Detection of a Novel Intraneuronal Pool of Insoluble Amyloid β Protein that Accumulates with Time in Culture

The amyloid-β peptide (Aβ) is produced at several sites within cultured human NT2N neurons with Aβ1-42 specifically generated in the endoplasmic reticulum/intermediate compartment. Since Aβ is found as insoluble deposits in senile plaques of the AD brain, and the Aβ peptide can polymerize into insoluble fibrils in vitro, we examined the possibility that Aβ1-40, and particularly the more highly amyloidogenic Aβ1-42, 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 Aβ that accounted for over half of the total intracellular Aβ. Aβ1-42 was more abundant than Aβ1-40 in this pool, and most of the insoluble Aβ1-42 was generated in the endoplasmic reticulum/intermediate compartment pathway. High levels of insoluble Aβ were also detected in several nonneuronal cell lines engineered to overexpress the amyloid-β precursor protein. This insoluble intracellular pool of Aβ 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 Aβ 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 Aβ deposition in the AD brain.