Research ArticleCell Biology

SERP1 is an assembly regulator of γ-secretase in metabolic stress conditions

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Science Signaling  17 Mar 2020:
Vol. 13, Issue 623, eaax8949
DOI: 10.1126/scisignal.aax8949
  • Fig. 1 SERP1 stimulates γ-secretase activity for Aβ generation.

    (A and B) Amount of Aβ40 and Aβ42 in the media of SY5Y-APPswe cells transfected with pSERP1-HA (A) and HEK293T/APPswe cells transfected with SERP1 shRNA (B) and treated with 10 nM Compound E (Comp.E) for 24 hours, measured with an ELISA kit. (C) Conditioned media from pSERP1-HA–transfected cells was subjected to immunoprecipitation (IP) assay using preimmune serum (Pre) or Aβ antibody (4G8), and the immunoprecipitates and whole-cell lysates (WCL) were analyzed with Western blotting (top). The immunoprecipitated Aβ abundance was quantified. Data are means ± SD; n = 3 experiments. *P < 0.05 and ***P < 0.001 by unpaired t test. (D and E) HEK293T cells were transfected with pSERP1-HA (D) or SERP1 shRNA (E) or the respective control, and 1% CHAPS-soluble membrane fractions were subjected to enzyme assay using fluorogenic substrate in the presence or absence of Compound E. Data are means ± SD; n = 5 (D) and n = 3 (E). *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired t test.

  • Fig. 2 SERP1 reduces NotchΔE processing and NICD target gene expression.

    (A and B) WT and SERP1 KO MEFs were transfected with pSC100-GFP (A) or pNotchΔE-GFP (NΔE-GFP) (B) and then treated with Compound E (10 nM) for another 24 hours. Fluorescence signals were observed by confocal microscopy. Scale bars, 10 μm. Images are representative of three experiments. (C) HEK293T cells were transfected with pNΔE-GFP and pSERP1 for 24 hours with or without Compound E (1 μM). Cell extracts were analyzed by Western blotting (left), and the amount of NICD in control and SERP1-overexpressed lanes without Compound E (middle two lanes) was quantitated by densitometry analysis (right). (D) Total RNA from SERP1-HA–overexpressed HEK293T cells was analyzed by RT-PCR (left), and the signals on the blots were quantitated (right). (E and F) HeLa cells were transfected for 48 hours with pSC100-GFP, pAPH1A-RFP, and either pSERP1-HA or SERP1 shRNA (E) or with pNΔE-GFP, pAPH1A-RFP, and either pSERP1-HA or SERP1 shRNA (F). Fluorescence signals were observed under a confocal microscope (top). Scale bars, 10 μm. Pearson’s correlation coefficients (PCCs) of SC100-GFP/APH1A-RFP [(E), bottom] and NΔE-GFP/APH1A-RFP [(F), bottom] images (random) were measured using ImageJ software. Data are means ± SD; n = 4 (C) and n = 3 (D to F). *P < 0.05 and **P < 0.01 by unpaired t test.

  • Fig. 3 SERP1 increases the amounts of APH1A/NCT subcomplex and γ-secretase complex.

    (A and B) Amount of γ-secretase holoenzyme in response to SERP1 abundance. Digitonin (1%)–soluble crude membrane fractions prepared from HEK293T cells transfected with pSERP1-HA (A) or psgSERP1 (B) were separated by blue native (BN)-PAGE or SDS-PAGE and analyzed by Western blotting (left), and the signals on the blots were quantitated by densitometry analysis (right). (C and D) Formation of APH1A/NCT subcomplex in response to SERP1 abundance. Digitonin (1%)–soluble membrane fractions prepared from PS1/2 DKO MEF cells transfected with pSERP1-HA (C) or psgSERP1 (PS1/2 DKO/SERP1 KO MEFs) (D) were separated by BN-PAGE and analyzed by Western blotting (left). The signals on the blots were quantitated by densitometry analysis (right). (E to G) Localization of γ-secretase components to lipid rafts in response to SERP1 abundance. HEK293T cells were transfected with pSERP1-HA for 24 hours and solubilized in 1% CHAPS lysis buffer. Equal amount of the soluble lysates was subjected to discontinuous (5, 35, and 45%) sucrose gradient fractionation assay (top, fraction 1; bottom, fraction 12), and each fraction was analyzed by Western blotting (E). DRM fractions (fractions 3 to 5) and non-DRM fractions (fractions 10 to 12) were collected and then analyzed by Western blotting (“D,” DRM; “N,” non-DRM) (F). The relative intensity of PS1-NTF was measured by densitometry analysis (G). Data in all panels are means ± SD; n = 3. *P < 0.05 by unpaired t test.

  • Fig. 4 SERP1 forms a complex with γ-secretase through interacting with APH1A/NCT.

    (A) Mouse cortical tissue lysates were subjected to immunoprecipitation assay with immunoglobulin G (IgG) or antibody to SERP1, followed by Western blotting. L.C., light chain of immunoglobulin. (B) Detection of endogenous SERP1 in γ-secretase subcomplex in membrane fractions prepared from WT and PS1/2 DKO MEFs. Samples were separated by BN-PAGE and analyzed by Western blotting. (C and D) Assessment of SERP1-HA interaction with APH1A and NCT. HEK293T cells were cotransfected with pSERP1-HA and pAPH1A-FLAG (C) or pNCT-V5 (D), and cell lysates (1% Triton X-100) were analyzed by immunoprecipitation assay. H.C., heavy chain of immunoglobulin. (E) PS1-Myc and SERP1-HA–overexpressed HEK293T cells were solubilized in either 1% CHAPS or 1% Triton X-100 lysis buffer and analyzed by immunoprecipitation assay. Blots in (A) to (E) are representative of three experiments. (F) Schematic diagram of SERP1 deletion and chimeric mutants. TMD, transmembrane domain. (G) HEK293T cells were transfected with pNCT-V5 and pSERP1 mutants and then analyzed by immunoprecipitation assay. Blots are representative of three experiments. H.C., heavy chain of immunoglobulin. (H) BiFC assay showing the interactions between SERP1 and NCT in HeLa cells. Scale bars, 10 μm. Images are representative of three experiments. (I) The effect of SERP1 C-terminal region on γ-secretase subcomplex. Membrane fractions prepared from SERP1 WT or SERP1ΔC-overexpressed PS1/2 DKO MEFs were separated by BN-PAGE (top). The amount of APH1A was quantified (bottom). Data are means ± SD; n = 3. *P < 0.05 by unpaired t test.

  • Fig. 5 ER stress increases γ-secretase activity and Aβ generation via SERP1.

    (A and B) HEK293T cells were cotransfected with pAPPswe-FLAG and either pSuper-neo or SERP1-shRNA (shSERP1), incubated in 0.5 μM thapsigargin (Tg) for another 24 hours, and the conditioned medium was analyzed for Aβ40 (A) and Aβ42 (B) using an ELISA kit. (C) HEK293T/shSERP1 cells were transfected with pC99-GVP and pUAS-luciferase, treated with 1 μM thapsigargin for 24 hours, and luciferase reporter assay was performed. (D and E) HEK293-APP695 (D) and HEK293T (E) cells were transfected with pshSERP1 and treated with 0.5 μM thapsigargin or 0.5 μM tunicamycin (Tuni) for 24 hours. Membrane fractions were subjected to AICD generation assay [(D), left] and Western blotting [(E), top]. The signals on the blots were quantitated by densitometry analysis [(D), right; (E), bottom]. (F) Effect of SERP1 on the formation of γ-secretase complex during ER stress. Crude membrane fractions prepared from HeLa/shCtrl and HeLa/shSERP1 cells after treatment with thapsigargin (0.5 μM for 24 hours) were separated by BN-PAGE for Western blot analysis (left), and the signals on the blots were quantitated by densitometry analysis (right). Data are means ± SD; n = 3 experiments (A to C, E, and F) or n = 4 experiments (D). *P < 0.05 and **P < 0.01 by unpaired t test.

  • Fig. 6 SERP1 contributes to Aβ generation in STZ-induced diabetic AD mouse model.

    (A) Effect of STZ treatment on serum glucose levels in 3x Tg-AD mice. High-titer lentiviral vectors expressing shSERP1 or shCtrl (as a control) were intracranially injected into the hippocampi of 3x Tg-AD mice (2 months old). After 19 days of the daily intraperitoneal injection with vehicle (Veh.) or STZ, serum, glucose levels were measured (n = 5 for control and shSERP1 with vehicle and n = 6 for control and shSERP1 with STZ). (B and C) Total amounts of Aβ40 and Aβ42 were measured by ELISA in the hippocampal extracts of each group (n = 5 for each group). (D) Digitonin (1%)-soluble membrane fractions prepared from control and diabetic AD model mice were subjected to BN-PAGE or SDS-PAGE and then analyzed by immunoblotting (left). The amount of PS1-NTF and SERP1 was quantified by densitometry analysis (right). Data in (A) to (D) are means ± SEM; n = 5. *P < 0.05 and **P < 0.01 by unpaired t test. (E) The relative amount of SERP1 was measured in the hippocampi of patients with AD. Hippocampal extracts from normal (control; n = 6) and AD patient (n = 8) postmortem brain tissues were analyzed by Western blotting and densitometry analysis. Data are means ± SEM. *P < 0.05, ***P < 0.001, or not significant (n.s.), by unpaired t test. (F) Immunohistochemical detection of SERP1 and APH1A in the hippocampi of patients with AD. Scale bars, 20 μm. Images are representative of three experiments.

  • Fig. 7 A proposed model on APP cleavage regulation by SERP1.

    Increased SERP1 under ER stress interacts with the APH1A/NCT subcomplex and stabilizes it, leading to increased abundance of γ-secretase complex and its abundance incorporation into lipid rafts. We propose that increased γ-secretase complex in lipid rafts enables more interaction with APP than with Notch, thereby promoting the generation of Aβ.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/623/eaax8949/DC1

    Fig. S1. SERP1 expression influences AICD generation.

    Fig. S2. SERP1 decreases the interaction of PS1 and NΔE.

    Fig. S3. SERP1 regulates the amounts of γ-secretase subunits.

    Fig. S4. SERP1 interacts with γ-secretase complex.

    Fig. S5. The subcomplex of γ-secretase is dissociated by 1% Triton X-100.

    Fig. S6. SERP1 colocalizes with APH1A.

    Fig. S7. The C terminus of SERP1 is critical for γ-secretase activity.

    Fig. S8. ER stress regulates the amount of SERP1 via IRE1 pathway.

    Fig. S9. SERP1 and γ-secretase complex are up-regulated in diabetic mouse brains.

    Fig. S10. Hyperglycemia increases the abundance of SERP1 and γ-secretase complex in SH-SY5Y cells.

    Fig. S11. SERP1 abundance is increased in the parietal lobes of patients with Braak stage VI AD.

    Fig. S12. Immunohistochemical detection of SERP1, SYP, or GFAP in the hippocampi of patients with AD.

    Table S1. Top hits from the genome-wide functional screen.

    Data file S1. Genome-wide functional screen data.

  • The PDF file includes:

    • Fig. S1. SERP1 expression influences AICD generation.
    • Fig. S2. SERP1 decreases the interaction of PS1 and NΔE.
    • Fig. S3. SERP1 regulates the amounts of γ-secretase subunits.
    • Fig. S4. SERP1 interacts with γ-secretase complex.
    • Fig. S5. The subcomplex of γ-secretase is dissociated by 1% Triton X-100.
    • Fig. S6. SERP1 colocalizes with APH1A.
    • Fig. S7. The C terminus of SERP1 is critical for γ-secretase activity.
    • Fig. S8. ER stress regulates the amount of SERP1 via IRE1 pathway.
    • Fig. S9. SERP1 and γ-secretase complex are up-regulated in diabetic mouse brains.
    • Fig. S10. Hyperglycemia increases the abundance of SERP1 and γ-secretase complex in SH-SY5Y cells.
    • Fig. S11. SERP1 abundance is increased in the parietal lobes of patients with Braak stage VI AD.
    • Fig. S12. Immunohistochemical detection of SERP1, SYP, or GFAP in the hippocampi of patients with AD.
    • Table S1. Top hits from the genome-wide functional screen.
    • Legend for data file S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Genome-wide functional screen data.

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