Elsevier

Free Radical Biology and Medicine

Volume 65, December 2013, Pages 488-496
Free Radical Biology and Medicine

Original Contribution
Impaired synthesis and antioxidant defense of glutathione in the cerebellum of autistic subjects: Alterations in the activities and protein expression of glutathione-related enzymes

https://doi.org/10.1016/j.freeradbiomed.2013.07.021Get rights and content

Highlights

  • Our previous studies have shown a brain region-specific deficit of glutathione in autism.

  • Here, we have studied the mechanism of glutathione redox imbalance in the cerebellum from subjects with autism.

  • The activity of glutathione cysteine ligase (GCL), an enzyme for glutathione synthesis, was impaired in autism.

  • The protein expression of its modulatory subunit GCLM was decreased in autism.

  • The regulation of GCL activity was affected in autism.

  • The activities of glutathione peroxidase and glutathione S-transferase were decreased in autism.

Abstract

Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence in autism suggests a deficit in glutathione (GSH), a major endogenous antioxidant. It is not known whether the synthesis, consumption, and/or regeneration of GSH is affected in autism. In the cerebellum tissues from autism (n=10) and age-matched control subjects (n=10), the activities of GSH-related enzymes glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), and glutamate cysteine ligase (GCL) involved in antioxidant defense, detoxification, GSH regeneration, and synthesis, respectively, were analyzed. GCL is a rate-limiting enzyme for GSH synthesis, and the relationship between its activity and the protein expression of its catalytic subunit GCLC and its modulatory subunit GCLM was also compared between the autistic and the control groups. Results showed that the activities of GPx and GST were significantly decreased in autism compared to that of the control group (P<0.05). Although there was no significant difference in GR activity between autism and control groups, 40% of autistic subjects showed lower GR activity than 95% confidence interval (CI) of the control group. GCL activity was also significantly reduced by 38.7% in the autistic group compared to the control group (P=0.023), and 8 of 10 autistic subjects had values below 95% CI of the control group. The ratio of protein levels of GCLC to GCLM in the autism group was significantly higher than that of the control group (P=0.022), and GCLM protein levels were reduced by 37.3% in the autistic group compared to the control group. A positive strong correlation was observed between GCL activity and protein levels of GCLM (r=0.887) and GCLC (r=0.799) subunits in control subjects but not in autistic subjects, suggesting that regulation of GCL activity is affected in autism. These results suggest that enzymes involved in GSH homeostasis have impaired activities in the cerebellum in autism, and lower GCL activity in autism may be related to decreased protein expression of GCLM.

Introduction

Autism belongs to a group of neurodevelopmental disorders known as the autism spectrum disorders (ASDs), which include pervasive developmental disorder-not otherwise specified (PPD-NOS) and Asperger's disorder. Autism is a heterogeneous disorder characterized by impairments in social and communicative behaviors, as well as by repetitive and stereotypic patterns of behavior [1]. The symptoms of ASDs are typically present before the age of 3 years. The prevalence of ASDs increased considerably over the past several decades. Recently, the Centers for Disease Control and Prevention (CDC) reported that 1 in 88 children is affected with autism in the United States [2]. The cause and pathological mechanism of autism are still elusive, although genetic and environmental factors, oxidative stress, mitochondrial dysfunction, and immune abnormalities have been suggested to play important roles in ASDs [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13].

Oxidative stress occurs when the generation of reactive oxygen species (ROS) exceeds the antioxidant ability of the cell. ROS are generated endogenously during oxidative metabolism and energy production by mitochondria [14], [15]. ROS levels dramatically increase under certain pathological conditions and due to the effect of environmental factors. They are unstable and may attack and damage vital components of the cell, such as polyunsaturated fatty acids, proteins, and nucleic acids. It is well known that ROS play a vital role in aging and neurodegenerative diseases. Convergent evidence from our and other groups suggests that oxidative stress may also play a central role in the development and clinical manifestation of autism [4], [5], [7], [9], [16], [17], [18], [19], [20], [21]. Several systematic reviews and meta-analysis of the published literature in this field have shed light on the role of oxidative stress in autism [5], [6], [8], [11], [22], [23], [24], [25].

Antioxidant system includes protective mechanisms by enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx), and by nonenzymatic antioxidants such as glutathione (GSH), vitamins E and C, metallothionein, and phenolic compounds. An impaired antioxidant system can result in cell membrane damage, alterations in membrane fluidity and permeability, and oxidative stress. GSH, a tripeptide containing a free thiol group, is a nonenzymatic antioxidant that plays a crucial role in antioxidant defense and detoxification of xenobiotics as well as their metabolites. GSH is the most important endogenous scavenger of environmental toxins and ROS. It is also involved in the maintenance of essential thiol status, storage of cysteine, and modulation of cell differentiation, proliferation, and apoptosis [26]. GSH is oxidized to GSSG (oxidized form of glutathione) by GPx, and GSSG is reversed to GSH when glutathione reductase (GR) catalyzes the reaction. GSH is the predominant form, and GSSG content is less than 1–1.2% of GSH [27]. Studies from our and other groups have reported low levels of GSH, increased levels of GSSG, and decreased ratio of GSH/GSSG in the brain tissues [7], [28], blood [16], [18], [29], [30], [31], [32], [33], [34], and lymphoblastoid cells from autistic subjects [35]. These findings implicate GSH depletion and glutathione-redox imbalance in individuals with autism.

GPx, one of important antioxidant enzymes, functions in scavenging and inactivating hydrogen and lipid peroxides to protect the body against oxidative stress. Glutathione-S-transferase (GST) is another antioxidant detoxification enzyme, which is involved in the detoxification of oxidized products by conjugating GSH to electrophilic centers in many toxic substrates to form nontoxic products. The human GST is a superfamily, and consists of at least eight isoforms and 16 subunits. In the brain, the isoenzymes Alpha, Mu, and Pi show high levels of expression. Although a few studies have reported alterations in the activities of GPx [30], [36], [37], [38], [39], [40], GST [34], and GR [34] in the blood from individuals with autism, their status in the brain is not known in autism. Furthermore, it is not known whether GSH synthesis is affected in autism. In the cytosol, the GSH synthesis includes two consecutive ATP-dependent enzymatic reactions. First, glutamate is coupled with cysteine to form γ-glutamylcysteine (γGC), in a reaction catalyzed by glutamate cysteine ligase (GCL), the key rate-limiting enzyme of GSH biosynthesis. Then, γGC is coupled with glycine to form GSH, and this reaction is catalyzed by glutathione synthetase (GS). GSH synthesis is regulated by multifactors. The major determinants are the availability of cysteine as well as GCL, which is composed of a heavy or catalytic subunit (GCLC, Mr~73,000) and a light or modulatory subunit (GCLM, Mr~30,000) [26].

There is general consensus from neuroimaging and postmortem neuropathological studies that dysfunction in the cerebellum may result in autistic symptoms [41], [42]. Loss of Purkinje and granule cells throughout the cerebellar hemispheres in autism has been reported [43], [44], [45]. We recently reported that the levels of total GSH and reduced GSH as well as GSH/GSSG redox ratio are significantly decreased in the cerebellum and temporal cortex of autistic subjects compared to the age-matched control subjects [7]. Such alterations in glutathione status were brain region-specific in autism, and were not observed in the frontal, parietal and occipital cortex [7]. The mechanism of glutathione-redox imbalance in autism is not known. Using the postmortem cerebellum tissues from control and autistic subjects, we compared the activities of GPx, GR, GST, and GCL enzymes, as well as the protein levels of GCLC and GCLM subunits in this study. To our knowledge, this is the first study to analyze whether synthesis, consumption, and/or regeneration of GSH are affected in the brain of subjects with autism.

Section snippets

Materials

Samples of frozen postmortem cerebellum tissues of brain from autistic and age-matched control subjects were obtained from the National Institute of Child Health and Human Development (NICHD) Brain and Tissue Bank for Developmental Disorders at the University of Maryland. Demographics of autistic and control subjects, including age, postmortem interval (PMI), and cause of death are summarized in Table 1. Donors with autism fit the diagnostic criteria of the Diagnostic and Statistical Manual-IV,

GPx, GST, and GR activities in the cerebellum of autistic subjects

The activities (mean±SE and 95% CI) of GPx, GST, and GR in the cerebellum from autistic and age-matched control subjects are represented in Table 2, and scattered plots are shown in Fig. 1. The activities of GPx and GST in the autistic group were significantly lower by 9.7% (P=0.031) and 14.1% (P=0.035), respectively, than that of the control group. If a 95% CI of control was taken as the reference range, 7 of 10 (70%) autistic subjects had GPx activities below the 95% CI of control group (Fig.

Discussion

Autism is a multifactorial disorder that is influenced by environmental and genetic factors. Clinical investigations from many groups have suggested that oxidative stress may impact the occurrence and severity of autism through interaction of environmental factors and genetically susceptible alleles [4], [5], [6], [8]. Antioxidants, GSH in particular, are essential for neuronal survival during the early critical period [47], [48]. GSH is the most important endogenous antioxidant, and plays an

Acknowledgments

Human brain tissues were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD. This work was supported by funds from the Department of Defense Autism Spectrum Disorders Research Program AS073224P2, the Autism Research Institute, and the NYS Office for People with Developmental Disabilities.

References (93)

  • S.J. James et al.

    Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism

    Am. J. Clin. Nutr.

    (2004)
  • G.A. Mostafa et al.

    Oxidative stress in Egyptian children with autism: relation to autoimmunity

    J. Neuroimmunol.

    (2010)
  • D.A. Geier et al.

    Biomarkers of environmental toxicity and susceptibility in autism

    J. Neurol. Sci.

    (2009)
  • Y. Al-Gadani et al.

    Metabolic biomarkers related to oxidative stress and antioxidant status in Saudi autistic children

    Clin. Biochem.

    (2009)
  • S. Sogut et al.

    Changes in nitric oxide levels and antioxidant enzyme activities may have a role in the pathophysiological mechanisms involved in autism

    Clin. Chim. Acta

    (2003)
  • O. Yorbik et al.

    Investigation of antioxidant enzymes in children with autistic disorder

    Prostaglandins Leukot. Essent. Fatty Acids

    (2002)
  • S.P. Pasca et al.

    High levels of homocysteine and low serum paraoxonase 1 arylesterase activity in children with autism

    Life Sci.

    (2006)
  • C.C. White et al.

    Fluorescence-based microtiter plate assay for glutamate-cysteine ligase activity

    Anal. Biochem.

    (2003)
  • R. Dringen

    Metabolism and functions of glutathione in brain

    Prog. Neurobiol.

    (2000)
  • M. Raffa et al.

    Reduced antioxidant defense systems in schizophrenia and bipolar I disorder

    Prog Neuropsychopharmacol. Biol. Psychiatry

    (2012)
  • H. Ono et al.

    Plasma total glutathione concentrations in healthy pediatric and adult subjects

    Clin. Chim. Acta

    (2001)
  • V.V. Dukhande et al.

    Reduced glutathione regenerating enzymes undergo developmental decline and sexual dimorphism in the rat cerebral cortex

    Brain Res.

    (2009)
  • J.J. Haddad et al.

    L-Gamma-glutamyl-L-cysteinyl-glycine (glutathione; GSH) and GSH-related enzymes in the regulation of pro- and anti-inflammatory cytokines: a signaling transcriptional scenario for redox(y) immunologic sensor(s)? Mol

    Immunol.

    (2005)
  • P. Goines et al.

    Van de Water, J. Autoantibodies to cerebellum in children with autism associate with behavior

    Brain Behav. Immun.

    (2011)
  • X. Ming et al.

    Genetic variant of glutathione peroxidase 1 in autism

    Brain Dev.

    (2010)
  • C.S. Huang et al.

    Amino acid sequence and function of the light subunit of rat kidney gamma-glutamylcysteine synthetase

    J. Biol. Chem.

    (1993)
  • N. Yan et al.

    Amino acid sequence of rat kidney gamma-glutamylcysteine synthetase

    J. Biol. Chem.

    (1990)
  • C.S. Huang et al.

    Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase

    J. Biol. Chem.

    (1993)
  • Y. Yang et al.

    Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(−/−) knockout mouse. Novel model system for a severely compromised oxidative stress response

    J. Biol. Chem.

    (2002)
  • T. Stojkovic et al.

    Risperidone reverses phencyclidine induced decrease in glutathione levels and alterations of antioxidant defense in rat brain

    Prog. Neuropsychopharmacol. Biol. Psychiatry

    (2012)
  • S.J. James et al.

    Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism

    Am. J. Clin. Nutr.

    (2009)
  • A. Shinohe et al.

    Increased serum levels of glutamate in adult patients with autism

    Prog. Neuropsychopharmacol. Biol. Psychiatry

    (2006)
  • D.K. Kinney et al.

    Prenatal stress and risk for autism

    Neurosci. Biobehav. Rev.

    (2008)
  • M.T. Miller et al.

    Autism associated with conditions characterized by developmental errors in early embryogenesis: a mini review

    Int. J. Dev. Neurosci.

    (2005)
  • M.J. Hitchler et al.

    An epigenetic perspective on the free radical theory of development

    Free Radic. Biol. Med.

    (2007)
  • P.G. Wells et al.

    Molecular and biochemical mechanisms in teratogenesis involving reactive oxygen species

    Toxicol. Appl. Pharmacol.

    (2005)
  • J.H. Wu et al.

    Glutathione and glutathione analogues; therapeutic potentials

    Biochim. Biophys. Acta

    (2013)
  • M. Wingate et al.

    Prevalence of autism spectrum disorders—autism and developmental disabilities monitoring network, 14 sites, United States, 2008

    MMWR Surveill. Summ.

    (2012)
  • J. Hallmayer et al.

    Genetic heritability and shared environmental factors among twin pairs with autism

    Arch. Gen. Psychiatry

    (2011)
  • A. Chauhan et al.

    Brain region-specific glutathione redox imbalance in autism

    Neurochem. Res.

    (2012)
  • J.K. Kern et al.

    Evidence of toxicity, oxidative stress, and neuronal insult in autism

    J. Toxicol. Environ. Health B Crit. Rev.

    (2006)
  • A. Chauhan et al.

    Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism

    J. Neurochem.

    (2011)
  • A. Chauhan et al.

    Mitochondrial respiratory chain defects in autism and other neurodevelopmental disorders

    J. Pediatr. Biochem

    (2012)
  • D.A. Rossignol et al.

    A review of research trends in physiological abnormalities in autism spectrum disorders: immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures

    Mol. Psychiatry

    (2012)
  • D.A. Rossignol et al.

    Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis

    Mol. Psychiatry

    (2012)
  • Cited by (72)

    • The histamine H3R and dopamine D2R/D3R antagonist ST-713 ameliorates autism-like behavioral features in BTBR T+tf/J mice by multiple actions

      2021, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      Accordingly, previous clinical observations revealed the increase in protein and lipid peroxidation products such as nitrotyrosine both in the periphery and CNS of ASD patients [30,32–36]. In addition, several other studies showed dysregulations in enzymatic and non-enzymatic antioxidants, e.g. consistent decreased levels of glutathione in brain and blood of patients diagnosed with ASD [30,37–39]. Notably, a recent preclinical study revealed that oxidant-antioxidant balance plays an essential role in the severity of ASD-like repetitive behaviors in BTBR mice, demonstrating BTBR mice as a useful model in exploration of antioxidant intervention strategies that have translational value [30].

    • The glutathione system in Parkinson's disease and its progression

      2021, Neuroscience and Biobehavioral Reviews
      Citation Excerpt :

      This issue is important for redox signaling, redox-sensitive enzyme regulation, the adequate capacity of antioxidant/detoxification, cell-cycle proliferation and differentiation, and gene transcription of antioxidant response elements (ARE) (Fig. 2) (Biswas et al., 2006). Pathways responsible for intracellular GSH homeostasis comprise de novo synthesis of GSH, direct uptake, and GSH redox cycling (Gu et al., 2013, 2015). Most of the hepatic production of GSH in the absence of oxidative stress is excreted into the plasma, while GSSG is preferentially excreted into bile (Purucker and Wernze, 1990).

    View all citing articles on Scopus
    View full text