The amyloid precursor protein (APP) and its mammalian homologs, APLP1, APLP2, have been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a cell surfacelocalized pool. In the brain, all APPs are restricted to neurons; however, their precise localization at the plasma membrane remained enigmatic. Employing a variety of subcellular fractionation steps, we isolated two synaptic vesicle (SV) pools from rat and mouse brain, a pool consisting of synaptic vesicles only and a pool comprising SV docked to the presynaptic plasma membrane. Immunopurification of these two pools using a monoclonal antibody directed against the 12 membrane span synaptic vesicle protein2 (SV2) demonstrated unambiguously that APP, APLP1 and APLP2 are constituents of the active zone of murine brain but essentially absent from free synaptic vesicles. The specificity of immunodetection was confirmed by analyzing the respective knock-out animals. The fractionation experiments further revealed that APP is accumulated in the fraction containing docked synaptic vesicles. These data present novel insights into the subsynaptic localization of APPs and are a prerequisite for unraveling the physiological role of all mature APP proteins in synaptic physiology.
The hallmarks of Alzheimer’s disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aβ-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network.
Synapses are focal hot spots for signal transduction and plasticity in the brain. A synapse comprises an axon terminus, the presynapse, the synaptic cleft containing extracellular matrix proteins as well as adhesion molecules, and the postsynaptic density as target structure for chemical signaling. The proteomes of the presynaptic and postsynaptic active zones control neurotransmitter release and perception. These tasks demand short- and long-term structural and functional dynamics of the synapse mediated by its proteinaceous inventory. This review addresses subcellular fractionation protocols and the related proteomic approaches to the various synaptic subcompartments with an emphasis on the presynaptic active zone (PAZ). Furthermore, it discusses major constituents of the PAZ including the amyloid precursor protein family members. Numerous proteins regulating the rearrangement of the cytoskeleton are indicative of the functional and structural dynamics of the pre- and postsynapse. The identification of protein candidates of the synapse provides the basis for further analyzing the interaction of synaptic proteins with their targets, and the effect of their deletion opens novel insights into the functional role of these proteins in neuronal communication. The knowledge of the molecular interactome is also a prerequisite for understanding numerous neurodegenerative diseases.
The amyloid precursor protein (APP) has previously been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a presynaptic active zone-localized pool. By analyzing homozygous APP knockout mice we evaluated the impact of APP on synaptic vesicle protein abundance at synaptic release sites. Following immunopurification of synaptic vesicles and the attached presynaptic plasma membrane, individual proteins were subjected to quantitative Western blot analysis. We demonstrate that APP deletion in knockout animals reduces the abundance of the synaptic vesicle proteins synaptophysin, synaptotagmin-1, and SV2A at the presynaptic active zone. Conversely, deletion of the additional APP family members, APLP1 and APLP2 resulted in an increase in synaptophysin, synaptogamin-1, and SV2A abundance. When transmembrane APP is lacking in APPsα-KI/APLP2-KO mice synaptic vesicle protein abundance corresponds to that in APP -KO mice. Deletion of the synaptic vesicle protein 2 (SV2) A and B had no effect on APP and synaptophysin abundance but decreased synaptotagmin-1. Our data suggest that APP controls the abundance of synaptic vesicle proteins at the presynaptic release sites and thus impacts synaptic transmission.
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