Research ArticleAlzheimer’s Disease

Familial Alzheimer’s disease–associated presenilin 1 mutants promote γ-secretase cleavage of STIM1 to impair store-operated Ca2+ entry

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Science Signaling  06 Sep 2016:
Vol. 9, Issue 444, pp. ra89
DOI: 10.1126/scisignal.aaf1371
  • Fig. 1 γ-Secretase attenuates CCE.

    (A) Cytoplasmic Ca2+ concentration ([Ca2+]i) in SH-SY5Y cells stably expressing empty vector (CTRL), PS1WT, or PS1-M146L. CCE was triggered by depleting ER Ca2+ stores using 50 μM carbachol in Ca2+-free HBSS (white bar), followed by readdition of 2 mM Ca2+ to the extracellular solution (dark gray bar). (B) Experiments like those described in (A) were performed with cells pretreated with 1 μM DAPT for 48 hours. In (A) and (B), gray lines depict individual cellular [Ca2+]i; colored lines depict the mean [Ca2+]i. (C and D) Summary of peak [Ca2+]i and initial rate of [Ca2+]i change during CCE with or without DAPT treatment. (E and H) Analogous CCE experiments performed in human skin fibroblasts from FAD patients harboring PS1-M146L or PS1-A246E mutation or from two age-matched individuals without PS1 mutation. (F and G) Summary of peak [Ca2+]i and initial rate of Ca2+ entry during CCE with or without DAPT treatment. Experiments were repeated three times for each cell line, with 30 cells analyzed in each experiment. Bars indicate means ± SEM. See also table S1. *P < 0.01, unpaired Student’s t test within group. #P < 0.01, one-way analysis of variance (ANOVA) with Tukey’s tests compared to CTRL and PS1WT groups in SH-SY5Y or CTRL1 and CTRL2 groups in human fibroblasts.

  • Fig. 2 PS1 interacts with STIM1.

    (A) PS1 and STIM1 in SH-SY5Y cells coexpressing STIM1 with empty vector, PS1WT, or PS1-M146L. Input (1/20) shown in the first three lanes, IgG immunoprecipitates (IP: IgG) shown in the middle three lanes, and STIM1 immunoprecipitates (IP: STIM1) shown in the last three lanes. The graphs summarize band intensities normalized to input from three separate experiments. *P < 0.01, one-way ANOVA with Bonferroni’s test compared to empty vector control. IB, immunoblot. (B) Coimmunoprecipitation of endogenous PS1 with STIM1 antibody in mouse primary cortical neurons. Input (1/20) shown in the first lane, IgG immunoprecipitates shown in the second lane, and STIM1 immunoprecipitates shown in the last lane. Experiments were repeated three times, and band intensities of coimmunoprecipitated proteins were normalized with their respective input intensities. (C) Representative micrographs showing the interaction between endogenous PS1 and STIM1 by in situ PLA in SH-SY5Y cells. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). In situ PLA performed in CRISPR-mediated PS1KO or STIM1KO SH-SY5Y cells as negative controls. Images were taken at ×20 magnification. Scale bar, 10 μm. Bars represent means ± SEM from three separate experiments, with 15 cells analyzed in each group. See also table S2. #P < 0.01, one-way ANOVA with Bonferroni’s test compared to PLA performed in normal SH-SY5Y cells using STIM1 and PS1 antibodies.

  • Fig. 3 FAD-linked PS1 mutant impairs STIM1 oligomerization.

    (A) FRET microscopy depicting oligomerization of STIM1 in SH-SY5Y cells stably expressing empty vector (CTRL), PS1WT, or PS1-M146L. FRET signals were triggered by carbachol-induced ER Ca2+ depletion in Ca2+-free buffer. FRET signals were normalized (NFRET) by CFP donor and YFP acceptor fluorescence intensities. (B) Representative images depicting localization and fluorescence intensities of CFP, YFP, and FRET channels at time = 5 min of cells from the experiment shown in (A). Arrowheads indicate translocation of CFP- or YFP-tagged STIM1 after ER Ca2+ store depletion. Scale bar, 10 μm. (C) Summary of NFRET at 5 min. Experiments were repeated five times, and data are means ± SEM. *P < 0.01 as compared to either the CTRL or PS1WT group, using one-way ANOVA with Tukey’s multiple comparisons.

  • Fig. 4 FAD-linked PS1 mutant attenuates STIM1-ORAI1 interaction.

    (A) Representative epifluorescence and TIRF images depicting the interaction of STIM1-GFP and ORAI1-mCherry in SH-SY5Y cells stably expressing empty vector (CTRL), PS1WT, or PS1-M146L before or after ER Ca2+ store depletion by 3 μM thapsigargin (TG) in the absence of extracellular Ca2+. Analogous experiments were performed with cells pretreated with 1 μM DAPT for 48 hours. (B) Quantification of STIM1-ORAI1–colocalized puncta in TIRF microscopy of SH-SY5Y cell lines shown in (A) normalized to their respective TIRF footprints before or after ER Ca2+ depletion. (C and D) ER Ca2+ depletion and TIRF microscopy experiments similar to (A) were performed in SH-SY5Y cells with endogenous PS1 knocked out (PS1KO) or native SH-SY5Y cells. Data are means ± SEM from three separate experiments, with 12 cells analyzed in each cell line and for each treatment. See also table S3. *P < 0.01 by Student’s t test compared to the respective DMSO treatment group. #P < 0.01 by one-way ANOVA with Tukey’s tests compared to either the CTRL and PS1WT group. #’P < 0.01 by Student’s t test compared to DMSO-treated native SH-SY5Y cells.

  • Fig. 5 FAD-linked PS1 mutant enhances γ-secretase cleavage of STIM1.

    (A) Schematic depiction of the cellular localization and orientation of STIM1 and APP, and sequence alignment of the transmembrane domains of STIM1 and APP. Aβ40 and Aβ42 cleavage sites are indicated. Orientation of transmembrane domains reflects native topologies. (B) Representative immunoblots of STIM1 in STIM1KO SH-SY5Y or cells transduced with empty vector (CTRL), PS1WT, or PS1-M146L treated with the proteasome inhibitor MG132, the γ-secretase inhibitor DAPT, or both, respectively. Arrow indicates STIM1-immunoreactive band detected with longer exposure. Actin served as the loading control. Experiments were repeated five times. Bars indicate means ± SEM. *P < 0.01 and #P < 0.01 compared with respective DMSO or MG132 treatment group within the same cell line using Student’s t test. (C) In vitro fluorogenic assay of γ-secretase activity of solubilized SH-SY5Y cells for APP, STIM1, or STIM1 in reversed orientation [STIM1 (Rev)] peptides in the presence of increasing concentrations of DAPT. Itgβ1 and NPR-A transmembrane peptides served as controls. Experiments were repeated five times. (D) Summary of proteolysis of synthetic transmembrane peptides of (C) by γ-secretase activity in SH-SY5Y cells stably expressing empty vector (CTRL), PS1WT, or PS1-M146L. Experiments were repeated five times. Bars indicate means ± SEM. #P < 0.01 compared to CTRL and PS1WT groups using one-way ANOVA with Tukey’s multiple comparisons.

  • Fig. 6 Mutant PS1 reduces the number of mature dendritic spines in cultured hippocampal neurons.

    (A) Confocal images of spine morphology of cultured rat hippocampal neurons transfected with CTRL (empty vector), PS1WT, or PS1-M146L together with EGFP as a transfection marker. Enlarged image in lower panel depicts a representative dendrite (white box). Scale bars, 10 μm (white) or 2 μm (red). (B) Effect of DAPT treatment (48 hours) on spine morphology. (C) Effect of STIM1 overexpression on spine morphology. (D) Summary of spine density and population in indicated cultured rat hippocampal neurons. Experiments were repeated three times for each group, with a total of 36 dendrites analyzed for each group. See also table S4. Bars indicate means ± SEM. #P < 0.01 compared with CTRL and PS1WT groups using one-way ANOVA with Tukey’s multiple comparison. *P < 0.01 in neurons treated with 1 μM DAPT or with STIM1 overexpression compared to control group using one-way ANOVA with Bonferroni’s multiple comparisons.

  • Fig. 7 Hypothetic model of spine destabilization induced by mutant PS1–associated CCE attenuation.

    STIM1 is a type I transmembrane protein that senses Ca2+ concentrations in the ER. It oligomerizes and translocates to near–plasma membrane regions and interacts with and activates ORAI1 to sustain extracellular Ca2+ entry upon ER Ca2+ store depletion. The transmembrane (TM) domain of STIM1 is highly similar to that of APP and is cleaved by γ-secretase. In normal neurons, PS1WT modulates CCE by inactivating STIM1 through γ-secretase cleavage (left panel). In FAD-PS1 neurons, mutant PS1 enhances γ-secretase cleavage of STIM1, resulting in the reduction of functional STIM1 oligomers with consequent diminished ORAI1 channel activation, leading to attenuated CCE. Sustained CCE reduction may lead to spine deformity and result in memory loss in AD.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/444/ra89/DC1

    Fig. S1. Mutant PS1 or PS1 knockout does not grossly affect STIM1 or ORAI1 abundance in SH-SY5Y cells.

    Fig. S2. Impaired CCE in mutant FAD-PS1–expressing cells or PS1 knockout cells.

    Fig. S3. PS1 interacts with STIM1 but not with CFTR.

    Fig. S4. In situ PLA demonstrates that endogenous PS1 and STIM1 interact.

    Fig. S5. FAD-linked mutant PS1 impairs STIM1 puncta formation.

    Fig. S6. Mutant PS1 attenuates the STIM1-ORAI1 interaction in fibroblasts from FAD patients.

    Fig. S7. In vitro fluorogenic γ-secretase cleavage assay.

    Table S1. The effects of PS1 on CCE.

    Table S2. The amount of PLA red fluorescent dots in SH-SY5Y cells.

    Table S3. The effects of PS1 on the STIM1-ORAI1 interaction after ER Ca2+ depletion by thapsigargin.

    Table S4. The effects of PS1 on spine stability in isolated rat hippocampal neurons.

  • Supplementary Materials for:

    Familial Alzheimer's disease–associated presenilin 1 mutants promote γ-secretase cleavage of STIM1 to impair store-operated Ca2+ entry

    Benjamin Chun-Kit Tong, Claire Shuk-Kwan Lee, Wing-Hei Cheng, Kwok-On Lai, J. Kevin Foskett, King-Ho Cheung*

    *Corresponding author. Email: kingho.cheung{at}hku.hk

    This PDF file includes:

    • Fig. S1. Mutant PS1 or PS1 knockout does not grossly affect STIM1 or ORAI1 abundance in SH-SY5Y cells.
    • Fig. S2. Impaired CCE in mutant FAD-PS1–expressing cells or PS1 knockout cells.
    • Fig. S3. PS1 interacts with STIM1 but not with CFTR.
    • Fig. S4. In situ PLA demonstrates that endogenous PS1 and STIM1 interact.
    • Fig. S5. FAD-linked mutant PS1 impairs STIM1 puncta formation.
    • Fig. S6. Mutant PS1 attenuates the STIM1-ORAI1 interaction in fibroblasts from FAD patients.
    • Fig. S7. In vitro fluorogenic γ-secretase cleavage assay.
    • Table S1. The effects of PS1 on CCE.
    • Table S2. The amount of PLA red fluorescent dots in SH-SY5Y cells.
    • Table S3. The effects of PS1 on the STIM1-ORAI1 interaction after ER Ca2+ depletion by thapsigargin.
    • Table S4. The effects of PS1 on spine stability in isolated rat hippocampal neurons.

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    Citation: B. C.-K. Tong, C. S.-K. Lee, W.-H. Cheng, K.-O. Lai, J. Kevin Foskett, K.-H. Cheung, Familial Alzheimer's disease&ndashl;associated presenilin 1 mutants promote γ-secretase cleavage of STIM1 to impair store-operated Ca2+ entry. Sci. Signal. 9, ra89 (2016).

    © 2016 American Association for the Advancement of Science

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