Involvement of protein kinase C beta1-serotonin transporter system dysfunction in emotional behaviors in stressed mice

https://doi.org/10.1016/j.neuint.2020.104826Get rights and content

Highlights

  • Mice exposed to swim stress developed emotional impairment.

  • Phosphorylated PKCβI and SERT were decreased in the PFCs of stressed mice.

  • PKC activator ameliorated behavior and phosphorylated protein levels.

  • PKC activator also decreased cell surface SERT and increased serotonin turnover.

  • PKCβI-SERT phosphorylation was involved in development of emotional impairment.

Abstract

Selective serotonin reuptake inhibitors are the first-line antidepressants for treating major depressive and post-traumatic stress disorders. These inhibitors directly bind to the serotonin transporter (SERT). Protein kinase C (PKC) is a key regulator of SERT functions as it can attenuate SERT activity through phosphorylation and its subsequent internalization. However, whether PKC-regulated SERT functions are involved in emotional impairment in a mouse model of stress remains unclear. Using a mouse model of swim-induced stress, we investigated whether the PKC-SERT system is involved in emotional impairment and tried to identify the PKC isoforms involved in this mechanism. Mice exposed to swim stress showed enhanced immobility and decreased social interaction times compared to those in swim stress-naive mice. Moreover, significant decreases in phosphorylated PKCβI and SERT levels were observed in the prefrontal cortex of stressed mice compared to those of swim stress-naive mice. No changes in levels of other phosphorylated PKC isoforms were observed between the two groups. Phorbol 12-myristate 13-acetate (a PKC activator) administration significantly attenuated enhanced immobility and decreased social interaction time in stressed mice and increased the serotonin turnover. Further, the PKC activator increased levels of phosphorylated PKCβI or SERT and decreased cell surface localization of SERT in stressed mice. Contrary to this, chelerythrine (a PKC inhibitor) administration exacerbated the immobility and sociality of mice exposed to mild stress. Our results suggest that PKCβI activation attenuates emotional impairment by suppressing SERT function in stressed mice. Thus, PKCβI may be a key target for the development of new treatment strategies for emotional impairment in stress-related disorders.

Introduction

Selective serotonin reuptake inhibitors (SSRIs) are widely used as the first-line treatment for depressive and anxiety symptoms in major depressive disorder (MDD), post-traumatic stress disorder (PTSD), and anxiety disorder (AD). These inhibitors can bind directly to serotonin transporter (SERT) expressed on the neuronal membrane (Diallinas, 2008; Zhou et al., 2009; Locher et al., 2017). SERT dysfunction may increase the vulnerability to depression, and SERT expression and activity are related to the pathophysiology of depression, depressive-like behaviors, and treatment response (Tang et al., 2014; Kong et al., 2015; Fakhoury, 2016). Depressive symptoms are aggravated by alterations in SERT expression or localization (i.e. increased SERT on the cell surface) following exposure to environmental stressors, illustrating the relationship between depressive or anxiety symptoms associated with stress and SERT expression (Caspi et al., 2003; Eley et al., 2004; Yalcin et al., 2008; Bruchas et al., 2011). SERT activity is attenuated by protein kinase C (PKC) through its phosphorylation and subsequent internalization (Ramamoorthy and Blakely, 1999). Human SERT (hSERT) protein has five phosphorylation sites [serine (Ser)13, Ser149, Ser277, threonine (Thr)603, and Thr616], three of which (Ser149, Ser277, and Thr603) are known targets of PKC (Sørensen et al., 2014). Phosphorylation of Ser277 is the initial phase of SERT down-regulation and alters its intrinsic transport properties (Jayanthi et al., 2005). Subsequent phosphorylation of Thr603 is involved in SERT internalization with a less direct effect on transport function (Jayanthi et al., 2005). Although PKC is a key regulator of SERT activity, the functional relationship between PKC, SERT activity, and emotional impairment remains unclear.

Several studies reported low PKC levels in central and peripheral tissues in MDD patients (Shelton et al., 2009) and animal models of stress (Chen et al., 2012). Thus, the PKC signaling pathway may be involved in regulating stress-induced depressive symptoms. PKC belongs to a multigene family of related Ser/Thr kinases that sit at the crossroads of many signal transduction pathways (Steinberg, 2008). Eleven isoforms of PKC are known—α, βI, βII, γ, δ, ε, η, θ, μ, ζ, and ι/λ—the activation of which is regulated by several different mechanisms (Liu and Heckman, 1998). Postmortem analysis of patients with MDD showed down-regulation of PKCβI and PKCε in the prefrontal cortex (PFC) (Shelton et al., 2009).

Developing in vitro models to study stress-related disorders such as MDD or PTSD is difficult because of the wide spectrum of disruptions that characterizes these conditions. However, various animal models have been developed based on the hypothesis that stress-related disorders are caused by stressors (e.g. swim, restraint, social defeat, etc.) (Tong et al., 2017). Although these animal models are widely used for testing novel compounds to treat stress-related disorders, the molecular basis of the development of these disorders is not clearly understood. Physical stress has been implicated in both the development and manifestation of depressive or anxiety symptoms (Tsunekawa et al., 2008) that may share common elements of the underlying pathophysiology (Boyer, 2000). Repeated stress can accelerate disease progression, cause neuronal degeneration, and lead to MDD or stress-related disorders (Nestler et al., 2002). Tsunekawa et al. (2008) reported that swim stress induces behavioral abnormalities, such as the enhanced immobility in the swim test and impaired social behaviors in social interaction test, suggesting increased depressive and anxiety symptoms, which can be correlated to human PTSD (Tsunekawa et al., 2008).

Elucidating the mechanisms regulating SERT expression and functions during stress will significantly enhance our understanding of the neurobiological basis of emotional function. It will also help in developing new therapeutic strategies for stress-related disorders. The present study investigated the role of the PKC-SERT system in emotional behaviors and tried to identify the PKC isoforms involved in regulating this mechanism using a mouse model of swim-induced stress.

Section snippets

Animals

Changes in SERT function and expression induced by swim stress in male C57BL/6J mice (Schindler et al., 2012) and in SERT metabolism (Mouri et al., 2012) have been reported. In the present study, we used male C57BL/6J mice, which were obtained from Japan SLC Inc. (RRID: MGI: 5488963, Shizuoka, Japan). Eight-week-old mice were used for experiments. The mice were housed in groups of 4–5 per plastic cage without any elements of housing enrichment. Food (CE2, Clea Japan Inc., Tokyo, Japan) and

Mice exposed to swim stress developed emotional impairment

We investigated whether swim stress induced emotional impairment in mice. Mice exposed to swim stress (15 min) showed significantly prolonged immobility (p < 0.01, n = 16; Fig. 2A) and shortened social interaction (p < 0.01, n = 14; Fig. 2B) times compared to those in swim stress-naive (n = 16 and n = 14, respectively) mice. Moreover, mice exposed to mild swim stress (1 min, n = 12) also showed a significantly prolonged immobility time compared to that in naive mice (p < 0.01; Fig. 2A).

Discussion

Several studies have demonstrated that stress causes profound and complex alterations in serotonin and dopamine transmission of the mesolimbic and mesocortical systems (Holmes et al., 2003; Tang et al., 2014; Loseva et al., 2015). The mesocortical serotonergic system is involved in depressive symptoms in MDD, PTSD, and AD, which can be induced by stressful experiences (Warden et al., 2012; Boyarskikh et al., 2013; Teissier et al., 2015). Physical stressors can be used to induce depressive-like

Conclusion

The results of our study showed that PKCβI activation attenuated emotional impairment in stressed mice, which was correlated with increased SERT phosphorylation and internalization. Thus, PKCβI may be a key regulator for the development of new treatment strategies for emotional symptoms in stress-related disorders.

Authorship contributions

Category 1

Participated in research design: T. Ito, Y. Noda, A. Mouri.

Conducted experiments: T. Ito, Y. Noda, A. Mouri, Y. Hiramatsu, M. Uchida, T. Mamiya.

Performed data analysis: T. Ito, Y. Noda, Y. Hiramatsu, A. Yoshimi, N. Ozaki.

Category 2

Wrote or contributed to the writing of the manuscript: T. Ito, Y. Noda, Y. Hiramatsu, A. Mouri.

Revising the manuscript critically for important intellectual content: T. Ito, Y. Noda, Y. Hiramatsu, M. Uchida, A. Yoshimi, T. Mamiya, A. Mouri, N. Ozaki.

Category

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgments

We thank Dr. Hirotake Hida, Mr. Kentaro Mori, Ms. Ayumi Ishihara, Mr. Fumiya Yamamoto, Ms. Mari Kimura, and all the staff members of the Faculty of Pharmacy, Meijo University who were involved in this study. Chinese hamster ovary (CHO) cells expressing rat serotonin transporter (rSERT) (CHO-rSERT) were kindly gifted by Prof. Shigeo Kitayama (Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan). We also thank Editage (www.editage.com) for English

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