Detection of a novel intraneuronal pool of insoluble amyloid β protein that accumulates with time in culture

The amyloid-beta peptide (A beta) is produced at several sites within cultured human NT2N neurons with A beta 1-42 specifically generated in the endoplasmic reticulum/intermediate compartment. Since A beta is found as insoluble deposits in senile plaques of the AD brain, and the A beta peptide can polymerize into insoluble fibrils in vitro, we examined the possibility that A beta 1-40, and particularly the more highly amyloidogenic A beta 1-42, accumulate in an insoluble pool within NT2N neurons. Remarkably, we found that formic acid extraction of the NT2N cells solubilized a pool of previously undetectable A beta that accounted for over half of the total intracellular A beta. A beta 1-42 was more abundant than A beta 1-40 in this pool, and most of the insoluble A beta 1-42 was generated in the endoplasmic reticulum/intermediate compartment pathway. High levels of insoluble A beta were also detected in several nonneuronal cell lines engineered to overexpress the amyloid-beta precursor protein. This insoluble intracellular pool of A beta was exceptionally stable, and accumulated in NT2N neurons in a time-dependent manner, increasing 12-fold over a 7-wk period in culture. These novel findings suggest that A beta amyloidogenesis may be initiated within living neurons rather than in the extracellular space. Thus, the data presented here require a reexamination of the prevailing view about the pathogenesis of A beta deposition in the AD brain.