Abstract
A hallmark of Alzheimer's disease (AD) pathology is the appearance of senile plaques, which are composed of β-amyloid (Aβ) peptides. Aβ is produced by sequential cleavages of amyloid precursor protein (APP) by β- and γ-secretases. These cleavages take place in endosomes during intracellular trafficking of APP through the endocytic and recycling pathways. Genome-wide association studies have identified several risk factors for late-onset AD, one of which is CD2-associated protein (CD2AP), an adaptor molecule that regulates membrane trafficking. Although CD2AP's involvement in APP trafficking has recently been reported, how APP trafficking is regulated remains unclear. We sought to address this question by investigating the effect of CD2AP overexpression or knockdown on the intracellular APP distribution and degradation of APP in cultured COS-7 and HEK293 cells. We found that overexpression of CD2AP increases the localization of APP to Rab7-positive late endosomes, and decreases its localization to Rab5-positive early endosomes. CD2AP overexpression accelerated the onset of APP degradation without affecting its degradation rate. Furthermore, nutrient starvation increased the localization of APP to Rab7-positive late endosomes, and CD2AP overexpression stimulated starvation-induced lysosomal APP degradation. Moreover, the effect of CD2AP on the degradation of APP was confirmed by CD2AP overexpression and knockdown in primary cortical neurons from mice. We conclude that CD2AP accelerates the transfer of APP from early to late endosomes. This transfer in localization stimulates APP degradation by reducing the amount of time before degradation initiation. Taken together, these results may explain why impaired CD2AP function is a risk factor for AD.
Highlights
A hallmark of Alzheimer’s disease (AD) pathology is the appearance of senile plaques, which are composed of -amyloid (A) peptides
We found that overexpression of CD2-associated protein (CD2AP) increases the localization of amyloid precursor protein (APP) to Rab7-positive late endosomes, and decreases its localization to Rab5-positive early endosomes
Because endogenous APP was mainly found at the perinuclear region in either Neuro-2a cells or primary neurons, we focused on APP in the cytoplasmic endosomes hereafter
Summary
CD2AP is localized to endosomal vesicles and regulates membrane trafficking [21, 30]. To control for possible effects of protein overexpression, we compared the expression levels of APP in the presence and absence of EGFP in COS-7 cells (Fig. S6, a and b) and cultured primary neurons (Fig. S6, c and d). We examined the effect of CD2AP knockdown on APP protein expression Both shRNAs, shCD2AP #1 and #2, decreased CD2AP expression as shown by immunoblotting (Fig. 5, e and g) and immunofluorescent staining (Fig. S5, b and c), and these shRNAs increased the expression level of APP by ϳ1.25- and 1.32-fold, respectively, in Neuro-2a cells (Fig. 5f). The rates of APP decrease were similar thereafter, in the presence or absence of CD2AP (Fig. 6c) We repeated this CHX-chase experiment and overexpressed EGFP as a control. The expression levels of APP were not affected by EGFP (Fig. S7), suggesting that CD2AP promotes the commitment of APP to a degradation pathway
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