Intracellular Aβ1-42 Aggregates Stimulate the Accumulation of Stable, Insoluble Amyloidogenic Fragments of the Amyloid Precursor Protein in Transfected Cells

Abstract

We have analyzed the effect of internalized amyloid beta-protein (A beta) 1-42 aggregates on the metabolism of the amyloid precursor protein (APP) in stably transfected 293 cells. The amount of potentially amyloidogenic fragments of APP immunoprecipitated by anti-carboxyl-terminal APP and anti-A beta antibodies is dramatically enhanced by the treatment of the cells with A beta 1-42, which is resistant to degradation, but not A beta 1-28, which does not accumulate in cells. This accumulation of amyloidogenic carboxyl-terminal fragments is specific, since there is relatively little effect of A beta 1-42 on the amount of the nonamyloidogenic alpha-secretase carboxyl-terminal fragment. The amyloidogenic fragments accumulate in the same nonionic detergent-insoluble fraction of the cell that contains the internalized A beta 1-42. Western analysis indicates that a subset of the amyloidogenic fragments react with antibodies that recognize a conformation of A beta that is specifically associated with aggregated forms of A beta, suggesting that the adoption of this aggregation-related conformation may be an early event which precedes the final processing that produces A beta. Pulse-chase analysis of the [35S]Met-labeled 16-kDa amyloidogenic fragment indicates that it is relatively stable in A beta 1-42-treated cells, with a half-life of approximately 50 h. This fragment is degraded with a half-life of 30 min in control cells treated with A beta 1-28. In contrast, the turnover of the nonamyloidogenic alpha-secretase product is not significantly altered by the presence of A beta 1-42. The continuous uptake of A beta 1-42 from the medium is not required for the stimulation of amyloidogenic fragment accumulation, suggesting that the presence of intracellular A beta 1-42 aggregates establishes a new pathway for APP catabolism in cells which leads to the long term stability of the fragments. If these amyloidogenic fragments of APP ultimately give rise to A beta, then the production of A beta may be an autocatalytic, "runaway" process in cells containing A beta 1-42 nuclei. It is conceivable that the accumulation of insoluble APP and amyloidogenic fragments of APP in response to A beta 1-42 aggregates may mimic the pathophysiology of dystrophic neurites, where the accumulation of intracellular APP and APP fragments has been documented by immunohistochemistry.

Aβ(1–40) Prevents Heparanase-catalyzed Degradation of Heparan Sulfate Glycosaminoglycans and Proteoglycans in Vitro

“…This hypothesis is supported by experiments that suggest that A␤ peptides stimulate the accumulation of amyloidogenic fragments of ␤APP inside cells (9). When A␤(1-42) is added to the media of cultured cells, it is internalized in a concentrationdependent manner (9,27) and accumulates in an aggregated, protease-resistant form within late endosomes or secondary lysosomes (27). Even at low concentrations of A␤(1-42) in the medium (100 g/ml), more than 10 pg of peptide was internalized per cell (27), suggesting that the concentration of A␤(1-42) inside cells is more than sufficient to affect HSPG catabolism by heparanases.…”

“…This will result in an increased level of HSPGs, which could stabilize the aggregates and prevent them from being degraded by proteases (1,14). This hypothesis is supported by experiments that suggest that A␤ peptides stimulate the accumulation of amyloidogenic fragments of ␤APP inside cells (9). When A␤(1-42) is added to the media of cultured cells, it is internalized in a concentrationdependent manner (9,27) and accumulates in an aggregated, protease-resistant form within late endosomes or secondary lysosomes (27).…”

“…Even at low concentrations of A␤(1-42) in the medium (100 g/ml), more than 10 pg of peptide was internalized per cell (27), suggesting that the concentration of A␤(1-42) inside cells is more than sufficient to affect HSPG catabolism by heparanases. Interestingly, if the cells were transfected with ␤APP to increase the levels of expressed precursor, the exogenously added A␤(1-42) stimulated the production of A␤-containing fragments from the precursor protein, which also accumulated inside the cell (9). It was postulated that the amyloidogenic fragments produced from ␤APP by normal processes interacted with the A␤(1-42) aggregates, and this association allowed them to escape the normal degradation pathways (9).…”

“…Production of A␤ from the amyloid precursor protein is thought to occur primarily in the endosomal-lysosomal pathway (7)(8)(9). HSPGs are initially degraded to short glycosaminoglycan chains in the same intracellular location (10 -12), so it is possible that this is where the A␤-HSPG interaction necessary for plaque formation occurs.…”

Amyloid beta peptide directly inhibits PKC activation

“…These results indicate that treatment of Ah inhibited activation of PKCa and PKCq in the cell. We tested whether extracellular Ah entered the cell, as was previously reported (Bahr et al, 1998;Bi et al, 2002;Ida et al, 1996;Knauer et al, 1992;Nagele et al, 2002;Yang et al, 1995;Yazawa et al, 2001). Immunocytochemical and Western blot analyses confirmed localization of Ah 1 -42 peptides inside the cells that were treated with exogenous Ah 1 -42, but not in cells that were not treated with Ah 1 -42.…”