The Regulatory Mechanism And Physiological Function Of Proteolytic Cleavage Of CNTNAP2

Contactin-associated protein-like 2（CNTNAP2）gene is a widely validated autism-susceptibility gene,Cntnap2-deficient mice(Cntnap2^-/-)also show core autism-relevant behaviors.Cntnap2^-/-is often used in the development of new treatment methods and drugs for autism.However,the molecular mechanisms of how CNTNAP2deletions/mutations lead to autism are still unclear.Numerous typeⅠsingle transmembrane proteins in the nervous system undergo proteolytic cleavage and participate in a variety of neuropathological processes.CNTNAP2 is also a type I single transmembrane protein,and sequence analysis showed that it contains a potentialγ-secretase recognition site.In this study,the regulatory mechanism and physiological function of CNTNAP2 proteolytic cleavage were studied at the cellular and animal levels.Objective:This study focuses on the regulatory mechanism and physiological function of CNTNAP2 proteolytic cleavage,and proposes a new mechanism of CNTNAP2 regulating autism-related behaviors.It not only contributes to explain the mechanism of CNTNAP2 mutation leading to autism,but also opens up new avenues for the functional research of similar molecules.Methods:We demonstrated that CNTNAP2 undergoes two steps of proteolytic cleavage by sequence comparison and biochemical analysis,and analyzed the role of the proteolytic product in autism-related behaviors in Cntnap2^-/-mice using stereotaxic injection of recombination adeno-associated virus and a series of behavioral analysis.Then,the potential target gene regulated by the proteolytic products of CNTNAP2 were screened by RNA-seq,and their regulatory relationships were certified by real-time fluorescence quantitative PCR and western blotting.Further,we analyzed the autism-related behavioral phenotypes in target gene knockout mice and the role of target gene in autism-related behaviors of Cntnap2^-/-mice through stereotaxic injections and behavioral analysis.Subsequently,we demonstrated the involvement of CNTNAP2 interacting protein in the regulation of the target gene by immunoprecipitation,luciferase reporter system and chromatin immunoprecipitation.Finally,we used nuclear/cytoplasmic protein extraction and immunofluorescence experiments to clarify the molecular mechanism of the proteolytic product regulating target gene expression,and elucidated the specific signaling pathway in the proteolytic product of CNTNAP2 regulating target genes,which involved in autism-related behavior regulation.Results:1.CNTNAP2 underwent two steps of proteolytic cleavage and was first cleaved in extracellular juxtamembrane domain releasing a soluble extracellular fragment and a membrane-tethered 20KDa C-terminal fragment（CTF,～20KDa）.The CTF was further processed byγ-secretase to generate the CNTNAP2 intracellular C-terminal domain（CICD）;2.Overexpression of CICD by recombination adeno-associated virus（AAV）injection in the m PFC of Cntnap2^-/-mice rescued their autism-related deficiency,including impaired social interaction and repetitive behaviors,and improved them to the comparable levels of wild-type mice;3.Necdin,a Prader-Willi syndrome（PWS）related gene,is the most notably changed gene in Cntnap2^-/-mice（significantly down-regulated）.CNTNAP2 or CICD can positively regulate Necdin expression,and overexpression of CICD by AAV injection in the m PFC of Cntnap2^-/-mice normalized the Necdin protein to the level of wild-type mice;4.Necdin^-p/+mmice exhibited social deficiency without repetitive behaviors,and overexpression of Necdin by AAV injection in the m PFC of Cntnap2^-/-mice rescued the social deficit,but had no effect on repetitive behavior;5.CICD interacted with CASK through C-terminus PDZ binding motif and promoted the nuclear distribution of CASK to regulate gene expression,such as Necdin.This process is associated with autism-related behaviors in Cntnap2^-/-mice.Conclusion:Our results strongly demonstrate that CNTNAP2underwent two steps of proteolytic cleavage process generate the CNTNAP2 intracellular C-terminal domain（CICD）.CICD promoted the nuclear distribution of CASK and regulated the transcription of downstream genes,such as Necdin.This process was strongly associated with autism-related behaviors in Cntnap2^-/-mice,and Necdin^-p/+mmice exhibited social deficiency.Overexpression of CICD or Necdin by AAV injection in the m PFC of Cntnap2^-/-mice completely or partially rescued the autism-related behaviors deficit.Our results revealed a new pathogenesis of autism,and highlighted the role of the CNTNAP2-CASK-Necdin signaling pathway in autism etiology.