Increased oxidative stress in the cerebellum and peripheral immune cells leads to exaggerated autism-like repetitive behavior due to deficiency of antioxidant response in BTBR T + tf/J mice

Highlights

• BTBR mice have increased peripheral/cerebellar oxidative stress as compared to C57 mice.

• BTBR mice have deficiency of antioxidant response after induction of oxidative stress.

• Increased oxidative stress is associated with autism-like behavior in BTBR mice.

Abstract

Autism is a neurodevelopmental disorder that affects social cognitive abilities resulting in communication or sensory deficits, and stereotyped behaviors in millions of people worldwide. Oxidant-antioxidant imbalance contributes significantly to the neurobehavioral dysregulations and severity of symptoms in patients with autism, however it has not been explored earlier whether it affects autism-like behavior directly. Therefore, we investigated oxidant-antioxidant balance in peripheral immune cells (neutrophils and CD3+ T cells) and cerebellum of BTBR T + tf/J (BTBR) mice which show autism-like behavior and the social C57BL/6 J (C57) mice. Further, we utilized buthionine sulfoximine (BSO), a glutathione depleting agent to assess the impact of oxidant-antioxidant dysregulation on autism-like behavior. Our study shows that BTBR mice have increased lipid/protein oxidation products in cerebellum and neutrophils/CD3+ T cells along with increased NADPH oxidase (NOX2) and inducible nitric oxide synthase (iNOS) expression. This was concurrent with lower levels of glutathione and enzymatic antioxidants such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the cerebellum and peripheral immune cells. BSO administration led to further lowering of glutathione with a concurrent upregulation of iNOS, and NOX2 in cerebellum and peripheral immune cells. However, there was deficiency of an adaptive antioxidant response which was associated with exaggerated repetitive behaviors in BTBR mice. On the other hand, C57 mice also had increased oxidative stress after BSO treatment, however there was an enzymatic antioxidant response both in cerebellum and periphery. Overall, this study suggests that BTBR mice have increased oxidative stress with a deficient enzymatic antioxidant response that is associated with autism-like repetitive behaviors.

Introduction

Autism affects millions of people worldwide and is usually diagnosed around the age of two with a higher prevalence in boys than girls (4:1 ratio). It is a multifaceted disorder that affects social cognitive abilities resulting in communication or emotional deficits, and stereotyped behaviors, which may be a significant impediment to the daily living of the patients and their families (Levy et al., 2009; Baron-Cohen, 2004). The treatment cost of autistic disorders is huge in USA and Europe which make them a significant public health concern, therefore it is necessary to gain a better understanding of the biological underpinnings leading to the etiopathogenesis or severity of autism (Baird et al., 2006; Nevison, 2014; State and Neuroscience, 2012).

Dysregulations in the peripheral and central nervous system (CNS) immune cells of both adaptive and innate origin are involved in the pathogenesis of autism. These cells release several inflammatory mediators which have shown to be associated with autistic behavior both in the human and BTBR T + tf/J mice (BTBR). Several previous reports have shown that BTBR mice exhibit peripheral and CNS abnormalities which are similar to the patients of autism (Careaga et al., 2015; Onore et al., 2013; Nadeem et al., 2017a; 2017b; Bjorklund et al., 2016). For example, behavioral patterns are dysregulated in BTBR mice which are similar to human autistic subjects such as deficits in social communication and repetitive self-grooming behavioral pattern. This contrasts with the background strain, i.e. C57BL/6 J mice (C57), that exhibit high sociability and normal grooming behavioral pattern (Gibney and Drexhage, 2013; Crawley, 2007; McFarlane et al., 2008; Moy and Nadler, 2008). Immune system dysregulations involving greater pro-inflammatory cytokine release and activation of various immune populations are also similar in nature to human autistic patients (Careaga et al., 2015; Bjorklund et al., 2016; Nadeem et al., 2018b). However, an important aspect of the human autistic subjects is the presence of oxidative stress in the periphery and CNS which has not been explored in detail earlier in BTBR mice.

Oxidant-antioxidant imbalance in patients with autism contributes significantly to the pathogenesis and severity of autism along with inflammatory cytokines, which together are thought to be responsible for neurobehavioral dysregulation. Previous reports have shown increased protein and lipid peroxidation products such as nitrotyrosine and TBARS both in the periphery and CNS of autistic patients (El-Ansary and Al-Ayadhi, 2012; Frye and James, 2014; James et al., 2009; Chauhan et al., 2004; Sajdel-Sulkowska et al., 2009). Other studies have shown dysregulations in enzymatic and non-enzymatic antioxidants in brain and blood of autistic subjects. Particularly glutathione has been shown to consistently decreased in autistic children both in the brain and blood (Adams et al., 2009; Chauhan et al., 2012; Gu et al., 2013). However, whether oxidant-antioxidant dysregulations exist in BTBR mice needs a thorough investigation. Further, it is not known whether alteration of oxidant-antioxidant balance in BTBR mice affects their behavioral pattern.

Glutathione is one of the central regulators of oxidant-antioxidant balance in different cells including immune and neuronal cells. It helps in the detoxification of oxidants directly through scavenging different oxidants or indirectly by providing reducing equivalents to the enzymatic antioxidant network (Dringen et al., 2015; Gu et al., 2015). Several earlier studies have shown that environmental pollutants that cause oxidative stress by depletion of glutathione are closely associated with severity of the disease in autistic subjects. For example, mercury levels are closely associated with severity of symptoms in autistic children and oxidative stress (Geier et al., 2009; Adams et al., 2009; Hodgson et al., 2014; Kern et al., 2016). However, it has not been investigated if glutathione depletion produces neurobehavioral changes in BTBR mice. Therefore, we utilized an agent which depletes glutathione, i.e. buthionine sulfoximine (BSO) to assess its effect on oxidant-antioxidant balance and behavioral patterns of BTBR and C57 mice. This information can strengthen use of BTBR mice as a model of autism-like behavior especially for those studies which plan to delineate the role of oxidative stress or antioxidants in conjunction with autism.

Our study shows that BSO treated BTBR mice have augmented oxidant stress along with lack of proper antioxidant response which could account for exaggerated autism-like behavior. Overall, this study suggests that BTBR mice display oxidant-antioxidant imbalance similar to autistic subjects and can be utilized for the translational purpose involving an antioxidant intervention.