Research paper
Jmjd3 is involved in the susceptibility to depression induced by maternal separation via enhancing the neuroinflammation in the prefrontal cortex and hippocampus of male rats

https://doi.org/10.1016/j.expneurol.2020.113254Get rights and content

Highlights

  • MS induced short and long term depressive-like behaviors and neuroinflammation.

  • Infant and adult MS rats had higher levels of Jmjd3 in the PFC and HIP.

  • MS rats treated with LPS in adulthood displayed serious behavioral dysfunction and neuroinflammation.

  • Jmjd3 is involved in MS-induced susceptibility to depressive disorders.

Abstract

Adverse childhood experience is a major risk factor for the onset of depression in adulthood. Neuroinflammation characterized by microglial activation and cytokine secretion is involved in susceptibility to depression induced by early life stress. Jumonji domain-containing protein 3 (Jmjd3), a trimethylated lysine 27 in histone 3 (H3K27me3) demethylase, can be activated by nuclear factor-kappa B (NF-κB), further regulating the expression of pro-inflammatory cytokines and resulting in neuroinflammation. However, its involvement in susceptibility to early life stress-related depression is unknown. In the current study, maternal separation (MS) was utilized as a model of early life stress and systemic lipopolysaccharide (LPS) administration in adulthood was used as a later-life challenge. Depressive- and anxiety-like behaviors and memory impairment were detected by behavioral tests. Microglial activation, pro-inflammatory cytokine expression, and NF-κB, Jmjd3, and H3K27me3 expression were detected in the prefrontal cortex and hippocampus in both infant and adult rats. Meanwhile, the Jmjd3 inhibitor GSK-J4 was used as an intervention in vivo and in vitro. Our results showed that MS induced depression-like behaviors and synchronously caused microglial activation, pro-inflammatory cytokine over-expression, NF-κB and Jmjd3 over-expression, and decreased H3K27me3 expression in infant rats. All these alterations could also be detected in adulthood. Seven-day LPS administration in adult rats induced similar changes of behaviors and biomarkers. Interestingly, compared with rats not exposed to MS, MS-exposed rats receiving LPS administration developed more severe depression-like behaviors and neuroinflammatory status, higher levels of NF-κB and Jmjd3 expression, and lower levels of H3K27me3 expression. In addition, LPS induced microglial activation, pro-inflammatory cytokine expression and increased Jmjd3 expression in vitro. Furthermore, GSK-J4 treatment alleviated these alterations in vivo and in vitro. Thus, our data indicate that Jmjd3 is involved in the susceptibility to depression induced by MS via enhancement of neuroinflammation in the prefrontal cortex and hippocampus of rats.

Introduction

As the most widespread neuropsychiatric disorder, depression affects about 16% of the global population. At present, one in six men and one in four women suffer from depression in their lifetime, causing a huge disease burden worldwide (Kessler et al., 2010; Parker and Brotchie, 2010).

Among the risk factors for the onset of depression, stress, especially early life stress (ELS), has the most robust effects. Clinical research identified maltreatment in childhood including neglect and abuse as the most common cause of abnormal brain development (Teicher and Samson, 2016). This causes long-lasting behavioral alterations and predisposes the individuals to psychiatric diseases such as depression in adulthood (Green et al., 2010; Widom, 2007). In line with clinical research, numerous laboratory studies have confirmed that ELS exposure in rodents induces long-lasting depression-like behaviors which persist into adulthood (Wang et al., 2017a; Xu et al., 2017a). Maternal separation (MS) is a validated rodent model of ELS that has been reported to induce depression-like behaviors by disturbing hypothalamic–pituitary–adrenal (HPA) axis function (Karsten and Baram, 2013; van Bodegom et al., 2017; Zhang et al., 2012) and activating inflammatory responses (Diz-Chaves et al., 2013; Roque et al., 2016). Thus, we have used the MS paradigm as an early-life adverse experience to examine the long-lasting effects of MS on depression-like behaviors and neuroinflammation induced by later-life challenges.

Many studies have revealed that the neuroinflammatory state is responsible for the pathogenesis of many mental disorders including depression (Jeon and Kim, 2016). Cytokines, as the pivotal mediators of neuroinflammation, can change neurochemical processes of the brain and thereby have huge impacts on behaviors (Avitsur et al., 2013; Kurosawa and Seki, 2016). Our previous study and others demonstrated that lipopolysaccharide (LPS) administration induces an increase of pro-inflammatory cytokine (e.g. interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and IL-1β) and depression-like behaviors in mice (Diz-Chaves et al., 2013; Hennessy et al., 2019; Zhao et al., 2015). Meanwhile, it has been widely reported that ELS increases the expression of pro-inflammatory cytokine and enhances the brain's response to an inflammatory challenge in adulthood. (Miller and Chen, 2010; Roque et al., 2016; Wang et al., 2017a). These findings showed that ELS, inflammation, and depression are closely related, which is the social signal transduction theory of depression (Hennessy et al., 2011; Slavich and Irwin, 2014). Microglia, as the dominant immune cells in the brain, are the main source of cytokines (Colton, 2009). Our previous studies and others have demonstrated that stress induces the microglial M1 activation (classical activation) phenotype with release of pro-inflammatory cytokine (Gong et al., 2018; Hinwood et al., 2012; Nakagawa and Chiba, 2014; Wang et al., 2018; Xu et al., 2017b). More importantly, during critical growth periods of the brain, normal microglial function is essential for successful developmental processes such as synaptic maturation, neurogenesis, axonal growth, and myelination (Tay et al., 2017). Numerous studies have demonstrated ELS exposure induces abnormal microglial activation, which leads to structural and behavioral alterations that persist into adulthood (Diz-Chaves et al., 2013; Johnson and Kaffman, 2018; Roque et al., 2016). Calcia and Nusslock put forward an interesting theory that microglia have the function of “psychological immune memory”, which means microglia are primed by ELS and remain in pre-activated states for years. Therefore, primed microglia have an exaggerated response to later-life challenges such as stress, infection and high-fat diet, thereby increasing the susceptibility to mental disorders (Calcia et al., 2016; Nusslock and Miller, 2016). Accordingly, we have assumed in the present study that the activation of microglia, especially with the M1 phenotype and cytokine expression following MS, plays a critical role in susceptibility to depression.

Epigenetic studies have found that histone methylation is an essential mechanism in the regulation of gene expression. Environmental stressors could interfere histone lysine methylation, further bringing about alterations in brain transcriptomes in mood disorders (De Santa et al., 2007; Peter and Akbarian, 2011). Jumonji domain-containing protein 3 (Jmjd3), also known as lysine-specific demethylase (KDM6B), specifically demethylates the trimethylated lysine 27 in histone 3 (H3K27me3) which is affiliated with transcriptional repression. Thus, several studies have reported that Jmjd3 as a chromatin modifier that is involved in macrophage activation and plays a critical role in many inflammatory process by potentiating the transcription of pro-inflammatory cytokine (Yu et al., 2017). Molecular biological studies have revealed that Jmjd3 can be induced by many inflammatory agents. For example, Jmjd3 expression can be induced by nuclear factor-kappa B (NF-κB) in LPS-stimulated macrophages and microglia (De Santa et al., 2009; De Santa et al., 2007; Przanowski et al., 2014). Meanwhile, knockdown of Jmjd3 alleviated the LPS-induced inflammatory cascade and down-regulates pro-inflammatory cytokine production (Liu et al., 2018b). Moreover, the Jmjd3 inhibitor GSK-J4 alleviates the inflammatory response by reducing the expression of pro-inflammatory cytokines in vivo and in vitro (Das et al., 2017; Kruidenier et al., 2012; Pan et al., 2018). However, it is not known whether Jmjd3 is involved in the neuroinflammatory state induced by MS.

In the present study, we hypothesized that MS is able to induce microglial activation and polarization and result in neuroinflammation in the prefrontal cortex (PFC) and hippocampus (HIP), which are primarily responsible for susceptibility to depression in adulthood. Meanwhile, Jmjd3, a regulator of pro-inflammatory cytokine production, was hypothesized to be involved in MS-induced susceptibility to depression. In order to verify our hypothesis, the MS protocol was used as stressful event in critical developmental period, and systemic LPS administration in adulthood was utilized as later-life environmental challenge. The depression-like behaviors, microglial activation and polarization, pro-inflammatory cytokine expression as well as NF-κB, Jmjd3, and H3K27me3 expression, were all assessed in both in infanthood and adulthood. Further, GSK-J4 treatment was used in adult rats to explore whether inhibition of Jmjd3 could reverse the alterations. In addition, the effects of LPS and GSK-J4 on microglial activation and polarization, pro-inflammatory cytokine expression, as well as Jmjd3 and H3K27me3 expression were tested in cultured BV-2 microglial cells (supplement).

Section snippets

Animals

Pregnant Wistar rats (3 months old) were purchased from the Experimental Animal Center of Shandong University at least three days before parturition. Each pregnant rat was housed in an individual cage and kept in a cage with its own pups after birth. Eighty male pups were used in this study. On postnatal day (PND) 1, litters were culled to 5 male pups per dam. After weaning, pups were housed in groups of three or four per cage until adulthood. All animals were housed under standard laboratory

MS induced anhedonia-like behavior, anxiety-like behaviors and memory function in infant rats

Fig. 1A illustrates the results of SPT. MS exposure significantly reduced the percentage of sucrose consumption in MS group compared with control group (t = 4.490, p < .01).

Fig. 1B-C illustrates the results of the OFT. MS exposure decreased the number of crossings (t = 2.955, p < .01) (Fig. 1B) and rearings (t = 2.537, p < .05) (Fig. 1C) in MS group when compared with control group.

Fig. 1D shows the results of the EPM test. MS exposure also reduced the ratio entry in MS group compared with

Discussion

Present study detected MS, an early life stress, induced short- and long-term alterations of depression-like behaviors, pro-inflammatory cytokine expression, microglial activation, and expression of NF-κB, Jmjd3, and H3K27me3 in the PFC and HIP. Further, more severe depression-like behaviors, higher levels of pro-inflammatory cytokine, microglial activation, over-expression of NF-κB and Jmjd3, and lower levels of H3K27me3 expression in the PFC and HIP in LPS-treated adult MS rats were detected.

Conclusions

Our study revealed that MS as an early life stress induced short- and long-term depression-like behaviors, pro-inflammatory cytokine expression, microglial activation, increased levels of NF-κB and Jmjd3, and decreased levels of H3K27me3 in the PFC and HIP of rats. Meanwhile, MS exacerbated behavioral dysfunction and enhanced the inflammatory response when rats exposed to inflammatory challenge in adulthood. In addition, treatment with GSK-J4 (Jmjd3 inhibitor) relieved the depression-like

Authors' contribution

FP conceived and designed the experiments. RW performed most experiment and analyzed data. FP and RW wrote and refined the article. WW participated in the animal modeling and behavioral experiments. JJX, HRW and XQQ assisted in laboratory work and figures preparation. DXL and HJ supervised acquisition of results.

Ethical statement

In the handling and care of all animals, the international guiding principles for animal research, as stipulated by the World Health Organization (WHO) Chronicle (World Health Organization, 1985) and as adopted by the Laboratory Animal Center, Shandong University, were followed.

Declaration of Competing Interest

The authors have no conflict of interests to declare.

Acknowledgements

This work was supported by funding from National Natural Science Foundation of China (NO. 31771220; 31371036).

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