Caspr Interaction With Amyloid Precursor Protein Reduces Amyloid-β Generation In Vitro

Objective:To investigate the function of Caspr on the generation of Aβ in vitro, and therefore toexplore a novel function of Caspr in AD pathology.Methods:Immunofluorescence staining and Western-blot analysis were used to detect theexpression pattern of Caspr in the coronal cerebral cortex of APP/PS1mice. To investigatethe function of Caspr on expression of APP, we transfected PCMV-Caspr-myc intoHEK-APP cells and CHO cells. Cell lysates were subjected to Western-blot analysis andQuantitative real-time PCR（qPCR） to measure the protein and mRNA levels of APP.Colocalization between APP and Caspr were examined in Caspr-transfected HEK-APPcells and the coronal cortical sections of C57BL/6J mice by double immunofluoresencestaining. The association of endogenous Caspr and APP was examined in mouse brainhomogenates by co-immunoprecipitation (Co-IP). The function of Caspr on Aβ generationwas investigated on Caspr-transfected HEK-APP cells by ELISA. Cells viability wasdetermined by the MTT reduction assay.Results:Immunfluorescence staining showed that Caspr-immunoreactivity distributed in aclustering pattern around Aβ plaques in the cerebral cortex of7-month-old APP/PS1mice.Increased levels of Caspr were detected by immunoblotting analysis in the cerebral cortexof APP/PS1mice compared with wild-type mice. APP and CTFs levels were higher inAPP/PS1mice. Western blotting and Quantitative real-time PCR（qPCR）analysis showedthat Caspr overexpression reduced protein level of APP, while it had no effect on level ofAPP mRNA. Double immunofluorescence staining showed that Caspr colocalized withAPP in either Caspr-transfected HEK-APP cells or in the cerebral cortex of C57BL/6Jmice. Co-IP showed that Caspr associates with APP. ELISA showed that Caspr overexpression inhibited generation of Aβ40and Aβ42, while it did not change the ratio ofAβ42/Aβ40. MTT analysis indicated that overexpression of Caspr does not change cellviability.Conclusion:Caspr colocalizes and interacts with APP. Caspr decreases level of APP through apost-transcriptional mechanism, and therefore reduces generation of Aβ that formsβ-amyloid plaques. Thus, Caspr may play roles in AD pathology.