Functional analysis of juxta- and intra-membrane domains of murine APP by genome editing in Neuro2a cells

Amyloid-β precursor protein (APP) correlates with the pathogenesis of certain brain diseases, such as Alzheimer disease (AD). APP is cleaved by several enzymes to produce APP metabolites, including the amyloid beta peptide (Aβ), which accumulates in the brain of AD patients. However, the exact functions of APP metabolites remain elusive. In this study, using genome editing technology, we mutated juxta- and intra-membrane domains of murine APP in the mouse neuroblastoma cell line, Neuro2a. We identified several clones that expressed characteristic patterns of APP metabolites. Mutations in juxta- (deletion 673A), and intra-membrane (deletion 705-6LM) domains of APP, decreased overall levels of APP metabolites or decreased the level of α-secretase-cleaved carboxy-terminal fragment (αCTF), respectively. APP is known to influence neuronal differentiation; therefore, we used theses clones to dissect the function of APP metabolites during neuronal differentiation. One clone (CA), which expressed reduced levels of both FL-APP and αCTF, showed increased expression of the neuronal marker, β3-tubulin, and enhanced retinoic acid (RA)-induced neurite outgrowth. In contrast, a clone that expressed FL-APP, but was devoid of αCTF (CE), showed comparable expression of β3-tubulin and neurite outgrowth compared with normal Neuro2a cells. These data indicate that FL-APP is a suppressor of neurite outgrowth. Our data suggest a novel regulatory function of juxta- and intra-membrane domains on the metabolism and function of APP.