Rab5-stimulated up-regulation of the endocytic pathway increases intracellular beta-cleaved amyloid precursor protein carboxyl-terminal fragment levels and Abeta production.

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.

Alzheimer's disease-related overexpression of the cation-dependent mannose 6-phosphate receptor increases Abeta secretion: role for altered lysosomal hydrolase distribution in beta-amyloidogenesis.

Prominent endosomal and lysosomal changes are an invariant feature of neurons in sporadic Alzheimer's disease (AD). These changes include increased levels of lysosomal hydrolases in early endosomes and increased expression of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is partially localized to early endosomes. To determine whether AD-associated redistribution of lysosomal hydrolases resulting from changes in CD-MPR expression affects amyloid precursor protein (APP) processing, we stably transfected APP-overexpressing murine L cells with human CD-MPR. As controls for these cells, we also expressed CD-MPR trafficking mutants that either localize to the plasma membrane (CD-MPRpm) or to early endosomes (CD-MPRendo). Expression of CD-MPR resulted in a partial redistribution of a representative lysosomal hydrolase, cathepsin D, to early endosomal compartments. Turnover of APP and secretion of sAPPalpha and sAPPbeta were not altered by overexpression of any of the CD-MPR constructs. However, secretion of both human Abeta40 and Abeta42 into the growth media nearly tripled in CD-MPR- and CD-MPRendo-expressing cells when compared with parental or CD-MPRpm-expressing cells. Comparable increases were confirmed for endogenous mouse Abeta40 in L cells expressing these CD-MPR constructs but not overexpressing human APP. These data suggest that redistribution of lysosomal hydrolases to early endocytic compartments mediated by increased expression of the CD-MPR may represent a potentially pathogenic mechanism for accelerating Abeta generation in sporadic AD, where the mechanism of amyloidogenesis is unknown.