Previous studies have implicated the failure to degrade aggregated Abeta1-42 in late endosomes or secondary lysosomes as a mechanism for the accumulation of beta-amyloid in Alzheimer's disease. We examined the consequences of intracellular accumulation of Abeta1-42 on the integrity of the endosomal/lysosomal compartment by monitoring Lucifer Yellow fluorescence and the release of lysosomal hydrolases into the soluble, cytosolic fraction. In control cells, the Lucifer Yellow fluorescence is observed as punctate staining in a perinuclear distribution with no apparent cytoplasmic fluorescence, consistent with its localization in late endosomes or secondary lysosomes. After incubation with Abeta1-42 for 6 hr, a loss of lysosomal membrane impermeability is observed as evidenced by redistribution of the fluorescence to a diffuse, cytoplasmic pattern. The loss of lysosomal membrane impermeability is correlated with Abeta1-42 accumulation, since incubation of the cells with the nonaccumulating isoform of amyloid, Abeta1-40, does not induce leakage. The same results were obtained using the release of soluble lysosomal hydrolases, cathepsin D and beta-hexosaminidase, into the cytosol as an assay for the leakage of lysosomal contents. Together, our results suggest that the loss of lysosomal membrane impermeability may be an early event in Abeta pathogenesis, and provide an explanation for the miscompartmentalization of extracellular and cytoplasmic components observed in Alzheimer's disease (AD). The release of hydrolases may further cause the breakdown of the cytoskeleton and the blebbing of the plasma membrane, and the leakage of heparan sulfate glycosaminoglycans from the lysosome may ultimately promote the assembly of tau into neurofibrillary tangles (NFT).

Our early study indicates that intracellular A␤1-42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous A␤1-42 also leads to the accumulation of newly synthesized intracellular A␤. Here we describe that newly synthesized A␤, especially A␤n-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after A␤1-42 treatment. These results suggest that intracellular A␤ may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted A␤1-40, the solid-phase intracellular pathway preferentially produces A␤n-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular A␤ also share the same types of A␤ structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway.The major protein component of amyloid deposits associated with Alzheimer's disease (AD) 1 is a 39 -42-amino acid, selfassembling peptide known as the amyloid A␤ peptide. Although significant progress has been made in our understanding of the proteolytic processing of amyloid precursor protein (APP) and the secretion of soluble amyloid A␤ peptide, the mechanisms for the accumulation of insoluble amyloid deposits and their role in AD pathogenesis remains a matter of speculation. It is clear that at least two pathways for APP processing give rise to fragments bearing A␤ sequences at their amino termini: processing by ␣-secretase, which cleaves within the A␤ sequence, thereby precluding amyloid accumulation, and ␤-secretase processing, which generates carboxyl-terminal APP fragments containing the A␤ sequence. Amyloidogenic, ␤-secretase processing events may take place within several intracellular organelles, including the rough endoplasmic reticulum, trans-Golgi network, and lysosomes (1-7). Further processing of APP within the transmembrane domain by ␥-secretase releases soluble 3-and 4-kDa fragments containing all or part of the A␤ sequence (8). Recent evidence indicates that the familial AD amino acid substitutions within the APP transmembrane domain and presenilin favor the production of A␤1-42 form of A␤, which is preferentially localized within diffuse plaques and senile plaques in AD brain. This suggests that A␤1-42 is more closely associated with AD pathogenesis than shorter A␤ isoforms (9, 10).Biochemical studies of synthetic amyloid peptides have elucidated several important properties regarding their ability to assemble into the amyloid fibrils that characteristically accumulate in AD. Peptides that end at residue 42 aggregate much more rapidly than those ending at residue 39 or 40 (11, 12). The pH optimum for ␤-sheet forma...