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Zhang, Qing

University of British Columbia

Autism spectrum disorders (ASD) are a group of common neurodevelopmental disorders four times as prevalent in males as in females. Many genetic and environmental risk factors contribute to ASD, while the mechanisms underlying ASD pathogenesis remain elusive. Contactin-associated protein-like 2 (CNTNAP2) has been widely acknowledged as a risk gene for ASD. CNTNAP2 gene variants were frequently identified in ASD patients, and Cntnap2 knockout mice exhibited ASD-associated phenotypes. Dysregulations of CNTNAP2 could contribute to ASD pathogenesis, while the regulations of CNTNAP2 expression and modification remain largely unknown. In this study, we systematically investigated the transcriptional regulation, degradation, and proteolytic processing of human CNTNAP2. We cloned the human CNTNAP2 promoter and analyzed its transcriptional activities. We discovered that the RE1-Silencing Transcription Factor (REST) controls the neuronal-specific expression of CNTNAP2. Additionally, hypoxia, a risk factor for ASD, upregulates CNTNAP2 expression via interacting with the CNTNAP2 promoter. Next, we investigated the degradation pathway of CNTNAP2 and its C-terminal fragments (CTF). Two cell lines transfected with CNTNAP2 were treated with inhibitors for protein synthesis or degradation in dose- and time-dependent experiments. Results show that CNTNAP2 protein has a half-life of approximately 3-4 hours. CNTNAP2 and CTF are degraded via the ubiquitin-proteasome system and the macroautophagy-lysosomal pathway. Specifically, the lysosome pathway is a common mechanism of CNTNAP2 degradation in neuronal and non-neuronal cells. Furthermore, we demonstrated that CNTNAP2 undergoes sequential cleavages by furin, Alzheimer’s disease-associated α-secretase, and γ-secretase. Two α-secretase cleavage sites were identified at C-terminal L96 and I79. Moreover, the main α-secretase cleavage product C79 is further cleaved by γ-secretase to generate the CNTNAP2 intracellular domain (CICD). Finally, pathogenic CNTNAP2 mutations in ASD patients impair the α-cleavage of CNTNAP2 to generate C79, which may underlie the pathogenesis of ASD. In summary, this dissertation is the first study to reveal the fundamental regulations of CNTNAP2 expression and modifications. Dysregulations in transcription, degradation, and proteolysis of CNTNAP2 may underlie ASD pathogenesis related to CNTNAP2 disruptions.

Text

2024-09-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/82773

Neuroscience

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/