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      Ubiquitin-proteasome dependent degradation of GABA Aα1 in autism spectrum disorder

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          Abstract

          Background

          Although the neurobiological basis of autism spectrum disorder (ASD) is not fully understood, recent studies have indicated the potential role of GABA A receptors in the pathophysiology of ASD. GABA A receptors play a crucial role in various neurodevelopmental processes and adult neuroplasticity. However, the mechanism(s) of regulation of GABA A receptors in ASD remains poorly understood.

          Methods

          Postmortem middle frontal gyrus tissues (13 ASD and 13 control subjects) were used. In vitro studies were performed in primary cortical neurons at days in vitro (DIV) 14. The protein levels were examined by western blotting. Immunofluorescence studies were employed for cellular localization. The gene expression was determined by RT-PCR array and qRT-PCR.

          Results

          A significant decrease in GABA Aα1 protein, but not mRNA levels was found in the middle frontal gyrus of ASD subjects indicating a post-translational regulation of GABA A receptors in ASD. At the cellular level, treatment with proteasomal inhibitor, MG132, or lactacystin significantly increased GABA Aα1 protein levels and Lys48-linked polyubiquitination of GABA Aα1, but reduced proteasome activity in mouse primary cortical neurons (DIV 14 from E16 embryos). Moreover, treatment with betulinic acid, a proteasome activator significantly decreased GABA Aα1 protein levels in cortical neurons indicating the role of polyubiquitination of GABA Aα1 proteins with their subsequent proteasomal degradation in cortical neurons. Ubiquitination specific RT-PCR array followed by western blot analysis revealed a significant increase in SYVN1, an endoplasmic reticulum (ER)-associated degradation (ERAD) E3 ubiquitin ligase in the middle frontal gyrus of ASD subjects. In addition, the inhibition of proteasomal activity by MG132 increased the expression of GABA Aα1 in the ER. The siRNA knockdown of SYVN1 significantly increased GABA Aα1 protein levels in cortical neurons. Moreover, reduced association between SYVN1 and GABA Aα1 was found in the middle frontal gyrus of ASD subjects.

          Conclusions

          SYVN1 plays a critical role as an E3 ligase in the ubiquitin proteasome system (UPS)-mediated GABA Aα1 degradation. Thus, inhibition of the ubiquitin-proteasome-mediated GABA Aα1 degradation may be an important mechanism for preventing GABA Aα1 turnover to maintain GABA Aα1 levels and GABA signaling in ASD.

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          Most cited references25

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          GABA system dysfunction in autism and related disorders: from synapse to symptoms.

          Autism spectrum disorders (ASDs) are neurodevelopmental syndromes characterised by repetitive behaviours and restricted interests, impairments in social behaviour and relations, and in language and communication. These symptoms are also observed in a number of developmental disorders of known origin, including Fragile X Syndrome, Rett Syndrome, and Foetal Anticonvulsant Syndrome. While these conditions have diverse etiologies, and poorly understood pathologies, emerging evidence suggests that they may all be linked to dysfunction in particular aspects of GABAergic inhibitory signalling in the brain. We review evidence from genetics, molecular neurobiology and systems neuroscience relating to the role of GABA in these conditions. We conclude by discussing how these deficits may relate to the specific symptoms observed. Copyright © 2012 Elsevier Ltd. All rights reserved.
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            Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes.

            GABA(A) (gamma-aminobutyric acid(A)) receptors are molecular substrates for the regulation of vigilance, anxiety, muscle tension, epileptogenic activity and memory functions, which is evident from the spectrum of actions elicited by clinically effective drugs acting at their modulatory benzodiazepine-binding site. Here we show, by introducing a histidine-to-arginine point mutation at position 101 of the murine alpha1-subunit gene, that alpha1-type GABA(A) receptors, which are mainly expressed in cortical areas and thalamus, are rendered insensitive to allosteric modulation by benzodiazepine-site ligands, whilst regulation by the physiological neurotransmitter gamma-aminobutyric acid is preserved. alpha1(H101R) mice failed to show the sedative, amnesic and partly the anticonvulsant action of diazepam. In contrast, the anxiolytic-like, myorelaxant, motor-impairing and ethanol-potentiating effects were fully retained, and are attributed to the nonmutated GABA(A) receptors found in the limbic system (alpha2, alpha5), in monoaminergic neurons (alpha3) and in motoneurons (alpha2, alpha5). Thus, benzodiazepine-induced behavioural responses are mediated by specific GABA(A) receptor subtypes in distinct neuronal circuits, which is of interest for drug design.
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              GABA(A) receptor downregulation in brains of subjects with autism.

              Gamma-aminobutyric acid A (GABA(A)) receptors are ligand-gated ion channels responsible for mediation of fast inhibitory action of GABA in the brain. Preliminary reports have demonstrated altered expression of GABA receptors in the brains of subjects with autism suggesting GABA/glutamate system dysregulation. We investigated the expression of four GABA(A) receptor subunits and observed significant reductions in GABRA1, GABRA2, GABRA3, and GABRB3 in parietal cortex (Brodmann's Area 40 (BA40)), while GABRA1 and GABRB3 were significantly altered in cerebellum, and GABRA1 was significantly altered in superior frontal cortex (BA9). The presence of seizure disorder did not have a significant impact on GABA(A) receptor subunit expression in the three brain areas. Our results demonstrate that GABA(A) receptors are reduced in three brain regions that have previously been implicated in the pathogenesis of autism, suggesting widespread GABAergic dysfunction in the brains of subjects with autism.
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                Author and article information

                Contributors
                Journal
                Mol Autism
                Mol Autism
                Molecular Autism
                BioMed Central
                2040-2392
                2014
                1 September 2014
                : 5
                : 45
                Affiliations
                [1 ]Department of Psychiatry and Health Behavior, Medical College of Georgia, Georgia Regents University, 997 St. Sebastian Way, Augusta, GA 30912, USA
                [2 ]Department of Psychiatry, Weill Cornell Medical College, 21 Bloomingdale Rd, White Plains, NY 10605, USA
                Article
                2040-2392-5-45
                10.1186/2040-2392-5-45
                4228821
                25392730
                64298863-c679-42e7-ab74-3dcbaf9c5e87
                Copyright © 2014 Crider et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 19 June 2014
                : 11 August 2014
                Categories
                Research

                Neurosciences
                autism,gabaa receptor,ubiquitination,syvn1,neurons
                Neurosciences
                autism, gabaa receptor, ubiquitination, syvn1, neurons

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