Degradation of CNTNAP2 is mediated by both macroautophagy‐lysosome pathway and ubiquitin‐proteasome pathway

Abstract

AbstractBackgroundRecent studies suggested Contactin‐associated protein‐like 2 (CNTNAP2) as a novel risk gene for Alzheimer’s disease (AD). Reduced expression levels of CNTNAP2 were found in the brains of AD patients. However, the mechanisms underlying CNTNAP2 protein degradation remain elusive.MethodHEK and N2a cells transfected with human CNTNAP2 plasmid were treated with cycloheximide, lysosome inhibitors, and proteasome inhibitors. Immunoprecipitation was performed to study the interaction between CNTNAP2 and ubiquitin. Immunofluorescent staining was performed to investigate colocalization.ResultHuman CNTNAP2 protein has a half‐life of approximately 4 hours. CNTNAP2 protein has a glycosylated mature form on the cell membrane and an intracellular immature form. Mature CNTNAP2 was increased by lysosome inhibitors in a dose‐ and time‐dependent manner, while immature CNTNAP2 was increased by proteasome inhibitors dose‐ and time‐dependently. In addition, CNTNAP2 was co‐immunoprecipitated with ubiquitin and was increased by macroautophagy inhibitor 3‐MA. Immunofluorescent staining further showed the co‐localization of CNTNAP2 with ubiquitin and lysosome marker LAMP1.ConclusionHuman CNTNAP2 is degraded via both lysosome and proteasome pathways. Dysregulation of lysosome and ubiquitin‐proteasome pathways have been implicated in AD. Therefore, the present degradation study of a risk gene may deepen the current understanding of AD pathogenesis.