Amyloid Precursor Protein Modulates Itself Trafficking And Processing By Distinct N-terminal Domains

Alzheimer's disease(AD)is a devastating neurodegenerative disease affecting millions worldwide.Accumulating biochemical and genetic evidence establishes a pivotal role of the amyloid precursor protein(APP)in AD pathogenesis.Biochemically,accumulation of insoluble aggregates of the beta-amyloid(AP)peptides that are proteolytic cleavage products of APP forms the defining pathological hallmark of AD;genetically,both point mutations and duplications of wild-type APP which often show increased production of A? are linked to a subset of early onset of familial AD(FAD)and cerebral amyloid angiopathy.APP is a type I transmembrane protein,which resembles a cell surface receptor,and consists of a large ectodomain and a short cytoplasmic tail.?-and y-secretase liberate the neurotoxic A? by sequential cleavage of APP,whereas a-secretase prevents its generation by cleaving within the middle of A?.Furthermore,substantial evidence indicates that alterations in APP trafficking within the secretory and endocytic pathways directly impact the interaction of APP with its processing secretases and subsequent A?production.Although AP generation from APP extensively investigated,the N-terminal domain of APP contributions to the production of AP remains elusive.Utilizing the systematic deletion strategies,we have mapped and identified four novel candidates of APP motif,which could modulate APP itself processing,and therefore regulate the generation of A?.1)The deletion of the ACIDIC domain promotes a-secretase processing of APP whereas weakens its proteolysis by ?-secretase BACE1,and produces less extracellular A? and intracellular A?(iA?);2)Deleting the CAPPD domain promotes APP processing by BACE1 and a-secretase,which concomitantly increases A? and iA? generation.It seems that lack of the CAPPD domain enhances APP binding with BACE1.On the contrary,deleting the ?3 helix of the CAPPD domain greatly alleviates APP proteolysis by BACE1 and ?-secretase;3)After striking out the RC domain,APP boosts its interaction with BACE1 and further processing by BACE1 and a-secretase.Devoid of the RC domain possess two opposite effects for APP proteolysis,i.e.accumulation of A? while reduction of iA?;and,4)Removal of APP JMD domain restrains APP processing by BACE1,however improves its proteolysis by BACE1-independent ?-secretase.In addition,BACE1-independent P-secretase mediated cleavage sites of APP lie beyond the N-termini of A?,possibily localizing within APP RC domain,hence producing the longer forms of A? with extended N-termini.There are approximately three BACE1-independent ?-secretase cleavage sites,which could be modulated by modifying the APP JMD domain.Next,we sought to delineate the trafficking itinerary of the APP N-terminal deletion chimeras.We constructed a series of APP trafficking mutuants that limit APP processing to specific subcellular compartments.Our data reveals that the incomplete secretory and endocytic pathways impairs the production of both extracellular A? and intracellular iA?;moreover,iA? generation via an endocytosis-independent pathway.Hence,we employed iA? as an indicator of APP localization.It is suggested that the deletion of the ACIDIC or RC domain would retard APP exit of the TGN,while deleting the CAPPD domain would promote APP locating to the endosomes.In addition,we swapped the whole extracellular domain of APP with red fluorescent protein mCherry,and tagged the APP cytoplasmic tail with enhanced green fluorescent protein EGFP,thus generating the double fluorescent protein marked APP chimera mC99G.mC99G mimics the processing of APP by ?-secretase,?-secretase and ?-secretase.mC99G,C99G and APP695G share similiar subcellular distributions.Upon ?-secretase inhibitor DAPT treatment,although the mCherry signal of mC99G exhibits the same as APP695G,the EGFP signal of mC99G behaves much more like C99G,which accumulates enormously at the plasma membrane.Cell surface biotinylation indicates the CTFa-EGFP could be recruited at the cell surface,which suggests that the a-secretase mediated transformation of CTFP-EGFP to CTFa-EGFP ouccurs at the plasma membrane.Given the comparable level of metabolites of mC99G and C99G,it is suggested that the extracellular domain of APP maybe critical for itself trafficking and processing.Besides,mC99G could be utilized as a useful tool for APP processing research.As an application,we found that mC99G and its derivates secretes into extracellular milieu via exosomes.