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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

Up-regulation of PAT1 in primary neurons results in a strong colocalization with APP in the cytoplasm but not at the cell surface. A-B. PAT1-myc was overexpressed by transfection in primary neurons at 5 DIV. 24 h later cells were fixed by PFA 4% only (A) or followed by 0.2% Triton X 100 (B). Immunolabeling for PAT1-myc (Alexa-488) and endogenous APP (Cy3) was performed and analyzed by confocal microscopy. Two representative images in each condition are presented. In (A) immunolabeling was performed using the anti-myc tag polyclonal #06-549 and the anti-APP-Nter A4 antibody. In (B) the anti-myc tag MABE282 and the anti-APP-Cter polyclonal antibodies were used for immunolabeling. Quantification of colocalization using Volocity software was expressed by Pearson’s coefficient (upper panel). Data presented are the mean ± SEM of 3 independent experiments. Quantification of total APP in conditions of 0.2% Triton X 100 was performed in 23 transfected and 42 non-transfected cells (Ctrl) out of 3 independent experiments. Data are expressed in integrated density / cell in arbitrary units (AU) and represents the mean ± SEM (lower panel). Scale bar: 10 μm. C) Cell surface biotinylation was performed on PAT1-myc overexpressed cells comparatively to control cells (Ctrl). Experiments were performed on 10.106 cells 24 h after transfection. NCAM was used as internal control of membrane loading for cell surface biotinylation. The level of APP in biotinylated membranes was reported to NCAM and expressed in arbitrary units (AU). Data are the mean ± SEM of 3 independent experiments. Western blotting were performed using the anti-APP-Nter A4 and the anti-NCAM antibodies.
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Fig4: Up-regulation of PAT1 in primary neurons results in a strong colocalization with APP in the cytoplasm but not at the cell surface. A-B. PAT1-myc was overexpressed by transfection in primary neurons at 5 DIV. 24 h later cells were fixed by PFA 4% only (A) or followed by 0.2% Triton X 100 (B). Immunolabeling for PAT1-myc (Alexa-488) and endogenous APP (Cy3) was performed and analyzed by confocal microscopy. Two representative images in each condition are presented. In (A) immunolabeling was performed using the anti-myc tag polyclonal #06-549 and the anti-APP-Nter A4 antibody. In (B) the anti-myc tag MABE282 and the anti-APP-Cter polyclonal antibodies were used for immunolabeling. Quantification of colocalization using Volocity software was expressed by Pearson’s coefficient (upper panel). Data presented are the mean ± SEM of 3 independent experiments. Quantification of total APP in conditions of 0.2% Triton X 100 was performed in 23 transfected and 42 non-transfected cells (Ctrl) out of 3 independent experiments. Data are expressed in integrated density / cell in arbitrary units (AU) and represents the mean ± SEM (lower panel). Scale bar: 10 μm. C) Cell surface biotinylation was performed on PAT1-myc overexpressed cells comparatively to control cells (Ctrl). Experiments were performed on 10.106 cells 24 h after transfection. NCAM was used as internal control of membrane loading for cell surface biotinylation. The level of APP in biotinylated membranes was reported to NCAM and expressed in arbitrary units (AU). Data are the mean ± SEM of 3 independent experiments. Western blotting were performed using the anti-APP-Nter A4 and the anti-NCAM antibodies.

Mentions: Conversely, up-regulation of PAT1 was performed by transfection of PAT1-myc in primary cortical neurons. Then, the cells were fixed 24 h later, and processed for myc and APP double immunolabeling. Cells overexpressing PAT1-myc have little APP at the cell surface as observed by immunolabeling performed after PFA fixation in absence of any further permeabilization (Figure 4A). However when immunolabeling was performed in conditions of additional permeabilization with Triton X 100, more endogenous APP within the cytoplasm colocalized with PAT1-myc in transfected cells (Figure 4B), as indicated by Pearson’s coefficient. A significant increase of Pearson’s coefficient (p < 0.001) was observed in conditions of additional permeabilization comparatively to conditions of PFA fixation only (Figure 4A-B). Cell surface biotinylation of transfected neurons showed a significant lower ratio of APP/NCAM than in the control conditions, confirming immunocytochemical data (Ctrl vs PAT1-myc: p = 0.006) (Figure 4C). The total APP level detected in transfected cells by immunolabeling in conditions of additional permeabilization with Triton X 100, was not significantly different to that of non-transfected cells suggesting that the overexpression of PAT1-myc does not reduce the total level of APP (Figure 4B). Consequently, the decrease of APP at the cell surface is induced by the overexpression of PAT1-myc and not by changes in APP total levels under these conditions.Figure 4


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)

Up-regulation of PAT1 in primary neurons results in a strong colocalization with APP in the cytoplasm but not at the cell surface. A-B. PAT1-myc was overexpressed by transfection in primary neurons at 5 DIV. 24 h later cells were fixed by PFA 4% only (A) or followed by 0.2% Triton X 100 (B). Immunolabeling for PAT1-myc (Alexa-488) and endogenous APP (Cy3) was performed and analyzed by confocal microscopy. Two representative images in each condition are presented. In (A) immunolabeling was performed using the anti-myc tag polyclonal #06-549 and the anti-APP-Nter A4 antibody. In (B) the anti-myc tag MABE282 and the anti-APP-Cter polyclonal antibodies were used for immunolabeling. Quantification of colocalization using Volocity software was expressed by Pearson’s coefficient (upper panel). Data presented are the mean ± SEM of 3 independent experiments. Quantification of total APP in conditions of 0.2% Triton X 100 was performed in 23 transfected and 42 non-transfected cells (Ctrl) out of 3 independent experiments. Data are expressed in integrated density / cell in arbitrary units (AU) and represents the mean ± SEM (lower panel). Scale bar: 10 μm. C) Cell surface biotinylation was performed on PAT1-myc overexpressed cells comparatively to control cells (Ctrl). Experiments were performed on 10.106 cells 24 h after transfection. NCAM was used as internal control of membrane loading for cell surface biotinylation. The level of APP in biotinylated membranes was reported to NCAM and expressed in arbitrary units (AU). Data are the mean ± SEM of 3 independent experiments. Western blotting were performed using the anti-APP-Nter A4 and the anti-NCAM antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Up-regulation of PAT1 in primary neurons results in a strong colocalization with APP in the cytoplasm but not at the cell surface. A-B. PAT1-myc was overexpressed by transfection in primary neurons at 5 DIV. 24 h later cells were fixed by PFA 4% only (A) or followed by 0.2% Triton X 100 (B). Immunolabeling for PAT1-myc (Alexa-488) and endogenous APP (Cy3) was performed and analyzed by confocal microscopy. Two representative images in each condition are presented. In (A) immunolabeling was performed using the anti-myc tag polyclonal #06-549 and the anti-APP-Nter A4 antibody. In (B) the anti-myc tag MABE282 and the anti-APP-Cter polyclonal antibodies were used for immunolabeling. Quantification of colocalization using Volocity software was expressed by Pearson’s coefficient (upper panel). Data presented are the mean ± SEM of 3 independent experiments. Quantification of total APP in conditions of 0.2% Triton X 100 was performed in 23 transfected and 42 non-transfected cells (Ctrl) out of 3 independent experiments. Data are expressed in integrated density / cell in arbitrary units (AU) and represents the mean ± SEM (lower panel). Scale bar: 10 μm. C) Cell surface biotinylation was performed on PAT1-myc overexpressed cells comparatively to control cells (Ctrl). Experiments were performed on 10.106 cells 24 h after transfection. NCAM was used as internal control of membrane loading for cell surface biotinylation. The level of APP in biotinylated membranes was reported to NCAM and expressed in arbitrary units (AU). Data are the mean ± SEM of 3 independent experiments. Western blotting were performed using the anti-APP-Nter A4 and the anti-NCAM antibodies.
Mentions: Conversely, up-regulation of PAT1 was performed by transfection of PAT1-myc in primary cortical neurons. Then, the cells were fixed 24 h later, and processed for myc and APP double immunolabeling. Cells overexpressing PAT1-myc have little APP at the cell surface as observed by immunolabeling performed after PFA fixation in absence of any further permeabilization (Figure 4A). However when immunolabeling was performed in conditions of additional permeabilization with Triton X 100, more endogenous APP within the cytoplasm colocalized with PAT1-myc in transfected cells (Figure 4B), as indicated by Pearson’s coefficient. A significant increase of Pearson’s coefficient (p < 0.001) was observed in conditions of additional permeabilization comparatively to conditions of PFA fixation only (Figure 4A-B). Cell surface biotinylation of transfected neurons showed a significant lower ratio of APP/NCAM than in the control conditions, confirming immunocytochemical data (Ctrl vs PAT1-myc: p = 0.006) (Figure 4C). The total APP level detected in transfected cells by immunolabeling in conditions of additional permeabilization with Triton X 100, was not significantly different to that of non-transfected cells suggesting that the overexpression of PAT1-myc does not reduce the total level of APP (Figure 4B). Consequently, the decrease of APP at the cell surface is induced by the overexpression of PAT1-myc and not by changes in APP total levels under these conditions.Figure 4

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