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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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Quantitative immunodetection of hippocampal PAZ constituents derived from wild type and NexCre-cDKO mice.Bar graphs are mean ± SEM, n = 3. *p<0.05, **p<0.01, n.s. difference not significant. The control (APLP2-KO, wt) was set to 100%. A representative Western blot is shown below each diagram. Equal amounts of protein (150 ng) were loaded per lane. APP (100±20%, 13±1%; 110 kDa), APLP1 (100±8%, 92±10%, 90 kDa), Munc18 (100±6%, 117±4%, 68 kDa), NCAM (100±11%, 94±28%, 140 and 180 kDa), SNAP25 (100±17%, 103±8%, 25 kDa), SV2A (100±2%, 127±7%, 86 kDa), synaptotagmin-1 (100±1%, 124±3%, Syt-1, 65 kDa), synaptophysin (100±7%, 97±14%, S-phys, 38 kDa), syntaxin1 (100±23%, 118±26%, 34 kDa), VAMP2 (100±1%, 106±4%, 18 kDa). Note that in NexCre-cDKO the deletion of APP is restricted to excitatory neurons.
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pcbi.1004832.g002: Quantitative immunodetection of hippocampal PAZ constituents derived from wild type and NexCre-cDKO mice.Bar graphs are mean ± SEM, n = 3. *p<0.05, **p<0.01, n.s. difference not significant. The control (APLP2-KO, wt) was set to 100%. A representative Western blot is shown below each diagram. Equal amounts of protein (150 ng) were loaded per lane. APP (100±20%, 13±1%; 110 kDa), APLP1 (100±8%, 92±10%, 90 kDa), Munc18 (100±6%, 117±4%, 68 kDa), NCAM (100±11%, 94±28%, 140 and 180 kDa), SNAP25 (100±17%, 103±8%, 25 kDa), SV2A (100±2%, 127±7%, 86 kDa), synaptotagmin-1 (100±1%, 124±3%, Syt-1, 65 kDa), synaptophysin (100±7%, 97±14%, S-phys, 38 kDa), syntaxin1 (100±23%, 118±26%, 34 kDa), VAMP2 (100±1%, 106±4%, 18 kDa). Note that in NexCre-cDKO the deletion of APP is restricted to excitatory neurons.

Mentions: Recently, we analyzed the impact of single KO of either APP, ALPL1 or APLP2 on selected PAZ proteins derived from individual mouse brains [37] and developed a novel experimental setup to isolate the native PAZ from distinct brain regions such as olfactory bulb, cerebellum and hippocampus [14]. Employing quantitative immunodetection we now analyzed the NexCre-cDKO for selected members of the hippocampal PAZ proteome including APP, APLP1, the synaptic vesicle proteins synaptophysin (S-phys), SV2A, synaptotagmin-1 (Syt-1) and the SNARE complex members vesicle-associated membrane protein 2 (VAMP2), synaptosomal-associated protein 25 (SNAP25), syntaxin-1 (Syt-1) whereby the single APLP2-KO served as control (Fig 2). In agreement with our previous analysis [7] APP protein expression is reduced to about 10% of control in hippocampus whereas APLP1 remains unaltered. Similarly, the abundance of synaptophysin, VAMP2, SNAP25, syntaxin1, Munc18, and the neural cell adhesion molecule NCAM is unchanged in the hippocampal PAZ as compared to control. SV2A and synaptotagmin-1 are slightly increased in mutant mice.


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)

Quantitative immunodetection of hippocampal PAZ constituents derived from wild type and NexCre-cDKO mice.Bar graphs are mean ± SEM, n = 3. *p<0.05, **p<0.01, n.s. difference not significant. The control (APLP2-KO, wt) was set to 100%. A representative Western blot is shown below each diagram. Equal amounts of protein (150 ng) were loaded per lane. APP (100±20%, 13±1%; 110 kDa), APLP1 (100±8%, 92±10%, 90 kDa), Munc18 (100±6%, 117±4%, 68 kDa), NCAM (100±11%, 94±28%, 140 and 180 kDa), SNAP25 (100±17%, 103±8%, 25 kDa), SV2A (100±2%, 127±7%, 86 kDa), synaptotagmin-1 (100±1%, 124±3%, Syt-1, 65 kDa), synaptophysin (100±7%, 97±14%, S-phys, 38 kDa), syntaxin1 (100±23%, 118±26%, 34 kDa), VAMP2 (100±1%, 106±4%, 18 kDa). Note that in NexCre-cDKO the deletion of APP is restricted to excitatory neurons.
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pcbi.1004832.g002: Quantitative immunodetection of hippocampal PAZ constituents derived from wild type and NexCre-cDKO mice.Bar graphs are mean ± SEM, n = 3. *p<0.05, **p<0.01, n.s. difference not significant. The control (APLP2-KO, wt) was set to 100%. A representative Western blot is shown below each diagram. Equal amounts of protein (150 ng) were loaded per lane. APP (100±20%, 13±1%; 110 kDa), APLP1 (100±8%, 92±10%, 90 kDa), Munc18 (100±6%, 117±4%, 68 kDa), NCAM (100±11%, 94±28%, 140 and 180 kDa), SNAP25 (100±17%, 103±8%, 25 kDa), SV2A (100±2%, 127±7%, 86 kDa), synaptotagmin-1 (100±1%, 124±3%, Syt-1, 65 kDa), synaptophysin (100±7%, 97±14%, S-phys, 38 kDa), syntaxin1 (100±23%, 118±26%, 34 kDa), VAMP2 (100±1%, 106±4%, 18 kDa). Note that in NexCre-cDKO the deletion of APP is restricted to excitatory neurons.
Mentions: Recently, we analyzed the impact of single KO of either APP, ALPL1 or APLP2 on selected PAZ proteins derived from individual mouse brains [37] and developed a novel experimental setup to isolate the native PAZ from distinct brain regions such as olfactory bulb, cerebellum and hippocampus [14]. Employing quantitative immunodetection we now analyzed the NexCre-cDKO for selected members of the hippocampal PAZ proteome including APP, APLP1, the synaptic vesicle proteins synaptophysin (S-phys), SV2A, synaptotagmin-1 (Syt-1) and the SNARE complex members vesicle-associated membrane protein 2 (VAMP2), synaptosomal-associated protein 25 (SNAP25), syntaxin-1 (Syt-1) whereby the single APLP2-KO served as control (Fig 2). In agreement with our previous analysis [7] APP protein expression is reduced to about 10% of control in hippocampus whereas APLP1 remains unaltered. Similarly, the abundance of synaptophysin, VAMP2, SNAP25, syntaxin1, Munc18, and the neural cell adhesion molecule NCAM is unchanged in the hippocampal PAZ as compared to control. SV2A and synaptotagmin-1 are slightly increased in mutant mice.

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