The Role of Transglutaminase 2 in GABAA Receptor Regulation in Autism
Objectives: Our long term goal is to understand the regulatory mechanism of GABAAR signaling in autism as a prerequisite to the development of therapeutic protocols that can be used to attenuate the disease process. The objective of this proposal is to determine the role of transglutaminase 2 (TG2) in the regulation of GABAAR α1 signaling. The specific hypothesis behind the study is that increased TG2-dependent degradation of GABAAR α1 inhibits GABAergic neurotransmission with autism-like phenotype in mice.
Methods: Human postmortem prefrontal cortex samples from ASD/Autism and control subjects were obtained from NICHD. Animal experiments were performed in C57BL/6 mice overexpressing Transglutaminase 2 in neurons and wild-type. In vitro studies were performed in isolated cortical neurons as well as HEK293 cell line. Protein levels were measured by western blotting, mRNA levels were measured by RT-PCR, TG2 activity was measured by transglutaminase activity assay kit from Sigma. Behavioral measures included open field, tail suspension, forced swim test, social interaction test, and marble bury test.
Results: We found that mice with neuronal TG2 overexpression (TG2+/+) show autism-like phenotype with reduced GABAAR α1 protein levels in the frontal cortex. Increases in mRNA, protein, and activity of TG2 were found in PFC of subjects with autism. Moreover, we observed a significant reduction in GABAAR α1 protein levels, but not mRNA in PFC of subjects with autism suggesting GABAAR α1 degradation at the posttranscriptional level. Interestingly, we found significant increases in calnexin in PFC of subjects with autism. Additional studies indicated a role of calnexin in GABAAR regulation in our model.
Conclusions: Our data suggests that calnexin functions downstream of TG2 and is involved in GABAAR α1 regulation in autism. We further demonstrate that TG2-dependent degradation of GABAAR induces autism-like phenotype in mice and that calnexin mediates TG2-induced GABAAR endocytosis. Given the important role of GABAAR in neuroplasticity, these findings on the regulatory mechanisms of GABAAR may provide avenues to develop newer therapeutics for autism and related neurodevelopmental disorders.