Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer beta-amyloid peptides

The excessive generation and accumulation of 40- and 42-aa beta-amyloid pep tides (A beta(40)/A beta(42)) in selectively vulnerable brain regions is a major neuropathological feature of Alzheimer's disease. A beta, derived by proteolytic cleavage from the beta-amyloid precursor protein (beta APP), is normally secreted. However, recent evidence suggests that significant le l evels of A beta also may remain inside cells, Here, we have investigated th e subcellular compartments within which distinct amyloid species are genera ted and the compartments from which they are secreted, Three experimental a pproaches were used: (i) immunofluorescence performed in intact cortical ne urons; (ii) sucrose gradient fractionation performed with mouse neuroblasto ma cells stably expressing wild-type beta APP(695) (N2a(695)); and (iii) ce ll-free reconstitution of A beta generation and trafficking from N2a695 cel ls. These studies demonstrate that: (i) A beta(40) (A beta(1-40) plus A bet a(x-40), where x is an NH2-terminal truncation) is generated exclusively wi thin the trans-Golgi Network (TGN) and packaged into post-TGN secretory ves icles; (ii) A beta(x-42) is made and retained within the endoplasmic reticu lum in an insoluble state; (iii) A beta(42) (AP(1-42) plus A beta(x-42)) is made in the TGN and packaged into secretory vesicles; and (iv) the amyloid peptides formed in the TGN consist of two pools (a soluble population extr actable with detergents and a detergent-insoluble form), The identification of the organelles in which distinct forms of A beta are generated and from which they are secreted should facilitate the identification of the proteo lytic enzymes responsible for their formation.