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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

Down-regulation of PAT1 increases APP at the cell surface of primary neurons. Neurons at 2 DIV were treated with either PAT1 siRNAs or GAPDH siRNAs comparatively to control cells (Ctrl) in absence of treatment. A-B: After 66 h the cells were fixed and processed for PAT1 immunocytodetection (A) or extracted for western blots (B). (A) Immunocytochemistry was analyzed by confocal microscopy and quantified by Image J. Data are expressed in integrated density/cell in arbitrary units (AU). Two representative immunolabelings of PAT1 of each condition are presented. (B) 40 μg of cell extracts were loaded for western blotting. The level of PAT1 or of APP reported to actin was expressed in arbitrary units (AU). Data in A and B are the mean ± SEM of 4 independent experiments and of 3 experiments for right panel in B. C-D: After 66 h of PAT1 siRNAs or GAPDH siRNAs, the cells were fixed by and processed for APP immunodetection (C) or for cell surface biotinylation (D). (C) APP immunocytochemistry was analyzed by confocal microscopy and quantified with Image J. Data are expressed in integrated density/cell in arbitray units (AU). Three representative images of each condition are presented. (D) Cell surface biotinylation was performed on 106 cells. 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. In the whole figure immunocytochemistry was performed using the anti-APP-Nter A4 antibody. The anti-APP Cter polyclonal antibody was used for western blotting in 2B (right panel) and the anti-APP-Nter A4 antibody was used in 2D. Scale bar: 10 μm.
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Fig2: Down-regulation of PAT1 increases APP at the cell surface of primary neurons. Neurons at 2 DIV were treated with either PAT1 siRNAs or GAPDH siRNAs comparatively to control cells (Ctrl) in absence of treatment. A-B: After 66 h the cells were fixed and processed for PAT1 immunocytodetection (A) or extracted for western blots (B). (A) Immunocytochemistry was analyzed by confocal microscopy and quantified by Image J. Data are expressed in integrated density/cell in arbitrary units (AU). Two representative immunolabelings of PAT1 of each condition are presented. (B) 40 μg of cell extracts were loaded for western blotting. The level of PAT1 or of APP reported to actin was expressed in arbitrary units (AU). Data in A and B are the mean ± SEM of 4 independent experiments and of 3 experiments for right panel in B. C-D: After 66 h of PAT1 siRNAs or GAPDH siRNAs, the cells were fixed by and processed for APP immunodetection (C) or for cell surface biotinylation (D). (C) APP immunocytochemistry was analyzed by confocal microscopy and quantified with Image J. Data are expressed in integrated density/cell in arbitray units (AU). Three representative images of each condition are presented. (D) Cell surface biotinylation was performed on 106 cells. 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. In the whole figure immunocytochemistry was performed using the anti-APP-Nter A4 antibody. The anti-APP Cter polyclonal antibody was used for western blotting in 2B (right panel) and the anti-APP-Nter A4 antibody was used in 2D. Scale bar: 10 μm.

Mentions: In order to investigate if PAT1 levels can modulate the trafficking of APP to the cell surface we down and up-regulated PAT1. Down-regulation of PAT1 was performed by the help of specific siRNAs. The decrease of PAT1 level in primary neurons was checked 66 h after PAT1 siRNAs addition both by immunocytochemical staining for PAT1 without Triton X 100 and immunoblots from cell extracts (Figure 2A-B). A significant decrease of PAT1 level was observed in primary neurons treated with PAT1 siRNAs for 66 h comparatively to control (ctrl) cells in absence of treatment (ctrl vs PAT1 siRNAs: p < 0.001) while no difference of PAT1 levels was observed using GAPDH siRNAs, as control siRNAs (ctrl vs GAPDH siRNAs: NS) (Figure 2A-B). The down-regulation of PAT1 or GAPDH did not modify the level of total APP in neuronal extracts (Figure 2B).Figure 2


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)

Down-regulation of PAT1 increases APP at the cell surface of primary neurons. Neurons at 2 DIV were treated with either PAT1 siRNAs or GAPDH siRNAs comparatively to control cells (Ctrl) in absence of treatment. A-B: After 66 h the cells were fixed and processed for PAT1 immunocytodetection (A) or extracted for western blots (B). (A) Immunocytochemistry was analyzed by confocal microscopy and quantified by Image J. Data are expressed in integrated density/cell in arbitrary units (AU). Two representative immunolabelings of PAT1 of each condition are presented. (B) 40 μg of cell extracts were loaded for western blotting. The level of PAT1 or of APP reported to actin was expressed in arbitrary units (AU). Data in A and B are the mean ± SEM of 4 independent experiments and of 3 experiments for right panel in B. C-D: After 66 h of PAT1 siRNAs or GAPDH siRNAs, the cells were fixed by and processed for APP immunodetection (C) or for cell surface biotinylation (D). (C) APP immunocytochemistry was analyzed by confocal microscopy and quantified with Image J. Data are expressed in integrated density/cell in arbitray units (AU). Three representative images of each condition are presented. (D) Cell surface biotinylation was performed on 106 cells. 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. In the whole figure immunocytochemistry was performed using the anti-APP-Nter A4 antibody. The anti-APP Cter polyclonal antibody was used for western blotting in 2B (right panel) and the anti-APP-Nter A4 antibody was used in 2D. Scale bar: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Down-regulation of PAT1 increases APP at the cell surface of primary neurons. Neurons at 2 DIV were treated with either PAT1 siRNAs or GAPDH siRNAs comparatively to control cells (Ctrl) in absence of treatment. A-B: After 66 h the cells were fixed and processed for PAT1 immunocytodetection (A) or extracted for western blots (B). (A) Immunocytochemistry was analyzed by confocal microscopy and quantified by Image J. Data are expressed in integrated density/cell in arbitrary units (AU). Two representative immunolabelings of PAT1 of each condition are presented. (B) 40 μg of cell extracts were loaded for western blotting. The level of PAT1 or of APP reported to actin was expressed in arbitrary units (AU). Data in A and B are the mean ± SEM of 4 independent experiments and of 3 experiments for right panel in B. C-D: After 66 h of PAT1 siRNAs or GAPDH siRNAs, the cells were fixed by and processed for APP immunodetection (C) or for cell surface biotinylation (D). (C) APP immunocytochemistry was analyzed by confocal microscopy and quantified with Image J. Data are expressed in integrated density/cell in arbitray units (AU). Three representative images of each condition are presented. (D) Cell surface biotinylation was performed on 106 cells. 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. In the whole figure immunocytochemistry was performed using the anti-APP-Nter A4 antibody. The anti-APP Cter polyclonal antibody was used for western blotting in 2B (right panel) and the anti-APP-Nter A4 antibody was used in 2D. Scale bar: 10 μm.
Mentions: In order to investigate if PAT1 levels can modulate the trafficking of APP to the cell surface we down and up-regulated PAT1. Down-regulation of PAT1 was performed by the help of specific siRNAs. The decrease of PAT1 level in primary neurons was checked 66 h after PAT1 siRNAs addition both by immunocytochemical staining for PAT1 without Triton X 100 and immunoblots from cell extracts (Figure 2A-B). A significant decrease of PAT1 level was observed in primary neurons treated with PAT1 siRNAs for 66 h comparatively to control (ctrl) cells in absence of treatment (ctrl vs PAT1 siRNAs: p < 0.001) while no difference of PAT1 levels was observed using GAPDH siRNAs, as control siRNAs (ctrl vs GAPDH siRNAs: NS) (Figure 2A-B). The down-regulation of PAT1 or GAPDH did not modify the level of total APP in neuronal extracts (Figure 2B).Figure 2

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