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PAT1 inversely regulates the surface Amyloid Precursor Protein level in mouse primary neurons.

Dilsizoglu Senol A, Tagliafierro L, Huguet L, Gorisse-Hussonnois L, Chasseigneaux S, Allinquant B - BMC Neurosci (2015)

Bottom Line: Using down and up-regulation of PAT1, we observed respectively an increase and decrease of APP at the cell surface.The increase of APP at the cell surface induced by low levels of PAT1 did not trigger cell death signaling.These data suggest that PAT1 slows down APP trafficking to the cell surface in primary cortical neurons.

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR 894, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France. aysdilsizoglu@yahoo.com.

ABSTRACT

Background: The amyloid precursor protein (APP) is a key molecule in Alzheimer disease. Its localization at the cell surface can trigger downstream signaling and APP cleavages. APP trafficking to the cell surface in neurons is not clearly understood and may be related to the interactions with its partners. In this respect, by having homologies with kinesin light chain domains and because of its capacity to bind APP, PAT1 represents a good candidate.

Results: We observed that PAT1 binds poorly APP at the cell surface of primary cortical neurons contrary to cytoplasmic APP. Using down and up-regulation of PAT1, we observed respectively an increase and decrease of APP at the cell surface. The increase of APP at the cell surface induced by low levels of PAT1 did not trigger cell death signaling.

Conclusions: These data suggest that PAT1 slows down APP trafficking to the cell surface in primary cortical neurons. Our results contribute to the elucidation of mechanisms involved in APP trafficking in Alzheimer disease.

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Related in: MedlinePlus

APP and PAT1 colocalize poorly at the cell surface of primary neurons. A) Neurons at 5 DIV were fixed in PFA 4% for 30 min (left panel). After fixation, an additional permeabilization in 0.2% Triton X 100 was performed (right panel). In both cases cells were immunolabeled for Giantin or PAT1. Immunocytochemistry was analyzed by confocal microscopy. One representative immunocytochemical staining out of 4 independent experiments is presented. Scale bar: 10 μm. B) APP and PAT1 double immunolabeling was performed in neurons at 5 DIV fixed with PFA 4% only (left panel) and followed by 0.2% Triton X 100 (right panel). Anti-APP Cter polyclonal and monoclonal PAT1 antibodies were used as primary antibodies. Pearson’s coefficient was evaluated following confocal microscopy analyses and quantifications in Volocity software. Data presented are the mean ± SEM of 4 independent experiments. C) Ratio of APP to PAT1 before (Input) and after immunoprecipitation of APP in total extracts (Total Co-IP) and after cell surface biotinylation (Cell surface Co-IP). Immunoprecipitation of APP was performed with the anti-APP Cter polyclonal antibody. 10.106 cells at 6 DIV were used for each condition. 40 μg of cell extracts before immunoprecipitation were loaded (Input). Control immunoprecipitation in absence of primary antibody is presented (Ctrl). Detection of APP in western blotting was performed using the anti-APP-Nter A4 antibody. Data presented are the mean ± SEM of 3 independent experiments. Representative immunoblot and histogram of the ratio of APP to PAT1 in arbitrary units (AU) are presented.
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Fig1: APP and PAT1 colocalize poorly at the cell surface of primary neurons. A) Neurons at 5 DIV were fixed in PFA 4% for 30 min (left panel). After fixation, an additional permeabilization in 0.2% Triton X 100 was performed (right panel). In both cases cells were immunolabeled for Giantin or PAT1. Immunocytochemistry was analyzed by confocal microscopy. One representative immunocytochemical staining out of 4 independent experiments is presented. Scale bar: 10 μm. B) APP and PAT1 double immunolabeling was performed in neurons at 5 DIV fixed with PFA 4% only (left panel) and followed by 0.2% Triton X 100 (right panel). Anti-APP Cter polyclonal and monoclonal PAT1 antibodies were used as primary antibodies. Pearson’s coefficient was evaluated following confocal microscopy analyses and quantifications in Volocity software. Data presented are the mean ± SEM of 4 independent experiments. C) Ratio of APP to PAT1 before (Input) and after immunoprecipitation of APP in total extracts (Total Co-IP) and after cell surface biotinylation (Cell surface Co-IP). Immunoprecipitation of APP was performed with the anti-APP Cter polyclonal antibody. 10.106 cells at 6 DIV were used for each condition. 40 μg of cell extracts before immunoprecipitation were loaded (Input). Control immunoprecipitation in absence of primary antibody is presented (Ctrl). Detection of APP in western blotting was performed using the anti-APP-Nter A4 antibody. Data presented are the mean ± SEM of 3 independent experiments. Representative immunoblot and histogram of the ratio of APP to PAT1 in arbitrary units (AU) are presented.

Mentions: We first observed in primary cortical neurons that paraformaldehyde (PFA) fixation at 4% caused little permeabilization at the cell surface allowing some PAT1 detection under the cell surface while additional permeabilization by 0.2% Triton X 100 allowed to detect more PAT1 in the cytoplasm (Figure 1A). The Giantin a Golgi marker is detected after additional permeabilization with 0.2% Triton X 100 only.Figure 1


PAT1 inversely regulates the surface Amyloid Precursor Protein level in mouse primary neurons.

Dilsizoglu Senol A, Tagliafierro L, Huguet L, Gorisse-Hussonnois L, Chasseigneaux S, Allinquant B - BMC Neurosci (2015)

APP and PAT1 colocalize poorly at the cell surface of primary neurons. A) Neurons at 5 DIV were fixed in PFA 4% for 30 min (left panel). After fixation, an additional permeabilization in 0.2% Triton X 100 was performed (right panel). In both cases cells were immunolabeled for Giantin or PAT1. Immunocytochemistry was analyzed by confocal microscopy. One representative immunocytochemical staining out of 4 independent experiments is presented. Scale bar: 10 μm. B) APP and PAT1 double immunolabeling was performed in neurons at 5 DIV fixed with PFA 4% only (left panel) and followed by 0.2% Triton X 100 (right panel). Anti-APP Cter polyclonal and monoclonal PAT1 antibodies were used as primary antibodies. Pearson’s coefficient was evaluated following confocal microscopy analyses and quantifications in Volocity software. Data presented are the mean ± SEM of 4 independent experiments. C) Ratio of APP to PAT1 before (Input) and after immunoprecipitation of APP in total extracts (Total Co-IP) and after cell surface biotinylation (Cell surface Co-IP). Immunoprecipitation of APP was performed with the anti-APP Cter polyclonal antibody. 10.106 cells at 6 DIV were used for each condition. 40 μg of cell extracts before immunoprecipitation were loaded (Input). Control immunoprecipitation in absence of primary antibody is presented (Ctrl). Detection of APP in western blotting was performed using the anti-APP-Nter A4 antibody. Data presented are the mean ± SEM of 3 independent experiments. Representative immunoblot and histogram of the ratio of APP to PAT1 in arbitrary units (AU) are presented.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4355975&req=5

Fig1: APP and PAT1 colocalize poorly at the cell surface of primary neurons. A) Neurons at 5 DIV were fixed in PFA 4% for 30 min (left panel). After fixation, an additional permeabilization in 0.2% Triton X 100 was performed (right panel). In both cases cells were immunolabeled for Giantin or PAT1. Immunocytochemistry was analyzed by confocal microscopy. One representative immunocytochemical staining out of 4 independent experiments is presented. Scale bar: 10 μm. B) APP and PAT1 double immunolabeling was performed in neurons at 5 DIV fixed with PFA 4% only (left panel) and followed by 0.2% Triton X 100 (right panel). Anti-APP Cter polyclonal and monoclonal PAT1 antibodies were used as primary antibodies. Pearson’s coefficient was evaluated following confocal microscopy analyses and quantifications in Volocity software. Data presented are the mean ± SEM of 4 independent experiments. C) Ratio of APP to PAT1 before (Input) and after immunoprecipitation of APP in total extracts (Total Co-IP) and after cell surface biotinylation (Cell surface Co-IP). Immunoprecipitation of APP was performed with the anti-APP Cter polyclonal antibody. 10.106 cells at 6 DIV were used for each condition. 40 μg of cell extracts before immunoprecipitation were loaded (Input). Control immunoprecipitation in absence of primary antibody is presented (Ctrl). Detection of APP in western blotting was performed using the anti-APP-Nter A4 antibody. Data presented are the mean ± SEM of 3 independent experiments. Representative immunoblot and histogram of the ratio of APP to PAT1 in arbitrary units (AU) are presented.
Mentions: We first observed in primary cortical neurons that paraformaldehyde (PFA) fixation at 4% caused little permeabilization at the cell surface allowing some PAT1 detection under the cell surface while additional permeabilization by 0.2% Triton X 100 allowed to detect more PAT1 in the cytoplasm (Figure 1A). The Giantin a Golgi marker is detected after additional permeabilization with 0.2% Triton X 100 only.Figure 1

Bottom Line: Using down and up-regulation of PAT1, we observed respectively an increase and decrease of APP at the cell surface.The increase of APP at the cell surface induced by low levels of PAT1 did not trigger cell death signaling.These data suggest that PAT1 slows down APP trafficking to the cell surface in primary cortical neurons.

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR 894, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France. aysdilsizoglu@yahoo.com.

ABSTRACT

Background: The amyloid precursor protein (APP) is a key molecule in Alzheimer disease. Its localization at the cell surface can trigger downstream signaling and APP cleavages. APP trafficking to the cell surface in neurons is not clearly understood and may be related to the interactions with its partners. In this respect, by having homologies with kinesin light chain domains and because of its capacity to bind APP, PAT1 represents a good candidate.

Results: We observed that PAT1 binds poorly APP at the cell surface of primary cortical neurons contrary to cytoplasmic APP. Using down and up-regulation of PAT1, we observed respectively an increase and decrease of APP at the cell surface. The increase of APP at the cell surface induced by low levels of PAT1 did not trigger cell death signaling.

Conclusions: These data suggest that PAT1 slows down APP trafficking to the cell surface in primary cortical neurons. Our results contribute to the elucidation of mechanisms involved in APP trafficking in Alzheimer disease.

Show MeSH
Related in: MedlinePlus