Failure in beta-amyloid degradation in the endosomal/lysosomal pathway results in intracellular beta-amyloid accumulation within lipid rafts

Abstract

Endothelin-converting enzymes (ECE)-1 and -2 are members of the M13 family of zinc-metalloproteases that plays a major role in Aβ catabolism. We have recently established that ECE-1 and ECE-2 are found in Aβ-generating compartments and degrade the peptide at intracellular sites of production. Based on the localization of different ECE-1 isoforms to the plasma membrane, recycling endosomes, and late endosomes, ECE-1 activity influences extracellular Aβ levels (mainly by degrading the peptide prior to secretion) as well as intracellular levels. ECE-2, however, is restricted to the endosomal/lysosomal pathway in neurons and regulates a pool of Aβ produced within the vesicles of this pathway, including autophagosomes. Treatment of neuronal cells with ECE inhibitors produces increases in both extracellular and intracellular Aβ. Levels of intracellular Aβ, however, stabilize over time after ECE inhibition suggesting that additional proteases contribute to intracellular Aβ removal SH-SY5Y cells stably transfected with wild type APP were used as a neuronal model. Inhibition of ECE and lysosomal enzymes was produced with specific protease inhibitors. Autophagic flux was inhibited with lysosomotropic agents. Levels of Aβ were measured by ELISA and immunofluorescence. Detergent resistant membranes were isolated by discontinuous gradient centrifugation after incubation in cold 1% Triton X-100. Western blot analysis was used to confirm the presence of specific lipid raft markers in the obtained fractions. Inhibition of ECEs with phosphoramidon produced intracellular accumulation of Aβ in the lumen of endosomal/lysosomal vesicles. Simultaneous inhibition of ECEs and cysteine protease activity withE64 doubled intracellular Aβ accumulation compared to ECE inhibition alone, without further altering secreted levels. With the combined treatment, there was also a selective accumulation of Aβ in CTFs-enriched lipid rafts. Interestingly,E64 alone did not have any impact in either extracellular or intracellular Aβ concentration in these cells. Our findings demonstrate that failure in Aβ degradation in the endosomal/lysosomal pathway can result in an accumulation of intracellular Aβ in lipid rafts. As intracellular acidic compartments and lipid rafts have been proposed as potential sites for Aβ aggregation, our cell model may prove helpful in understanding the initial of steps of the disease.