Abstract
APP processing and amyloid-β production play a central role in Alzheimer disease pathogenesis. APP has been considered a ubiquitously expressed protein. In addition to amyloid-β, α- or β-secretase-dependent cleavage of APP also generates soluble secreted APP (APPsα or APPsβ, respectively). Interestingly, APPsβ has been shown to be subject to further cleavage to create an N-APP fragment that binds to the DR6 death receptor and mediates axon pruning and degeneration under trophic factor withdrawal conditions. By performing APP immunocytochemical staining, we found that, unexpectedly, many antibodies yielded nonspecific staining in APP-null samples. Screening of a series of antibodies allowed us to identify a rabbit monoclonal antibody Y188 that is highly specific for APP and prompted us to re-examine the expression, localization, and stability of endogenous APP and APPsβ in wild-type and in APPsβ knock-in mice, respectively. In contrast to earlier studies, we found that APP is specifically expressed in neurons and that its expression cannot be detected in major types of glial cells under basal or neuroinflammatory conditions. Both APPsα and APPsβ are highly stable in the central nervous system (CNS) and do not undergo further cleavage with or without trophic factor support. Our results clarify several key questions with regard to the fundamental properties of APP and offer critical cellular insights into the pathophysiology of APP.
Highlights
Endogenous APP expression in central nervous system (CNS) has not been rigorously examined
Identification of a Specific Antibody for APP Immunostaining— A variety of APP antibodies are available either commercially or from academic research laboratories. These antibodies have been extensively used for biochemical and immunohistochemical assays, we found that when performing immunocytochemical staining and comparing wild-type with APP-null neuronal cultures, most of the antibody stainings were indistinguishable between the two cultures, suggesting nonspecific staining for endogenous APP
We tested a selection of APP antibodies from different origins and against different epitopes on fixed mouse primary hippocampal cultures (Table 1 and Fig. 1A) and identified a rabbit monoclonal antibody (Y188), raised against the C-terminal sequences of APP, that displayed strong staining in wild-type neurons but almost no Summary of APP antibodies and their specificity
Summary
Results: We characterized the expression, localization, and stability of endogenous APP and APPs using a highly specific antibody. Screening of a series of antibodies allowed us to identify a rabbit monoclonal antibody Y188 that is highly specific for APP and prompted us to re-examine the expression, localization, and stability of endogenous APP and APPs in wild-type and in APPs knock-in mice, respectively. In contrast to earlier studies, we found that APP is expressed in neurons and that its expression cannot be detected in major types of glial cells under basal or neuroinflammatory conditions. Both APPs␣ and APPs are highly stable in the central nervous system (CNS) and do not undergo further cleavage with or without trophic factor support.
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