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. 2017 Mar 15;10:59.
doi: 10.3389/fnmol.2017.00059. eCollection 2017.

An Aberrant Phosphorylation of Amyloid Precursor Protein Tyrosine Regulates Its Trafficking and the Binding to the Clathrin Endocytic Complex in Neural Stem Cells of Alzheimer's Disease Patients

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Free PMC article

An Aberrant Phosphorylation of Amyloid Precursor Protein Tyrosine Regulates Its Trafficking and the Binding to the Clathrin Endocytic Complex in Neural Stem Cells of Alzheimer's Disease Patients

Ebbe T Poulsen et al. Front Mol Neurosci. .
Free PMC article

Abstract

Alzheimer's disease (AD) is the most common cause of dementia and is likely caused by defective amyloid precursor protein (APP) trafficking and processing in neurons leading to amyloid plaques containing the amyloid-β (Aβ) APP peptide byproducts. Understanding how APP is targeted to selected destinations inside neurons and identifying the mechanisms responsible for the generation of Aβ are thus the keys for the advancement of new therapies. We previously developed a mouse model with a mutation at tyrosine (Tyr) 682 in the C-terminus of APP. This residue is needed for APP to bind to the coating protein Clathrin and to the Clathrin adaptor protein AP2 as well as for the correct APP trafficking and sorting in neurons. By extending these findings to humans, we found that APP binding to Clathrin is decreased in neural stem cells from AD sufferers. Increased APP Tyr phosphorylation alters APP trafficking in AD neurons and it is associated to Fyn Tyr kinase activation. We show that compounds affecting Tyr kinase activity and counteracting defects in AD neurons can control APP location and compartmentalization. APP Tyr phosphorylation is thus a potential therapeutic target for AD.

Keywords: APP; Alzheimer's disease; Fyn kinase; Presenilin mutations; Tyrosine phosphorylation.

Figures

Figure 1
Figure 1
APP binding to Clathrin and AP2 is compromised in AD neurons. (A) ELISA analysis for secreted Aβ42 (Ab42) and Aβ40 (Ab40) levels from controls (C18 and C16) and AD neurons carrying mutations in the PS1 gene (L286V, M146L, and A246E) after 4 and 6 weeks in culture. Aβ levels were assessed from media after 24 h of plating. Data are expressed as pg/μg (pg/ug) of total protein. Each data point is the mean ± SEM of triplicate determinations of four independent experiments. (B) Co-Immunoprecipitation (CoIP) analysis from controls (C18, C16) and AD neurons (LV, ML, AE). Samples were immunoprecipitated with rabbit anti-APP (clone Y188) and analyzed with mouse anti-Clathrin (clone X22), mouse anti-AP2 (clone AP6), and rabbit anti-AP1 (left panel). The right panel shows total levels of APP, Clathrin, AP2, and AP1 expression in the same samples. Densitometric analysis is reported below. Data from total lysate samples were normalized to the corresponding β–actin values and expressed as % of C18. Data from IP samples were normalized to the corresponding APP input band and expressed as % of C18. The data are representative of five independent experiments. Statistically significant differences were calculated by one-way ANOVA and Tukey's post hoc test.
Figure 2
Figure 2
The extent of Clathrin/APP and AP2/APP colocalization is reduced in NSCs from patients with AD. Confocal microscopy analysis of double staining using rabbit anti-APP and mouse anti-AP2 (A) and mouse anti-Clathrin (B) in control (C18) and AD neurons with PS1 mutations (L286V; M146L; A246E). Colocalization analysis is reported in (C). The (R) coefficient (Pearson's coefficient) was used for the quantitative and comparative analyses. The extent of colocalization was calculated in at least five separate fields per slide in 10 different slides for each NSCs. The data are expressed as mean ± SEM. Scale bars are 5 μm for APP/AP2 and 6 μm for APP/Clathrin colocalization. Scale bars in high-resolution pictures are 5 μm. Statistically significant differences were calculated by one-way ANOVA and Tukey's post hoc test.
Figure 3
Figure 3
Tyr kinase inhibitors restore APP colocalization with Clathrin and AP2 in NSCs from patients with AD. (A) IP analysis of control and PS1 neurons that were exposed, or not exposed, to Sunitinib and PP2. Control C18 and AD samples (LV, ML, AE) were immunoprecipitated with anti-pTyr agarose conjugated antibody (4G10) and analyzed with rabbit anti-APP (clone Y188). The image is representative of four independent experiments. Quantification is reported in (B). Data were normalized with pTyr pulled down levels (input) and expressed as % of C18. Statistically significant differences were calculated by one-way ANOVA for repeated measures followed by Tukey's post hoc test for multiple comparisons. Confocal microscopy analysis of double staining using rabbit anti-APP and mouse anti-AP2 (C) and mouse anti-Clathrin (D) in controls and in neurons carrying L286V or M146L mutation on PS1 gene following exposure to the Tyr kinase inhibitor Sunitinib. The panels are representative of four different experiments performed in triplicate. (E) reports quantitative analysis of APP colocalization to AP2 and Clathrin after 12 h of exposure to Sunitinib. (E) also reports colocalization analysis after PP2 exposure. The (R) coefficient (Pearson's coefficient) was used for the quantitative and comparative analyses. The extent of colocalization was calculated in five separate fields per slide in five different slides for each experimental point. The data are expressed as mean ± SEM. Scale bars 6 μm. High-resolution pictures scale bar is 4 μm.
Figure 4
Figure 4
Tyr phosphatase inhibitors reduce APP colocalization with Clathrin and AP2 and increase phosphorylation of APP Tyr in control neurons. Confocal microscopy analysis of double staining with rabbit anti-APP and mouse anti-AP2 (A) or mouse anti-Clathrin (B) in C18, L286V, and M146L neurons before and after Tyr phosphatase inhibitor (TC2153) exposure. The panels are representative of five different experiments performed in duplicate. (C) Colocalization analysis of APP to AP2 and Clathrin following incubation with TC2153 and BVT948 (BVT) inhibitors in C18 neurons and in AD neurons. The (R) coefficient (Pearson's coefficient) was used for the quantitative and comparative analyses. The extent of colocalization was calculated in five separate fields per slide in four different slides for each experimental point. The data are expressed as mean ± SEM. Scale bars 6 μm and 4 μm. Statistically significant differences were calculated by one-way ANOVA and Tukey's post hoc test. (D,E). IP analysis of C18 (D) and AD neurons (E) before and after exposure to TC2153 and BVT948 (TC, BVT). Samples were immunoprecipitated with anti-pTyr agarose conjugated antibody (4G10) and analyzed with rabbit anti-APP (clone Y188). Densitometric analysis is reported in (F). Data were normalized with IgG levels and expressed as % of C18. Statistically significant differences were calculated using Student's t-test.
Figure 5
Figure 5
APP/AP2 and APP/Clathrin binding analysis in cortical tissues and fibroblasts of Göttingen minipigs with a PS1 M146I mutation in the PS1 gene. (A) CoIP analysis of cortical tissues and fibroblasts from control (Ctrl) and PS1 M146I minipigs. Samples were immunoprecipitated with rabbit anti-APP and analyzed with mouse anti-Clathrin (Clath) and mouse anti-AP2. (B) WB analysis of total lysate from controls (WT and Ctrl) and PS1 M146I minipigs. Densitometric analysis is reported in (C). Data were normalized to the corresponding β-actin values and expressed as % of Ctrl. Data from CoIP samples were normalized to the corresponding APP input amount and expressed as % of Ctrl. Statistically significant differences were calculated using Student's t-test. (D,E) Confocal microscopy analysis of double staining with rabbit anti-APP and mouse anti-AP2 (D) and mouse anti-Clathrin (E) in fibroblasts from Ctrl and PS1M146I minipigs in the presence of TC2153 or Sunitinib. The panels are representative of five different experiments performed in duplicate in Ctrl and PS1 fibroblasts from two independent control (Ctrl 1-2) and three PS1 (PS1a-c) minipigs. Colocalization analysis is reported in (F). The (R) coefficient (Pearson's coefficient) was used for the quantitative and comparative analyses. The extent of colocalization was calculated in six separate fields per slide in four different slides for each cell line. Scale bars 1 μM and 5 μM in high magnification. Statistically significant differences were calculated by one-way ANOVA and Tukey's post hoc test. (G) APP C-terminal peptide affinity pull-down analysis of cortical tissues from WT and PS1 minipigs. The table only reports information related to Fyn-APP interactions. The full list of adaptors has been provided as additional information in Table 3. (H) IP analysis of cortical tissues and fibroblasts in control and PS1 M146I minipigs (PS1), exposed or not, to the TC2153 (TC). Samples were immunoprecipitated with rabbit anti-Fyn antibody and analyzed using mouse anti-APP. Data were normalized to the corresponding total Fyn input levels. Total lysate from the same samples processed for IP (H) were probed with Src pTyr416 (used to detect pFyn Tyr420 levels; Xu et al., 2015) pan Fyn antibodies and β actin. Fyn pTyr420 levels were normalized to the corresponding total Fyn levels and expressed as % of control (WT and Ctrl). β-actin was used as loading control (J). Densitometric analysis of panel (H,J) is reported in (I,K). Statistically significant differences were calculated by one-way ANOVA and Tukey's post hoc test.
Figure 6
Figure 6
The Tyr kinase Fyn binds APP in AD neurons: (A) WB analysis performed on total lysate from control (C18) and AD neurons before or after TC2153 (TC) exposure. Samples were probed with Src pTyr416 and Src pTyr527 antibodies used to analyse Fyn pTyr420 and Fyn pTyr531 levels and were normalized to the corresponding total Fyn levels and expressed as % of control. β-actin was used as loading control (B). (C) IP analysis of controls (C18 and C16) and AD neurons (LV, ML, and AE). Samples were immunoprecipitated with rabbit anti-pan Fyn antibody and analyzed using rabbit anti-Src pTyr416 and rabbit anti- Src pTyr527 antibodies (used to detect Fyn pTyr420 and pTyr531 levels; Xu et al., 2015) or mouse anti-APP (clone Y188). Data were normalized to the corresponding input Fyn levels, and expressed as % of the correspondent C18 values (D). Densitometric analysis of (A,C) is reported in (B,D). Statistically significant differences were calculated by one-way ANOVA and Tukey's post hoc test.
Figure 7
Figure 7
APP immunofluorescence is increased in TGN46 and Rab7 and decreased in EEA1-positive vesicles in AD neurons. Confocal microscopy analysis of double staining with rabbit anti-APP (clone Y188) and mouse anti-TGN46 (A) or mouse-anti EEA1 (B) or with rabbit anti-Rab7 (C) in C18 and AD neurons (L286V and M146L). (A) and (B) also report confocal microscopy analysis in the presence of TC2153. Colocalization analysis is reported in (D) and (E). The same analysis was conducted in fibroblasts from two independent controls (C1, C2) and three independent PS1 M146I minipigs (PS1a-c) (F). Data are expressed as % of C18 in AD neurons and as % of Ctrl1 in fibroblasts. The (R) coefficient (Pearson's coefficient) was used for the quantitative and comparative analyses. The extent of colocalization was calculated in at least five separate fields per slide in seven different slides for each experimental point. The data are expressed as mean ± SEM vs. Ctrl1. Scale bars 7 μM; high magnification 5 μM. Statistically significant differences were calculated using Student's t-test.

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References

    1. Bamberger M. E., Harris M. E., McDonald D. R., Husemann J., Landreth G. E. (2003). A cell surface receptor complex for fibrillar beta-amyloid mediates microglial activation. J. Neurosci. 23, 2665–2674. - PMC - PubMed
    1. Barbagallo A. P., Weldon R., Tamayev R., Zhou D., Giliberto L., Foreman O., et al. . (2010). Tyr(682) in the intracellular domain of APP regulates amyloidogenic APP processing in vivo. PLoS ONE 5:e15503. 10.1371/journal.pone.0015503 - DOI - PMC - PubMed
    1. Chen L. H., Kao P. Y., Fan Y. H., Ho D. T., Chan C. S., Yik P. Y., et al. . (2012). Polymorphisms of CR1, CLU and PICALM confer susceptibility of Alzheimer's disease in a southern Chinese population. Neurobiol. Aging 33, 210 e1–e7. 10.1016/j.neurobiolaging.2011.09.016 - DOI - PubMed
    1. Dumanchin C., Czech C., Campion D., Cuif M. H., Poyot T., Martin C., et al. . (1999). Presenilins interact with Rab11, a small GTPase involved in the regulation of vesicular transport. Hum. Mol. Genet. 8, 1263–1269. 10.1093/hmg/8.7.1263 - DOI - PubMed
    1. Dyrlund T. F., Poulsen E. T., Scavenius C., Sanggaard K. W., Enghild J. J. (2012). MS data miner. a web-based software tool to analyze, compare, and share mass spectrometry protein identifications. Proteomics 12, 2792–2796. 10.1002/pmic.201200109 - DOI - PubMed
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