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