14 March 2008. The proteolytic processing of the amyloid precursor protein (APP) to release β amyloid requires endocytosis, and new work in the March 12 issue of the Journal of Neuroscience implicates a novel endocytic pathway in the internalization of APP. The pathway, which requires flotillin and cholesterol to cluster APP, reportedly promotes amyloidogenic processing in neurons.
Flotillin proteins are associated with cholesterol-rich, detergent-resistant membrane domains known as lipid rafts. These structures have been suggested to be the meeting place for APP and the secretases that process it to Aβ. In the case of cell surface rafts, cleavage of APP does not appear to kick off until the complex is internalized.
In the new study, from the lab of Mikael Simons at the University of Goettingen in Germany, first authors Anja Schneider and Lawrence Rajendran use siRNA to knock down flotillin-2. The result is a reduction in APP internalization and Aβ production in both neuroblastoma cells and cultured primary neurons. Cholesterol depletion of cells had a similar effect. The flotillin-dependent internalization pathway was distinct from the traditional clathrin-mediated mechanism. For one, while endocytosis of APP did depend on clathrin, it did not use all the traditional accessory proteins. For another, the dependence on cholesterol set apart APP internalization from a traditional clathrin-mediated pathway.
High-resolution fluorescence microscopy revealed that flotillin-2 promoted APP clustering on the cell surface, suggesting a means by which the protein might enhance APP internalization. Consistent with this, the investigators showed that clustering by itself, achieved by cross-linking APP with antibody, was sufficient to promote internalization, even in cells lacking flotillin-2. Co-immunoprecipitation experiments suggested that the two proteins were in a complex in cells, and cholesterol depletion reduced their interaction.
“Together, our data suggest that cholesterol/flotillin-dependent clustering of APP may stimulate the internalization into a specialized clathrin-dependent endocytosis pathway to promote amyloidogenic processing,” the authors write. The current work jibes with a previous paper from some of the same researchers showing that Aβ accumulates intracellularly in flotillin-positive endosomal vesicles (Rajendran et al., 2007).—Pat McCaffrey.
Schneider A, Rajendran L, Honsho M, Gralle M, Donnert G, Wouters F, Hell SW, Simons M. Flotillin-Dependent Clustering of the Amyloid Precursor Protein Regulates Its Endocytosis and Amyloidogenic Processing in Neurons. J. Neurosci. 2008 March 12; 28(11):2874-2882. Abstract