Alzheimer Amyloid Protein Precursor Is Localized in Nerve Terminal Preparations to Rab5-containing Vesicular Organelles Distinct from Those Implicated in the Synaptic Vesicle Pathway

Abstract

In order to localize amyloid protein precursor (APP) in nerve terminals, we have immunoisolated vesicular organelles from nerve terminal preparations using antibodies to Rab5 and synaptophysin. These immunoisolates were then analyzed by electron microscopy and by immunoblotting. The synaptophysin immunoisolates represented a nearly homogeneous population of small synaptic vesicles, with less than 10% contamination by other organelles, and very little APP. In contrast, Rab5 immunoisolates contained, in addition to small synaptic vesicles, substantial numbers of large uni-and bilamellar vesicles and high levels of APP. Thus, it appears that nerve terminal APP is contained predominantly in large vesicular organelles, distinct from synaptic vesicles and from the synaptic vesicle recycling pathway. Amyloid protein precursor (APP)1 is a type I membranespanning glycoprotein which is ubiquitously expressed in mammalian cells (1). Proteolytic processing of APP results in the generation of an ϳ40-residue amyloid (A␤) fragment which accumulates in the brains of individuals with Alzheimer's disease (AD). A central role for A␤ in the pathogenesis of AD is indicated by the discovery that various mutations within or flanking the A␤ region of APP cosegregate with affected status in individuals from several families with autosomal dominant AD. For this reason, the localization, trafficking, and processing of APP has generated great interest.Immunocytochemical studies of APP in cultured cells and in brain tissue reveal that a predominant fraction of APP is localized to the endoplasmic reticulum and the Golgi apparatus (2, 3). The localization of APP to biosynthetic organelles can be explained in part by the very high rate of synthesis and turnover of this protein. In axonal and synaptic compartments of brain tissue, APP is localized to large vesicular structures (2,3). APP is also found on the surface of cultured cells, from where it can be internalized and converted to A␤ (4 -7). APP is transported by fast axonal transport in central and peripheral neurons (8,9). Soluble APP (APP s ) can be released at synapses, 2 although it is not known whether the soluble fragments are generated at the synapse. Pools of unprocessed APP are also transported retrogradely from axonal or synaptic compartments to neuronal cell bodies and dendrites (10, 11). It is not known which membrane trafficking pathways are involved in anterograde and retrograde transport of APP in neurons or whether APP-containing vesicles are related to the recycling pathway of synaptic vesicles within the nerve terminal.In an attempt to study the nature of the APP-containing vesicles in nerve terminals, we have isolated APP-containing membranes from synaptosomes of rat forebrain and from PC12 cells. To differentiate vesicles participating in the synaptic vesicle pathway from other endocytic vesicles, organelles were immunoisolated using immobilized monoclonal antibodies directed against either synaptophysin, synaptobrevin, or the endosomal GTPase, Rab5. Synaptophysin and s...

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