Ubiquitin-proteasome dependent degradation of GABA_Aα1 in autism spectrum disorder

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