Synaptic and Endosomal Localization of Active γ-Secretase in Rat Brain

Background: A key player in the development of Alzheimer's disease (AD) is the gamma-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. gamma-Secretase is crucial for the generation of the neurotoxic amyloid beta-peptide (A beta) but also takes part in the processing of many other substrates. In cell lines, active gamma-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active gamma-secretase in the affected organ of AD, namely the brain. Principal Findings: We show by subcellular fractionation of rat brain that high gamma-secretase activity, as assessed by production of A beta 40, is present in an endosome-and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high A beta 40 production and contained high amounts of the gamma-secretase components. Further purification of the synaptic vesicles verified the presence of the gamma-secretase components in these compartments. The localization of an active gamma-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated gamma-secretase inhibitor together with confocal microscopy. Significance: The information about the subcellular localization of gamma-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active gamma-secretase.