Late‐onset Alzheimer's disease‐associated gene, CPAMD8, increases B‐CTF and affects APP processing through regulation of the autophagy‐lysosome pathway

Abstract

AbstractBackgroundC3‐ and PZP‐like alpha‐2‐macroglobulin domain containing 8 (CPAMD8) has been shown to be associated with familial late‐onset Alzheimer disease (LOAD) in recent studies. However, the role of CPAMD8 in AD‐related phenotypes is currently unknown.MethodsTo determine whether CPAMD8 affects known AD‐related phenotypes, we used RNA‐sequencing data from AD patients and healthy controls from WUTSL and publicly available from the Mount Sinai Brain Bank (195 individuals). Differential expression (DE) analysis was performed using DESeq2 software. We investigated the association of CPAMD8 transcript levels with AD case–control status and CDR scores. CPAMD protein levels were quantified from AD cases and controls from the ADRC‐WUSTL cohorts (N=105). Mouse neuroblastoma (N2A) cells stably expressing wild‐type human APP695 (N2A‐APP) were transiently transfected with wild‐type (NM_015692) human CPAMD8 (RC213448, Origene). CPAMD8 mRNA and protein levels were confirmed by pPCR and western blot. The levels of Aβ40 and Aβ42 were measured in cell culture media by sandwich ELISA (Invitrogen).ResultsCPAMD8 is differential expressed in cases and controls and is associated with CDR score (p = 8.45×10‐3). Co‐expression gene network analysis place CPAMD8 in the amyloid pathway (p = 8.5×10‐5). A significant reduction in CPAMD8 protein levels was found in postmortem brain tissue from AD patients compared to controls (p = 2.1×10‐3). N2A‐APP cells expressing CPAMD8 exhibited a dose‐dependent reduction in extracellular Aβ40, Aβ42, sAPPα and sAPPβ levels, a decrease in full‐length APP, and a significant increase in intracellular levels of α and β APP C‐terminal fragments (CTF). No significant differences were detected in α‐secretase or γ‐secretase protein levels. However, inhibition of γ‐secretase induced much higher levels of LC3‐II, SQSTM1/p62 and APP CTFs, and lower levels of Aβ40 and Aβ42 in N2A‐APP cells expressing CPAMD8. A direct interaction between CPAMD8 and APP was confirmed by co‐immunoprecipitation. N2A‐APP cells, but not HEK293 cells, expressing CPAMD8 displayed a significant increase in LC3‐II and SQSTM1/p62. Pharmacological modulation of autophagy demonstrated an increased autophagic flux in N2A‐APP cells expressing CPAMD8.ConclusionsOur data provides evidence that the AD‐associated gene, CPAMD8, alters APP processing through the regulation of the autophagy‐lysosome pathway.