, n = 16; CA Src, n = 15; KD Src, n = 16; one-way ANOVA test). For cumulative distributions, the Kolmogorov–Smirnov test was used. (Scale bar, 20 pA and 1 s.) Error bars indicate SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

Our observations that NL2 association with Src, CaM, and MyoVa and their functional states were altered by chronic stress prompted us to characterize their roles in regulating NL2 synaptic expression and inhibitory synaptic transmission. We first investigated the role of enhanced Src activity in NL2 trafficking and inhibitory transmission in cultured hippocampal neurons and found that overexpression of constitutively active (CA) Src reduced NL2 surface expression and mIPSC frequency but not amplitude, while WT or kinase-dead Src had no significant effect (Fig. 2 F and G). We also interrogated the effect of CaM tyrosine phosphorylation (pCaM) and found that expression of phospho-mimetic mutant CaM YY/EE, but not the phospho-dead CaM YY/FF or WT CaM, decreased the interaction between MyoVa and NL2 in HEK293T cells (SI Appendix, Fig. S3 A and B). In addition, surface NL2 expression (Fig. 3A), inhibitory synaptic density (SI Appendix, Fig. S3C), and mIPSC frequency (Fig. 3B) were significantly decreased in cultured hippocampal neurons expressing CaM YY/EE, but not WT CaM or CaM YY/FF mutant. Furthermore, CRISPR-Cas9 mediated genetic inactivation of CaM genes significantly reduced mIPSC frequency but not amplitude in hippocampal neurons (Fig. 3C and SI Appendix, Fig. S3D), suggesting a critical role of CaM in inhibitory transmission. Notably, mIPSC frequency was rescued by coexpressing single-guide RNAs (sgRNA)-resistant WT CaM* or CaM YY/FF*, but not by CaM YY/EE* (Fig. 3C), indicating a negative role of pCaM in the regulation of GABAergic transmission. Finally, expression of dominant-negative MyoVa reduced the surface, but not total, NL2 expression (Fig. 3D), inhibitory synaptic density (SI Appendix, Fig. S3E), and mIPSC frequency in hippocampal neurons (Fig. 3E), indicating a crucial role of MyoVa in NL2 trafficking and inhibitory transmission. Taken together, these findings demonstrate that chronic stress enhances Src kinase activity, leading to increased pCaM expression. This, in turn, reduces the interaction between MyoVa and NL2, leading to a decrease in NL2 surface expression and inhibitory synaptic transmission.

Fig. 3.

Characterization of the chronic stress-sensitive signaling pathway. (A) Surface but not total NL2 expression was significantly reduced in hippocampal neurons expressing CaM YY/EE, while neurons expressing CaM WT or CaM YY/FF showed no significant difference in surface and total NL2 expression compared to the Control. [Control, n = 29; CaM WT, n = 20; CaM YY/EE, n = 30; and CaM YY/FF, n = 25; one-way ANOVA test (Scale bar, 5 μm.)] (B) mIPSC frequency but not amplitude was significantly reduced in neurons expressing CaM YY/EE, while CaM WT or CaM YY/FF overexpression showed no significant alterations in mIPSC frequency or amplitude compared to the Control. (Control, n = 28; CaM WT, n = 22; CaM YY/EE, n = 33; CaM YY/FF, n = 26; one-way ANOVA test, and the Kolmogorov–Smirnov test was used for cumulative distributions). (Scale bar, 20 pA and 1 s.) (C) Schematic of the CaM gRNA construct (CaM triple gRNA) used to knock out endogenous CaM 1, 2, and 3 from mouse hippocampal neurons. mIPSC frequency but not amplitude was significantly reduced in hippocampal neurons expressing CaM triple gRNA. However, coexpression of CaM WT* or CaM YY/FF* with the CaM triple gRNA led to a recovery in mIPSC frequency, whereas coexpression with CaM YY/EE* did not result in a recovery of mIPSC frequency. (Control, n = 15; CaM Triple gRNA, n = 15; CaM Triple gRNA + CaM WT*, n = 16; CaM Triple gRNA + CaM YY/EE*, n = 15; CaM Triple gRNA + CaM YY/FF*, n = 15; one-way ANOVA test, and the Kolmogorov–Smirnov test was applied for cumulative distributions). *Indicates gRNA-resistant CaM construct. (Scale bar, 20 pA, and 1 s.) (D) Surface but not total NL2 expression was significantly reduced in neurons expressing the DN MyoVa construct, as compared to the Control [Control, n = 27; DN MyoVa, n = 28; Student's t test (Scale bar, 5 μm.)] DN, dominant negative. (E) mIPSC frequency but not amplitude was significantly reduced in hippocampal neurons expressing DN MyoVa compared to the Control (Control, n = 14; DN MyoVa, n = 15; Student's t test; for cumulative distributions, the Kolmogorov–Smirnov test was used). (Scale bar, 20 pA, and 1 s.) Error bars indicate SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

Pharmacological Manipulation of the Stress-Sensitive Pathway Reverses High Anxiety Phenotypes in RS Mice.

We reasoned that if impairment of the NL2 trafficking pathway and, eventually, reduced inhibitory synaptic transmission were critical for the manifestation of anxiety-like phenotypes in RS mice, we would be able to, at least partially, ameliorate behavioral and synaptic deficits by restoring the altered NL2 trafficking pathway. To this end, we employed a pharmacological approach to suppress Src kinase activity in chronically stressed mice (RS). Specifically, we administered intraperitoneally a potent Src kinase inhibitor, PP2 (2.5 mg/kg) (32), and its inactive analog, PP3 (2.5 mg/kg) for 7 consecutive days, after 14-d RS (Fig. 4A). We then performed behavioral analysis to measure anxiety levels. While RS animals treated with either vehicle (0.9% NaCl + 1% Dimethyl sulfoxide (DMSO)) or PP3 showed elevated anxiety-like behaviors, the high anxiety-like behavior was significantly reduced in the mice treated with PP2 in the EPM, MBT, and FST tests (Fig. 4 B–D, SI Appendix, Fig. S4 A–C, and Movies S1 and S2), suggesting that PP2, through inhibiting Src kinase activity, acts as an anxiolytic reagent in RS mice and can ameliorate high anxiety-like behavioral phenotypes in these animals.

Fig. 4.

Pharmacological manipulation of the stress-sensitive pathway reverses elevated anxiety phenotypes in RS mice. (A) 1 and 2: Schematic experimental procedures of the RS and vehicle (Veh.), PP2, or PP3 administration in WT mice to perform experiments. (B) RS mice spent significantly less time and made very fewer entries in the open arm of the EPM, as compared to the Control; however, after PP2 injections, RS mice showed a comparable number of entries and time spent in the open arms as relative to the Control (Control + Veh., n = 18; RS + Veh., n = 20; Control + PP2, n = 18; RS + PP2, n = 18; two-way ANOVA test). (C) RS mice buried significantly more marbles and had strongly reduced latency to bury the first marble in the MBT, and importantly, upon PP2 administration, the marble burying and latencies were ameliorated in PP2 treated RS mice as compared to the Control (Control + Veh., n = 18; RS + Veh., n = 20; Control + PP2, n = 18; RS + PP2, n = 18; two-way ANOVA test). (D) RS mice showed significantly higher immobility and lower mobility times in the last 4 min of the FST as compared to the Control, and the phenotype was rescued upon PP2 administrations in RS mice (Control + Veh., n = 18; RS + Veh., n = 20; Control + PP2, n = 18; RS + PP2, n = 18; two-way ANOVA test). (E) Expression of active Src and tyrosine pCaM was significantly increased in hippocampal lysates of RS mice as compared to the Control, and upon PP2 treatment, the increased expression was significantly reduced in RS mice (n = 3; two-way ANOVA test). (F) Expression of inhibitory synaptic proteins (NL2, gephyrin, vGAT, and α2) but not PSD95 was strongly reduced in hippocampal synaptosomal fractions of RS mice as compared to the Control, and importantly, PP2 administration rescued expression of inhibitory synaptic proteins in RS mice. (n = 3; two-way ANOVA test). (G) mIPSCs frequency but not amplitude was strongly reduced in CA1 neurons in acute hippocampal slices prepared from RS mice as compared to the Control, and PP2 administration rescued the deficits in mIPSC frequency in RS mice [Control + Veh., n = 22; RS + Veh., n = 30; Control + PP2, n = 21; RS + PP2, n = 28; two-way ANOVA test, and the Kolmogorov–Smirnov test was used for cumulative distributions (Scale bar; 20 pA, and 1 s.)] Error bars indicate SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.

We wondered whether the PP2-mediated amelioration of anxiety-like behaviors observed in RS mice was due to inhibition of the elevated Src activity by PP2 administration and, subsequently, reduced pCaM and increased NL2 synaptic expression. To this end, we performed biochemical and electrophysiological experiments to measure the active Src and pCaM expression levels in the total hippocampal lysates, NL2 expression in hippocampal synaptosomal fractions, and inhibitory synaptic transmission in acute hippocampal slices prepared from RS mice treated with vehicle, PP2, or PP3. We found that treatment of PP2, but not vehicle or PP3, reduced the expression of active Src and pCaM in hippocampal lysates prepared from RS mice (Fig. 4E and SI Appendix, Fig. S4D). Importantly, in RS mouse hippocampal synaptosomal fractions, PP2, but not vehicle or PP3, treatment increased synaptic expressions of NL2, Gabra2 subunit, and GABAergic synaptic markers (i.e., gephyrin and vGAT) (Fig. 4F and SI Appendix, Fig. S4E). Finally, GABAergic synaptic transmission was markedly reduced in hippocampal CA1 neurons in RS mice. Notably, treatment with PP2, but not the vehicle, rescued the mIPSC frequency deficits in RS mice (Fig. 4G). Together, these data show that inhibition of elevated Src kinase activity in RS mice can reverse high anxiety-like phenotypes by restoring synaptic expression of NL2 and GABAergic synaptic transmission.

Validation of the Specificity of the Stress-Sensitive Pathway in Controlling Anxiety.

The reversal of elevated anxiety-like behaviors in RS mice by PP2 treatment suggests a powerful mechanism for potential therapeutic applications. Mechanistically, although reduced synaptic expression of NL2 and inhibitory synaptic transmission in RS mice were rescued by PP2 treatment, it remained unknown whether PP2 was acting on the Src-CaM-MyoVa-dependent transport pathway for synaptic expression of NL2. To test this hypothesis, we first performed an in-depth in vitro analysis of the MyoVa–NL2 interaction and identified that the five amino acids, P₇₇₈DDVP₇₈₂ (PDDVP) in the NL2 C tail, were required for binding to MyoVa, as the NL2 mutant lacking PDDVP was not co-IPed with MyoVa (SI Appendix, Fig. S5 A–F). Additionally, the surface expression of NL2 mutant lacking PDDVP was strongly reduced in the cultured hippocampal neurons (SI Appendix, Fig. S5G), showing a critical role of this MyoVa-binding domain (MBD) in transporting NL2 to the neuronal surface.

Next, to understand further the role of the MBD in NL2 surface expression and inhibitory transmission in vivo, we generated a mutant mouse line in which the five aa, PDDVP (MBD), were genetically deleted (NL2ΔPDDVP) using the CRISPR-Cas9-mediated homologous recombination (SI Appendix, Fig. S6 A–C). Co-IP assays in detergent-solubilized mouse hippocampal lysates showed that MyoVa was co-IPed by NL2 in WT, but not in NL2ΔPDDVP mice (Fig. 5A), indicating that these five aa in NL2 are essential for interaction with MyoVa in vivo. Fractionation experiments showed that genetic disruption of NL2 interaction with MyoVa significantly reduced synaptic, but not total, expression of NL2 as well as gephyrin, vGAT, and GABAAR α2 subunit (Fig. 5B and SI Appendix, Fig. S6E). Similarly, immunocytochemical and immunohistological experiments demonstrated that NL2 and GABAergic synaptic density were significantly reduced in the NL2ΔPDDVP hippocampal slices and cultured neurons (Fig. 5 C and D and SI Appendix, Fig. S6F). Finally, both mIPSC frequency and amplitude were significantly reduced in CA1 neurons in acute hippocampal slices prepared from NL2ΔPDDVP mice (Fig. 5E). NL2ΔPDDVP mice were born and survived at the expected Mendelian ratio and did not show obvious abnormalities (SI Appendix, Fig. S6D). The behavioral analysis demonstrated that NL2ΔPDDVP mice showed elevated anxiety-like phenotypes in the EPM, MBT, and FST tests (Fig. 5F and SI Appendix, Fig. S6 G and H). Next, we wondered whether RS and genetic disruption of the NL2-MyoVa interaction increased anxiety through common mechanisms. To this end, we induced RS in NL2ΔPDDVP mice and employed the EPM test to measure anxiety-like behaviors in these mutant mice and observed no significant difference between the Control NL2ΔPDDVP mice and RS-treated NL2ΔPDDVP mice (Fig. 5 G and H), indicating that impaired MyoVa-dependent trafficking of NL2 is a key molecular mechanism underlying high anxiety-like behaviors in RS mice.

Fig. 5.

Genetic disruption of the MyoVa–NL2 interaction reduces synaptic expression of NL2 and increases anxiety. (A) MyoVa was coimmunoprecipitated with NL2 in WT, but not NL2ΔPDDVP, hippocampal lysates. (n = 4, IP, immunoprecipitation; IB, immunoblot; IgG, immunoglobulin G). (B) Expression of inhibitory synaptic proteins (vGAT, gephyrin, NL2, and α2), but not excitatory synaptic protein PSD95, was strongly reduced in hippocampal synaptosomal fractions prepared from NL2ΔPDDVP KI mice as compared to Control mice (n = 3; two-way ANOVA test). (C) Surface but not total NL2 staining was strongly reduced in cultured hippocampal neurons prepared from NL2ΔPDDVP mice as compared to Control hippocampal neurons [WT Control, n = 12; NL2ΔPDDVP, n = 14; Student's t test (Scale bar, 5 μm.)] (D) Immunohistochemical images from hippocampal CA1 regions showed that the punctual densities of NL2, vGAT, and gephyrin were strongly reduced in NL2ΔPDDVP mice, as compared to the Control (n = 3; Student's t test). (E) mIPSC frequency and amplitude were significantly reduced in CA1 neurons in acute hippocampal slices prepared from NL2ΔPDDVP mice as compared to the Control [WT Control, n = 14; NL2ΔPDDVP, n = 15; Student's t test; the Kolmogorov–Smirnov test was used for the cumulative distributions (Scale bar, 20 pA, and 1 s.)] (F) NL2ΔPDDVP mice spent significantly less time and made fewer entries in the open arm of the EPM as compared to the Control (n = 12 for both WT control and NL2ΔPDDVP; Student's t test). (G) Schematic representation of the RS protocol performed in NL2ΔPDDVP mice. (H) RS did not exacerbate anxiety-like behavior in NL2ΔPDDVP mice, as evidenced by similar time spent and entries made in the open arms of the EPM as compared to the Control (n = 6 for both conditions; Student's t test). Error bars indicate SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01.

However, it remained unknown whether PP2-mediated inhibition of Src kinase activity acted through MyoVa-dependent transport of NL2 in neurons in RS mice. To this end, we performed PP2 treatment in NL2ΔPDDVP mice to test whether PP2 could reduce anxiety-like behaviors in these mice (Fig. 6A). We found that in the EPM test, NL2ΔPDDVP animals made significantly less time and fewer entries in the open arm of the EPM (Fig. 6B) as compared to the Control; however, PP2 administration in NL2ΔPDDVP mice did not reduce anxiety compared to vehicle-treated NL2ΔPDDVP mice, as they spent similar time and made fewer entries in the open arm of the EPM (Fig. 6B and Movie S3). Similarly, NL2ΔPDDVP mice buried a significantly higher number of marbles, and the latency to bury was also decreased compared to the Control. However, PP2 could not rescue these phenotypes in these NL2ΔPDDVP animals (Fig. 6C and Movie S4). Finally, in the FST, NL2ΔPDDVP mice were significantly more immobile than the Control, but PP2 treatment did not rescue the behavioral deficits in these NL2ΔPDDVP mice (Fig. 6D). Taken together, these data show that anxiety levels are significantly increased in NL2ΔPDDVP mice, but the Src kinase inhibitor, PP2, has no effect in reducing anxiety-like behaviors in these mice due to the disruption of MyoVa-dependent trafficking of NL2.

Fig. 6.

PP2 administration in NL2ΔPDDVP mice did not lead to the recovery of the enhanced anxiety-like phenotypes observed in these animals. (A) Schematic of the experimental procedure. (B) NL2ΔPDDVP mice spent significantly less time and fewer entries in the open arm of the EPM compared to the Control, and importantly, PP2 administration did not rescue the phenotype in NL2ΔPDDVP mice (n = 12 for every condition; two-way ANOVA test). (C) NL2ΔPDDVP mice buried a significantly high number of marbles with strongly reduced latency to bury the first marble in the MBT as compared to the Control. However, PP2 administration did not affect this phenotype in NL2ΔPDDVP mice (n = 12 for each condition; two-way ANOVA test). (D) NL2ΔPDDVP mice were significantly less mobile and had significantly high mobility in the last 4 min of the FST, and PP2 administration did not improve this behavioral phenotype in these animals compared to the Control (n = 12 for every condition; two-way ANOVA test). Error bars indicate SEM. ****P < 0.0001; ***P < 0.001.

Discussion

We have identified a signaling pathway that is highly sensitive to chronic stress (Fig. 7). In this pathway, enhanced Src kinase activity impairs MyoVa-dependent NL2 transport, leading to reduced synaptic expression of NL2. Importantly, inhibition of Src kinase activity by PP2 administration in chronically stressed mice increases synaptic abundance of NL2 by restoring the MyoVa–NL2 interaction and, consequently, reduces elevated anxiety-like behaviors in these mice. Consistently, in NL2ΔPDDVP mice in which the MyoVa–NL2 interaction is genetically abolished, RS-induced anxiety is occluded and PP2 administration cannot rescue reduced expression of synaptic NL2 and elevated anxiety-like behaviors, confirming that reduced synaptic NL2 and enhanced anxiety in RS mice are due to impaired interaction between MyoVa and NL2. For a more detailed discussion of the Src-CaM-MyoVa-NL2 pathway, please refer to the supplementary discussion (SI Appendix, Supplemental Discussion).

Fig. 7.

Schematic model illustrating the effect of RS and PP2 on the stress-sensitive Src-CaM-MyoVa-NL2 pathway in WT and NL2ΔPDDVP mice. (A) The schematic model shows that RS triggers a substantial increase in active Src activity in WT mice. This, in turn, leads to a significant increase of tyrosine phosphorylation of CaM, resulting in disruption of MyoVa-dependent NL2 surface trafficking. As a consequence, there is a reduction in GABAergic synaptic density and inhibitory synaptic transmission, leading to heightened anxiety-like behaviors in WT mice. Importantly, PP2 administration effectively ameliorates molecular and behavioral deficits induced by RS in WT mice. (B) Genetic ablation of the domain in the NL2 C terminus (PDDVP) that mediates the interaction with MyoVa in NL2ΔPDDVP mice reduces NL2 surface expression, resulting in decreased GABA_AR synaptic abundance and inhibitory synaptic transmission, leading to an enhanced anxiety-like phenotype. Importantly, RS does not exacerbate anxiety-like behaviors, and PP2 administration fails to rescue the deficits at the molecular and behavioral levels in these NL2ΔPDDVP mice.

In addition to Src, our work in NL2ΔPDDVP mice indicates a critical role of MyoVa interaction with NL2 in regulating anxiety-like behaviors in chronically stressed mice. Currently, this molecular pathway downstream of Src kinase has not been explored in clinical studies in the treatment of anxiety and depression. It is worth noting that MyoVa mutations have been shown to cause neurological deficits in humans and animal models (33, 34), and importantly, MyoVa brain-specific dominant-negative mutant mice show strong anxiety-like phenotypes and obsessive–compulsive behaviors (29). Together with that NL2 has been strongly implicated in anxiety (18–22), our identification of the Src-CaM-MyoVa-NL2 pathway might represent a key signaling cascade that is highly responsive to chronic stress on the one hand, and that can be utilized to reverse anxiety-like behaviors in stressed animals on the other hand.

Current FDA-approved medications for the treatment of anxiety and depression, including GABA_AR PAMs, and serotonin transporter inhibitors, primarily target vital receptors that are integral to various biological functions (8, 10, 11). Modulation of activities of these receptors and transporters by these medications may potentially impact a multitude of signaling pathways, extending beyond their intended roles in anxiety and depression management, which may contribute to a diverse range of side effects associated with their use (35, 36). Thus, it becomes imperative to identify the specific signaling pathways that are directly involved in anxiety and depressive behaviors (13, 37). From this perspective, our identification of the Src-CaM-MyoVa-NL2 pathway as a pivotal signaling cascade highly responsive to chronic stress and with the potential to reverse anxiety and depressive phenotypes in animal models provides an alternative option for management of anxiety and depression.

In summary, we have identified a signaling pathway that is highly responsive to chronic stress. Significantly, we have demonstrated the feasibility of manipulating this signaling pathway to reverse anxiety-like behaviors in the mice, providing preclinical evidence for application of this pathway in anxiety and depressive disorder management.

Limitations of the Study.

While our study underscores the pivotal role of the Src-CaM-MyoVa-NL2 pathway in anxiety- and depressive-like behaviors in mice, our data have not shown where this pathway operates in the brain to regulate anxiety- and depressive-like behaviors. Although we have characterized this pathway in the hippocampus, currently we cannot rule out the possibility of other brain regions also involved in the regulation of anxiety- and depressive-like behaviors through this pathway. Future work with more selective manipulation of the Src-CaM-MyoVa-NL2 pathway in specific cell types or brain regions will help understand the regulatory mechanisms for anxiety- and depressive-like behaviors.

Materials and Methods

Comprehensive materials and methods, encompassing materials, instruments, imaging protocols, as well as in vitro and in vivo biological assays, are delineated in SI Appendix. Important techniques and protocols employed in this manuscript are summarized below.

Animal Handling.

The animals were handled and maintained in accordance with the protocols approved by the institutional animal care and use committee at the NINDS, NIH. Adult C57BL/6J mice (6 to 8 wk) were purchased from the Charles River Laboratory. Timed-pregnant C57BL/6J mice were used for dissociated hippocampal neuronal cultures at E17.5 to E18.5. The NL2ΔPDDVP domain in-frame deletion mice were generated using CRISPR/Cas9 technology with sgRNAs that specifically target nucleotide sequences of the PDDVP motif in the NL2 genomic DNA in a C57BL/6J background. Mice of either sex were used and were randomly assigned for the experiments unless otherwise specified.

Stress Protocols for Anxiety Generation.

Chronic RS.

We used young adult male C57BL/6J (6 to 8 wk) mice for chronic RS. The test animals were physically restrained in the conical plastic restraining bags for 3 h (11.00 am to 1.00 pm) daily for 14 consecutive days, and the Control animals were present in their home cage situated in the same procedure room.

MS.

During the MS procedure, half of the pups were separated from the mother for 3 h (11.00 am to 1.00 pm) daily, in isolated cages placed in a quiet procedure room from P5 to P21. After the procedure, the pups were returned to their mother in home cages. After P21, the pups were weaned, and experiments were performed from P22 onward.

Behavior tests.

We performed the EPM test, MBT, and FST to assess behaviors in the rodents. Details of the test were described in supplementary materials.

Electrophysiology, biochemistry, imaging, cell culture, and transfection.

Please refer to supplementary methods for detailed information.

Supplementary Material

Appendix 01 (PDF)

Movie S1.

Male C57BL/6 mice (7–9 weeks old) were subjected to daily 3-hour restraint stress (RS) in plastic bags for 14 days. Following the RS period, the mice were treated with either Vehicle, PP2, or PP3 through intraperitoneal injections at a dose of 2.5mg/kg body weight, once daily for 7 consecutive days. After the treatment phase, the mice underwent an Elevated Plus Maze (EPM) experiment. Representative sample videos of the mice under each condition were recorded and presented in the trimmed video at a speed of 2X to illustrate the experimental outcomes.

Movie S2.

Male C57BL/6 mice (7–9 weeks old) were subjected to daily 3-hour restraint stress (RS) in plastic bags for 14 days. Following the RS period, the mice were treated with either Vehicle, PP2, or PP3 through intraperitoneal injections at a dose of 2.5mg/kg body weight, once daily for 7 consecutive days. After the treatment phase, the mice underwent Marble Bury Test (MBT) experiment. Representative sample videos of the mice under each condition were recorded and presented in the trimmed video at a speed of 2X to illustrate the experimental outcomes.

Movie S3.

Male WT Control and NL2ΔPDDVP mice (7–9 weeks old) were treated with either Vehicle or PP2 through intraperitoneal injections at a dose of 2.5mg/kg body weight, once daily for 7 consecutive days. After the treatment phase, the mice underwent an Elevated Plus Maze (EPM) experiment. Representative sample videos of the mice under each condition were recorded and presented in the trimmed video at a speed of 2X to illustrate the experimental outcomes.

Movie S4.

Male WT Control and NL2ΔPDDVP mice (7–9 weeks old) were treated with either Vehicle or PP2 through intraperitoneal injections at a dose of 2.5mg/kg body weight, once daily for 7 consecutive days. After the treatment phase, the mice underwent Marble Bury Test (MBT) experiment. Representative sample videos of the mice under each condition were recorded and presented in the trimmed video at a speed of 2X to illustrate the experimental outcomes.

Data, Materials, and Software Availability

The NL2 mass spectrometry proteomics raw data, peak list, and search results were submitted to the MASSIVE repository (38) and ProteomeXchange: PXD044155 (26). This paper does not report the original code.