The online version of this article (doi:10.1186/2040-2392-5-45) contains supplementary material, which is available to authorized users.
Amanda Crider, Chirayu D Pandya contributed equally to this work.
The authors declare that they have no competing interests.
AC carried out the gene as well as protein expression studies, and drafted the manuscript. CDP and DP performed the immunoprecipitation and immunofluorescence studies, and helped to draft the manuscript. AOA carried out the statistical analysis and helped to revise the manuscript. AP conceived of the study, and participated in its design and coordination and wrote the manuscript. All authors read and approved the final manuscript.
Although the neurobiological basis of autism spectrum disorder (ASD) is not fully understood, recent studies have indicated the potential role of GABAA receptors in the pathophysiology of ASD. GABAA receptors play a crucial role in various neurodevelopmental processes and adult neuroplasticity. However, the mechanism(s) of regulation of GABAA receptors in ASD remains poorly understood.
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.
A significant decrease in GABAAα1 protein, but not mRNA levels was found in the middle frontal gyrus of ASD subjects indicating a post-translational regulation of GABAA receptors in ASD. At the cellular level, treatment with proteasomal inhibitor, MG132, or lactacystin significantly increased GABAAα1 protein levels and Lys48-linked polyubiquitination of GABAAα1, but reduced proteasome activity in mouse primary cortical neurons (DIV 14 from E16 embryos). Moreover, treatment with betulinic acid, a proteasome activator significantly decreased GABAAα1 protein levels in cortical neurons indicating the role of polyubiquitination of GABAAα1 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 GABAAα1 in the ER. The siRNA knockdown of SYVN1 significantly increased GABAAα1 protein levels in cortical neurons. Moreover, reduced association between SYVN1 and GABAAα1 was found in the middle frontal gyrus of ASD subjects.
SYVN1 plays a critical role as an E3 ligase in the ubiquitin proteasome system (UPS)-mediated GABAAα1 degradation. Thus, inhibition of the ubiquitin-proteasome-mediated GABAAα1 degradation may be an important mechanism for preventing GABAAα1 turnover to maintain GABAAα1 levels and GABA signaling in ASD.
Additional file 1: Table S1: Postmortem brain tissue information. (DOCX 19 KB)13229_2014_136_MOESM1_ESM.docx
Additional file 2: Table S2: Descriptive statistics of study variables. (DOCX 16 KB)13229_2014_136_MOESM2_ESM.docx
Additional file 3: Table S3: Ubiquitination array data. (DOCX 27 KB)13229_2014_136_MOESM3_ESM.docx
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- Ubiquitin-proteasome dependent degradation of GABAAα1 in autism spectrum disorder
Chirayu D Pandya
Anthony O Ahmed
- BioMed Central
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