Abstract
We previously identified abnormalities of the endocytic pathway in neurons as the earliest known pathology in sporadic Alzheimer's disease (AD) and Down's syndrome brain. In this study, we modeled aspects of these AD-related endocytic changes in murine L cells by overexpressing Rab5, a positive regulator of endocytosis. Rab5-transfected cells exhibited abnormally large endosomes immunoreactive for Rab5 and early endosomal antigen 1, resembling the endosome morphology seen in affected neurons from AD brain. The levels of both Abeta40 and Abeta42 in conditioned medium were increased more than 2.5-fold following Rab5 overexpression. In Rab5 overexpressing cells, the levels of beta-cleaved amyloid precursor protein (APP) carboxyl-terminal fragments (betaCTF), the rate-limiting proteolytic intermediate in Abeta generation, were increased up to 2-fold relative to APP holoprotein levels. An increase in beta-cleaved soluble APP relative to alpha-cleaved soluble APP was also observed following Rab5 overexpression. BetaCTFs were co-localized by immunolabeling to vesicular compartments, including the early endosome and the trans-Golgi network. These results demonstrate a relationship between endosomal pathway activity, betaCTF generation, and Abeta production. Our findings in this model system suggest that the endosomal pathology seen at the earliest stage of sporadic AD may contribute to APP proteolysis along a beta-amyloidogenic pathway.
Highlights
In the brain parenchyma, the deposition of the small peptide A, a proteolyic fragment of the amyloid precursor protein (APP),1 is an invariant feature of Alzheimer’s disease (AD) [1]
In this study we have modeled a second aspect of AD-related endocytic pathway abnormalities, such as the apparent increase in endocytosis and early endosomal fusion seen in sporadic forms of the disease, by overexpressing Rab5 in cells
Along with the effector proteins rabaptin 5 [35,36,37] and early endosomal antigen 1 (EEA1) (38 – 40), Rab5 plays a key role in modulating early endosomal fusion and the membrane docking events involved in endocytosis and recycling [34, 41]
Summary
Cell Lines and Transfections—Murine fibroblast-like L cells stably transfected with human APP695 have been described (L/APP cells) [31, 42]. The cells grown on glass coverslips were washed and incubated for 30 min at 37 °C in phosphate-buffered saline containing 0.1% bovine serum albumin and 20 mM HEPES. Following the collection of conditioned medium, Triton X-100 lysates (1% Triton X-100, 140 mM NaC1, 25 mM Tris, pH 7.4, 0.5 mM EDTA, 1 mM EGTA, and protease inhibitors) were prepared and frozen for later determination of cell-associated APP metabolite levels. Cell-associated CTF levels were determined using a sandwich ELISA employing C1/6.1 as the capture antibody and JRF/〈N/25 as the detection antibody. Using an aliquot of the Triton X-100 cell lysate, an additional ELISA developed to detect APP holoprotein as well as the cell-associated CTFs (APP/total CTF ELISA) was run This ELISA uses C1/6.1 as the capture antibody, as does the CTF ELISA, and employs a second carboxylterminal APP antibody for detection (C2/7.1; see Table I). Throughout, ELISA measurements were determined from standard curves run on the same plate and represent the means of two or more wells
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