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APP Is a Context-Sensitive Regulator of the Hippocampal Presynaptic Active Zone.

Laßek M, Weingarten J, Wegner M, Mueller BF, Rohmer M, Baeumlisberger D, Arrey TN, Hick M, Ackermann J, Acker-Palmer A, Koch I, Müller U, Karas M, Volknandt W - PLoS Comput. Biol. (2016)

Bottom Line: Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell Biology and Neuroscience, Biologicum, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.

ABSTRACT
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.

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Scheme illustrating the regulatory role of APP in a context-sensitive manner at the hippocampal PAZ. R, regulator; M, mediator, C, central player.Color code: magenta, upregulation (in APP-mutants); green, downregulation (in APP-mutants); yellow, unaltered.
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pcbi.1004832.g009: Scheme illustrating the regulatory role of APP in a context-sensitive manner at the hippocampal PAZ. R, regulator; M, mediator, C, central player.Color code: magenta, upregulation (in APP-mutants); green, downregulation (in APP-mutants); yellow, unaltered.

Mentions: The application of network analysis by e.g. centrality and path length, visualization techniques, and functional analysis by subcommunity structures allowed insights into the changes of protein abundance following APP deletion. APP is highly interconnected in all networks analyzed and serves as a hub, implicating that it is important for the structure and function of the entire network. The high value for betweenness centrality indicates a bridging role of APP between the functional modules. The low values for the clustering coefficient supports the linking function of APP in the interaction network of the hippocampal PAZ. APP and its interactors are central in the information flow as indicated by the high value for edge betweenness. For example, APP interacts with bassoon physically [38] and is thereby embedded into the cytomatrix of the active zone (CAZ). Interactors of bassoon are in turn the ERC protein 2, piccolo and regulating synaptic membrane exocytosis protein 1. Deletion of APP leads to a decomposition and rearrangement of the entire network structure. This network is based on current database knowledge about physical interactions between proteins. Therefore APP deletion results in loss of network components. Interestingly, this is not reflected by a loss of biological functions. For example following APP deletion bassoon and its interactors are still part of the synaptic vesicle cycle. However they are no longer connected in cytoskeleton organization and calcium homeostasis subnetworks. In addition reorganization of the network results in changes in path lengths. For example in the synaptic vesicle cycle the vesicular proton pump vATPase and bassoon are linked via APP. Deletion of APP results in an increase in the shortest path length by three steps (interactions). Similarly, in calcium homeostasis loss of APP extends the shortest path length from extracellular cell adhesion (e.g. NCAM1) to trimeric G-proteins involved in intracellular signaling by three interactions. In contrast deletion of APP adds three additional interactions (4→7) from the homophilic cell-cell adhesion molecule 1, that acts in a calcium independent manner, to the actin-related protein complex. Based on our data, we hypothesize that APP functions as a regulator involved in all functional activities at the release sites. Depending on its respective microenvironment it may exert varying functions. This is illustrated in a scheme (Fig 9) which depicts the physical interaction between APP (regulator, R), mediators (M) and central players (C). Deletion of APP results in significant changes in the abundance of mediators but not of central players. For example the mediator of synaptic vesicle exocytosis α-synuclein [39] is downregulated in the mutants, however the central players the SNARE proteins VAMP2, SNAP25, and syntaxin1 [13] are not affected. In this context it is noteworthy to mention that deletion of SV2A does not affect the abundance of SNARE proteins but results in reduced SNARE complex formation [40]. Similarly, the mediators in calcium homeostasis calmodulin and neuromodulin are downregulated whereas the central player in learning and memory CaMKII as well as calcium-channels remain unaltered.


APP Is a Context-Sensitive Regulator of the Hippocampal Presynaptic Active Zone.

Laßek M, Weingarten J, Wegner M, Mueller BF, Rohmer M, Baeumlisberger D, Arrey TN, Hick M, Ackermann J, Acker-Palmer A, Koch I, Müller U, Karas M, Volknandt W - PLoS Comput. Biol. (2016)

Scheme illustrating the regulatory role of APP in a context-sensitive manner at the hippocampal PAZ. R, regulator; M, mediator, C, central player.Color code: magenta, upregulation (in APP-mutants); green, downregulation (in APP-mutants); yellow, unaltered.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4836664&req=5

pcbi.1004832.g009: Scheme illustrating the regulatory role of APP in a context-sensitive manner at the hippocampal PAZ. R, regulator; M, mediator, C, central player.Color code: magenta, upregulation (in APP-mutants); green, downregulation (in APP-mutants); yellow, unaltered.
Mentions: The application of network analysis by e.g. centrality and path length, visualization techniques, and functional analysis by subcommunity structures allowed insights into the changes of protein abundance following APP deletion. APP is highly interconnected in all networks analyzed and serves as a hub, implicating that it is important for the structure and function of the entire network. The high value for betweenness centrality indicates a bridging role of APP between the functional modules. The low values for the clustering coefficient supports the linking function of APP in the interaction network of the hippocampal PAZ. APP and its interactors are central in the information flow as indicated by the high value for edge betweenness. For example, APP interacts with bassoon physically [38] and is thereby embedded into the cytomatrix of the active zone (CAZ). Interactors of bassoon are in turn the ERC protein 2, piccolo and regulating synaptic membrane exocytosis protein 1. Deletion of APP leads to a decomposition and rearrangement of the entire network structure. This network is based on current database knowledge about physical interactions between proteins. Therefore APP deletion results in loss of network components. Interestingly, this is not reflected by a loss of biological functions. For example following APP deletion bassoon and its interactors are still part of the synaptic vesicle cycle. However they are no longer connected in cytoskeleton organization and calcium homeostasis subnetworks. In addition reorganization of the network results in changes in path lengths. For example in the synaptic vesicle cycle the vesicular proton pump vATPase and bassoon are linked via APP. Deletion of APP results in an increase in the shortest path length by three steps (interactions). Similarly, in calcium homeostasis loss of APP extends the shortest path length from extracellular cell adhesion (e.g. NCAM1) to trimeric G-proteins involved in intracellular signaling by three interactions. In contrast deletion of APP adds three additional interactions (4→7) from the homophilic cell-cell adhesion molecule 1, that acts in a calcium independent manner, to the actin-related protein complex. Based on our data, we hypothesize that APP functions as a regulator involved in all functional activities at the release sites. Depending on its respective microenvironment it may exert varying functions. This is illustrated in a scheme (Fig 9) which depicts the physical interaction between APP (regulator, R), mediators (M) and central players (C). Deletion of APP results in significant changes in the abundance of mediators but not of central players. For example the mediator of synaptic vesicle exocytosis α-synuclein [39] is downregulated in the mutants, however the central players the SNARE proteins VAMP2, SNAP25, and syntaxin1 [13] are not affected. In this context it is noteworthy to mention that deletion of SV2A does not affect the abundance of SNARE proteins but results in reduced SNARE complex formation [40]. Similarly, the mediators in calcium homeostasis calmodulin and neuromodulin are downregulated whereas the central player in learning and memory CaMKII as well as calcium-channels remain unaltered.

Bottom Line: Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell Biology and Neuroscience, Biologicum, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus