Chronic unpredictable stress negatively regulates hippocampal neurogenesis and promote anxious depression-like behavior via upregulating apoptosis and inflammatory signals in adult rats

Highlights

• Enhanced glial activation and neuroinflammation cause anxious depression in rats after CUS exposure.

• CUS mediated reduced adult hippocampal neurogenesis results in anxious depression.

• Increased apoptosis is observed in cortex and hippocampus of rat brain after CUS exposure.

• Downregulated p-Akt/Akt and upregulated p-Erk/Erk ratio may cause anxious depression.

Abstract

Psychological and physical stress play a pivotal role in etiology of anxiety and depression. Chronic psychological and physical stress modify various physiological phenomena, as a consequence of which oxidative stress, decreased neurotransmitter level, elevated corticosterone level and altered NSC homeostasis is observed. However, the precise mechanism by which chronic stress induce anxious depression and modify internal milieu is still unknown. Herein, we show that exposure to CUS increase oxidative stress, microgliosis, astrogliosis while it reduces hippocampal NSC proliferation, neuronal differentiation and maturation in adult rats. CUS exposure in rats reduce dopamine and serotonin level in cortex and hippocampus, which result in increased anxiety and depression-like phenotypes. We also found elevated level of NF-κB and TNF-α while decreased anti-inflammatory cytokine IL-10 level, that led to increased expression of Bax and cleaved Caspase-3 whereas down regulation of antiapoptotic protein Bcl2. Additionally, CUS altered adult hippocampal neurogenesis, increased gliosis and neuronal apoptosis in cerebral cortex and hippocampus which might be associated with reduced AKT and increased ERK signaling, as seen in the rat brain tissue. Taken together, these results indicate that CUS induce oxidative stress and neuroinflammation which directly affects NSC dynamics, monoamines levels and behavioral functions in adult rats.

Introduction

The maintenance of ‘internal milieu’ inside living beings with changing environment is termed as “homeostasis”(Ahmad et al., 2010). Selye in 1956 reported that any disturbance in homeostasis is termed as “Stress”(Selye, 1956). Stressors are factors that cause stress and the response of living beings against these stressors is termed as “stress response”. However, stress response is an adaptive process which is mediated by endocrine, nervous and immune system (Chrousos, 2009). It has been shown that prolonged stress conditions may lead to tissue damage and degeneration in brain (Ahmad et al., 2010; Schneiderman et al., 2005). Hypothalamic paraventricular nucleus (PVN), anterior pituitary and adrenal glands are set mediators of stress response (Arnett et al., 2016), collectively named as hypothalamic- pituitary-adrenal (HPA) axis, which is mainly regulated by corticotrophin-releasing factor (CRF) (Rivier and Vale, 1983; Vale et al., 1981). Downstream effector of HPA axis is corticosterone, which exert its effect through glucocorticoid (GCs) receptors (Rivier and Vale, 1983; Vale et al., 1981). Clinical and pre-clinical studies have demonstrated that development and progression of various neurodegenerative diseases is associated with elevated GC levels (Conrad, 2008). Hippocampal neurodegeneration in stressed and corticosteroid-treated animals has been reported (Vyas et al., 2016). Previous reports suggest that oxidative stress plays a significant role in pathophysiology of depression, (Bajpai et al., 2014), which might be due to sensitivity of the central nervous system (CNS) to free radicals (Black et al., 2015). Chronic stress exert detrimental effect on several cell functions, including antioxidant system (Ahmad et al., 2010; Salim, 2017; Schiavone et al., 2013; Tripathi et al., 2017; Zafir and Banu, 2009). Oxidative stress mediated changes in neurotransmitter synthesis, degradation and alteration in protein homeostasis in brain are mainly responsible for neurodegenerative and psychiatric disorders (Kanarik et al., 2008; Lucca et al., 2009). Moreover, balanced synthesis of monoamines in CNS is essential for normal brain functioning (Flugge et al., 2004). Due to different type of stressors, change in monoamine level has been reported in various brain regions including nucleus accumbens, hippocampus and cortex (Joels and Baram, 2009). Brain is primary target and more vulnerable to oxidative damage due to high energy demand (Salim, 2017; Zafir and Banu, 2009). Majority of studies have reported increased oxidative burden during physiologically unfavorable conditions, thus causing damage to central monoaminergic system (Ahmad et al., 2010; Lilja, 1979). However, the proportion of oxidation products in monoaminergic system of brain, that can agitate redox state during stressful conditions, is not much known. Oxidation products of dopamine (DA) and serotonin (5-HT) are assumed to play a major role in neurodegenerative disease progression (Biosa et al., 2018). It has been suggested that cell- mediated immune activation and their long-term sequels are prime mediators of neurodegeneration and reduced adult neurogenesis, that characterize depression (Halliwell, 2006; Maes et al., 2009).

In brain, adult neurogenesis occurs via generation of functional neurons from adult neural precursor cells. It is a multistep process which continues throughout life, but only in restricted brain regions (Winner and Winkler, 2015). In adult brain two distinct regions generate new functional neurons; the dentate gyrus (DG) of hippocampus and the subventricular zone (SVZ) of lateral ventricles (Bond et al., 2015). SVZ provides a path for new born neurons to migrate from rostral migratory stream to the olfactory region (Sun et al., 2010). Adult neurogenesis also take place in other brain regions like neo cortex, striatum, amygdala and substantia nigra (Bond et al., 2015; Gould, 2007). Importantly, adult neurogenesis takes place in a gliogenic environment (Gotz et al., 2016). Unlike neural stem cells (NSCs) of embryonic stage, adult NSCs in DG of hippocampus are surrounded by many glial cell types, such as mature oligodendrocytes, microglia and astroglia cells (Kriegstein and Alvarez-Buylla, 2009). Adult neurogenesis requires a specific additional mechanism of neuronal fate specification and maintenance (Gotz et al., 2016; Grande et al., 2013; Herrera et al., 1999). Astrocytes and microglia are probably prime regulators of brain’s microenvironment and play both beneficial and detrimental roles (Block et al., 2006; Dohi et al., 2010). Microglia and astrocytes are extensively found in neurogenic niches, such as SVZ and SGZ (Mosher et al., 2012). In hippocampal neurogenesis, NPCs in SGZ of the DG give rise to new born neuroblasts, among these a small portion of neuroblasts join the hippocampal circuitry as mature neurons. The remaining newborn cells undergo apoptosis, which are further cleared through phagocytosis by microglia (Diaz-Aparicio et al., 2020; Sierra et al., 2010). Hence, microglia play an important role in adult hippocampal neurogenesis (Sierra et al., 2010). It has been seen that phagocytic property of microglia remains constant, although many of the newly generated neurons diminish with age and in other pathological conditions (Sierra et al., 2010). According to an earlier report, microglia and astrocytes have been shown to exert mixed effect on adult neurogenesis (Guttenplan and Liddelow, 2019). At the time of neuronal stress, initially these glial cells play protective role by producing various metabolites and neurotrophins to support neurons (Ransohoff and Perry, 2009; Sochocka et al., 2017; Xu et al., 2020). However, in case of chronic pathological condition, these functionally plastic glial cells undergo morphological changes, subsequently transformed from their basal latent stage to activated ramified form (Burda and Sofroniew, 2014; Jakel and Dimou, 2017; Rock et al., 2004) and secrete proinflammatory cytokines and free radicals (Ransohoff and Perry, 2009).

It has been reported that disease or injury induce hyperactivation of glial cells resulting in generation of ROS and RNS (Hu et al., 1995; Singh et al., 2019). Production of ROS and RNS cause malfunctioning of activated glial cells, which further result in lipid peroxidation and inflammation, that ultimately worsen neurological disorders (Jou, 2008; Lee et al., 1993). Inflammation is usually associated with innate immune system, which occurs in response to infection or injury, and leads to increased levels of proinflammatory cytokines, such as interleukin (IL)-1β, interleukin (IL)- 6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ and decreased levels of anti-inflammatory cytokines (Zhang and An, 2007). Some of the evidences show the association of pathology of depression with inflammation of the peripheral and CNS (Fernandes et al., 2016; Miller and Raison, 2016). In CNS, cytokine network is made by neurons and glial cells, that produce cytokines and express cytokine receptors, additionally they also amplified cytokine signals, which has intense effect on neurotransmitter and corticotrophin releasing hormone (CRH) function, as well as on behavior (Dantzer, 2004; Raison et al., 2006; Raison and Miller, 2003). Earlier reports have shown presence of increased proinflammatory cytokines, chemokines, acute phase proteins, and cellular adhesion molecules in serum and cerebrospinal fluid (CSF) of depressed patients (Alesci et al., 2005; Maes, 1999; Sluzewska et al., 1995), while on the other hand, anti-inflammatory drugs as adjuvant therapy have shown positive effects in schizophrenia and depressive disorders (Al-Hakeim et al., 2015). Altered level of proinflammatory cytokines and chemokines in brain has been shown to affect neurotransmission, neuroendocrine activity and brain anatomy, which leads to neurotoxicity and induce emotional, cognitive, and behavioral changes (Haroon et al., 2012). Inflammatory and neurodegenerative hypothesis of depression show that enhanced neurodegeneration in depression is partly associated with chronic inflammatory response (Hurley and Tizabi, 2013; Maes et al., 2009). Neuroinflammation affects hypothalamus–pituitary– adrenal (HPA) axis and results in alteration of brain serotonin and adult neurogenesis in dentate gyrus of the hippocampus (Kim et al., 2016; Monje et al., 2011). Release of various neurotoxic cytokines, such as IL-6, IL1β, IL16, TNFα and IFN-γ etc. by pro-inflammatory microglial phenotypes and activated astrocytes has been shown to impair various stages of adult neurogenesis(Biscaro et al., 2012; Eddleston and Mucke, 1993; Wyss-Coray and Mucke, 2002), specifically causing reduction in NSC proliferation, neuronal differentiation and survival of adult newborn neurons (Choi et al., 2014; Iosif et al., 2006; Wu et al., 2012). One of the reports have shown reduced progenitor cell proliferation in IL-6 overexpressing transgenic mice(Vallieres et al., 2002). Although several studies have suggested that either endogenous or exogenous encounter of glucocorticoid in brain provide immunosuppressive effect (Kaye et al., 2000). However, in vivo evidences demonstrate that stress induced endogenous increase of glucocorticoids and exogenous glucocorticoid injection actually enhance immune function within the CNS. For example, increased proinflammatory cytokine level has been observed in rat brain and peripheral tissues after exposure to various stressors such as tail-shock stress, social isolation stress and immobilization stress in different experiments(Minami et al., 1991; Nguyen et al., 1998; Pugh et al., 1999). According to earlier reports, psychological and physical stress have potential to activate microglia in vivo through production of various inflammatory mediators, such as macrophage migration inhibitory factor (MIF), cyclooxygenase-2 (cox-2) nitric oxide, and 5-lipoxygenase (5-LO) (Madrigal et al., 2003; Niino et al., 2000; Uz et al., 1999). In addition to increase in inflammatory mediators and glucocorticoid level, chronic stress exposure cause excitotoxic damage that could be due to over-stimulation of the N-methyl-d-aspartate (NMDA) receptor and subsequent upsurge in extracellular glutamate, which ultimately results in dendritic atrophy, neuronal cell death and altered neurogenesis within the hippocampus (Stein-Behrens et al., 1994; Takahashi et al., 2002). Among above-described pathological conditions like microglia activation and diminished adult neurogenesis etc. due to chronic stress, some of these conditions are also seen in clinical data of MDD patients (Wang et al., 2018).

Emerging evidences demonstrate the involvement of Akt signaling in MDD (Beaulieu et al., 2009). An earlier clinical report has shown that phosphorylation of Akt decrease in hippocampus and PFC of suicide subjects (Dwivedi et al., 2010). ERK1/2 is considered as one of the leading signaling kinases associated with depression, an integrated transcriptome analysis has shown abnormalities in ERK1/2 signaling in PFC, derived from rat and humans, which demonstrate the crucial role of ERK1/2 signaling in vulnerability to develop depression (Malki et al., 2015). Oxidative stress induced neuronal apoptosis plays a key role in depression, according to previous reports, excessive free radicals mediated apoptosis reduce regional brain volume and neuronal cell body numbers (Shelton et al., 2011). Apoptosis is also reported in brain samples of suicidal subjects, where apoptotic outcomes are accompanied by a decrease in antiapoptotic and increase in proapoptotic mediators (Duman, 2009; Ilchibaeva et al., 2016; Kubera et al., 2011). Chronic stress has also been shown to dysregulate apoptosis in the DG (Lucassen et al., 2006; Pittenger and Duman, 2008). Many factors can influence hippocampal neurogenesis during adulthood (Balu and Lucki, 2009; Shohayeb et al., 2018). In this regard, few research articles have shown the deleterious effect of chronic exposure on both hippocampal neurogenesis and hippocampal-dependent behavior (Conrad, 2010; Joels et al., 2007; Mirescu and Gould, 2006). Chronic stress reduce proliferation, survival and neuronal differentiation of newly born cells (Joels et al., 2007; Koutmani and Karalis, 2015). Decreased hippocampal neurogenesis due to chronic stress markedly contributes to the mood related behavioral deficits in animals (Kempermann et al., 2008; Lagace et al., 2010; Pittenger and Duman, 2008).