Abstract
Innate immunity is associated with Alzheimer’s disease1, but the influence of immune activation on the production of amyloid-β is unknown2,3. Here we identify interferon-induced transmembrane protein 3 (IFITM3) as a γ-secretase modulatory protein, and establish a mechanism by which inflammation affects the generation of amyloid-β. Inflammatory cytokines induce the expression of IFITM3 in neurons and astrocytes, which binds to γ-secretase and upregulates its activity, thereby increasing the production of amyloid-β. The expression of IFITM3 is increased with ageing and in mouse models that express familial Alzheimer’s disease genes. Furthermore, knockout of IFITM3 reduces γ-secretase activity and the formation of amyloid plaques in a transgenic mouse model (5xFAD) of early amyloid deposition. IFITM3 protein is upregulated in tissue samples from a subset of patients with late-onset Alzheimer’s disease that exhibit higher γ-secretase activity. The amount of IFITM3 in the γ-secretase complex has a strong and positive correlation with γ-secretase activity in samples from patients with late-onset Alzheimer’s disease. These findings reveal a mechanism in which γ-secretase is modulated by neuroinflammation via IFITM3 and the risk of Alzheimer’s disease is thereby increased.
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Source data are provided with this paper. All other data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank A. Kentsis and R. Hendrickson for LC–MS/MS and proteomic analysis. We thank H. Brogdon and N. L. Nadon for mouse brains. We thank E. Sikora for Apoe genotyping, D. Yarilin for immunostaining, S. Fujisawa and Y. Romin for help with microscopy, and S. Shuldberg for human brain samples. We thank S. Wagner for providing NGP-97555. This work is supported by the JPB Foundation (Y.-M.L., P.G. and A.G.), the Fisher Center for Alzheimer’s Research Foundation (P.G.), Cure Alzheimer’s Fund (Y.-M.L), the National Institutes of Health R01NS096275 (Y.-M.L.), RF1AG057593 (Y.-M.L.), R01AG061350 (Y.-M.L.), R01AG046170 (B.Z.), RF1AG057440 (B.Z.) and R01AG057907 (B.Z.). We also acknowledge the MSK Cancer Center Support Grant/Core Grant (grant P30 CA008748) the ADRC parent grant (P30 AG062429), Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center of MSKCC, and the William Randolph Hearst Fund in Experimental Therapeutics.
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Contributions
J.-Y.H. and Y.-M.L. conceived the study. J.-Y.H., G.R.F. and Y.-M.L. planned, and J.-Y.H., G.R.F. and X.W. performed most of the experiments, and J.-Y.H., G.R.F. and Y.-M.L. analysed the data. X.W. and C.C. performed proteomics analysis. S.J.P. and E.W. cloned the constructs and S.J.P. expressed a recombinant substrate protein. E.W. performed a photolabelling study in primary neurons and Notch AlphaLISA assay in knockout cells. M.B. performed immnuofluorescence. T.L. perfused mouse brains and T.L., Y.Z. and Y.K. helped with primary neuronal culture. P.N. synthesized compounds. J.C.W. assisted with gene expression studies and analysed the large human brain dataset. J.T. cultured human iPS cell-derived neurons and astrocytes and performed immunofluorescence. L.G., A.M., C.M., X.Z., M.W. and B.Z. analysed gene expression in a large human brain dataset. Y.S. performed qPCR in mouse hippocampal cells and analysed the data. A.E.R. and B.T.E. performed SNP genotyping and analysed the data. K.R.S. and R.V. coordinated and provided 5xFAD transgenic tissue samples. I.T., R.A.R. and E.M. coordinated and provided the collection of human brain samples. G.R.F. summarized findings in illustrations. J.-Y.H., J.C.W., R.V., B.Z., D.S.J., E.M., P.G., A.G. and Y.-M.L. analysed the data. J.-Y.H. wrote the initial draft of the manuscript and, J.-Y.H., G.R.F. and Y.-M.L. revised the manuscript. All authors discussed and commented on the manuscript.
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Y.-M.L. is co-inventor of intellectual property (assay for gamma secretase activity and screening method for gamma secretase inhibitors) owned by MSKCC and licensed to Jiangsu Continental Medical Development.
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Extended data figures and tables
Extended Data Fig. 1 Identification of IFITM3 as γ-secretase binding protein.
a, LC–MS/MS analysis of the 15-kDa band identified four peptides that match with human IFITM3. b, Western blotting analysis of PS1-NTF protein labelled with E2012-BPyne (500 nM). c, Structures of imidazole GSMs, acid GSM and GSIs. d, Western blotting analysis of E2012-BPyne-labelled proteins in the absence or presence of imidazole GSMs, acid GSM and GSIs. Labelled proteins were captured and analysed by western blotting for IFITM3. e, Structures of GY6 and 163-BP-l-biotin. f, IFITM3 co-immunoprecipitates with γ-secretase subunits. CHAPSO-solubilized cell membranes were immunoprecipitated with anti-IFITM3 antibody and probed with antibodies against PS2-CTF and PEN-2. Rabbit IgG was used as a negative control. g, IFITM3 does not co-immunoprecipitate with SPP. CHAPSO-solubilized cell membranes were immunoprecipitated with a monoclonal anti-IFITM3 antibody (9D11) and probed with antibodies against SPP and IFITM3. Mouse IgG was used as a negative control. h, Analysis of the total protein level in wild-type MEF or PS1/PS2 double knockout MEF cells. The same amount of membrane proteins was loaded and analysed by western blotting. i, Ifitm3 mRNA expression levels were measured by RT–PCR in wild-type MEFs or PS1/PS2 double knockout MEF cells (n = 6). All western blotting images and graphs are representative of three independent experiments (except in a and d, which represent two replicates). Data are mean ± s.d. ns, not significant. P values were determined by two-sided Student’s t-test.
Extended Data Fig. 2 Effect of IFITM3 knockdown and knockout on γ-secretase.
a, Quantification of western blotting (Fig. 2a) showed that IFITM3 knockdown did not change protein expression levels of APP, NCT and PS1-NTF in HEK-APP wild-type cells (n = 3). b, Schematic representation of cell-free γ-secretase assay. γ-secretase is incubated with a recombinant APP substrate in the presence of 0.25% CHAPSO. Cleaved Aβ40 and Aβ42 species are measured with cleavage specific antibodies and AlphaLISA technology. c, Schematic model showing different GSM and GSI binding sites in γ-secretase: E2012 (imidazole GSM), GSM-1 (acid GSM), and L458 (transition state analogue inhibitor, GSI). d–f, Comparison of IC50 values of GSM-25 (EV: n = 9, KO: n = 8) for Aβ40 (****P < 0.0001) and Aβ42 (ns) (d), GSM-1 (n = 6) for Aβ40 (**P = 0.0039) and Aβ42 (ns) (e), and L458 (n = 3) for Aβ40 (ns) and Aβ42 (ns) (f) cleavages in the U138 EV or KO cell lines (n ≥ 3). g, IFITM3 knockdown (KD) does not affect expression of γ-secretase subunits. IFITM3 was knocked down by siRNA (6 pmol, n = 3) in HEK-Notch∆E cells and scramble siRNA (SC, n = 3) was used as a negative control. Cell lysates were probed by antibodies against NCT, PS1-NTF and IFITM3. β-Actin was used as a loading control. h, Effect of IFITM3 knockdown on γ-secretase activity. IFITM3 knockdown increased γ-secretase cleaved product NICD, analysed by western blotting. Cell lysates were probed by antibodies against c-Myc (NotchΔE) and NICD and a representative quantification of NICD (n = 8, ***P = 0.001) is shown (bottom). i, Cell-based NICD AlphaLISA assay (left) revealed an increase in NICD production with IFITM3 knockdown. Quantification of NICD (n = 8, ***P = 0.001) is shown in the right panel. j, Effect of IFITM3 knockout on γ-secretase activity. Knockout cells lines have increased γ-secretase activity as compared to the EV cell line. The NICD cleavage in vitro was measured by AlphaLISA assay (n = 3, **P = 0.0096). All western blotting images and graphs are representative of three independent experiments. Data are mean ± s.d. P values were determined by two-sided Student’s t-test.
Extended Data Fig. 3 Effect of ageing and the expression of APP/PS1 on the level of γ-secretase and IFITM3.
a, Western blotting for NCT and PS1-NTF (Fig. 3a) were quantified by Odyssey imaging (n = 5 mice pooled per group, except n = 4 for 28F, ns). b, Effect of ageing on subcellular localizations of IFITM3. A hemibrain from male wild-type C57BL/6 mouse at 4 and 28 months (n = 1 per group) were homogenized and layered on iodixanol gradient (2.5–30%). Fractions were collected from the top and resolved by western blotting for γ-secretase, IFITM3 and different subcellular markers. c, Western blotting for APP, NCT and PS1-NTF (Fig. 3e) were quantified by Odyssey imaging (n = 5 mice per group except n = 4 for WT at 12 months). APP: 3moWT-3mo5x: ****P < 0.0001, 3mo5x-12mo5x: ****P < 0.0001, 12moWT-12mo5x: ****P < 0.0001). NCT: 3moWT-12moWT: ****P < 0.0001, 3mo5x-12mo5x: ****P < 0.0001, 12moWT-12mo5x: ****P < 0.0001. PS1-NTF: 3moWT-12mo5x: ***P = 0.0004, 12moWT-12mo5x: ****P < 0.0001. d, Immunostaining of IFITM3 in mouse brains. Fluorescence microscopy of IFITM3 expression in 12-month-old PFA-perfused mice (WT, top; 5xFAD, bottom). Representative images of cortex, hippocampus and subiculum (left to right) show IFITM3 (green) and DAPI (blue). Scale bars, 1 mm, 200 μm, 100 μm (left to right). Total IFITM3 fluorescence area within the hippocampus and cortex of WT and 5xFAD was quantified using FIJI. Total IFITM3 was divided by tissue area and IFITM3 expression was normalized to average of WT (WT: n = 7, 5xFAD: n = 9) (cortex: **P = 0.0035, hippocampus: ****P < 0.0001). e, IFITM3 expression in astrocytes and microglia is upregulated in 5xFAD mice compared to wild-type mice. Fluorescence microscopy of IFITM3, GFAP (top) and IBA1 (bottom) expression in 12-month-old PFA-perfused mice (WT, top; 5xFAD, bottom). Representative images of the hippocampus and cortex show IFITM3 (red), GFAP (green, top), IBA1 (green, bottom), and DAPI (blue). Scale bar, 500 μm. Inset panels (left to right) show GFAP or IBA1 (green), IFITM3 (red) and merged staining. Scale bar, 50 μm. All western blotting images and graphs are representative of three independent experiments (except in b, which denotes two replicates). Data mean ± s.d. P values were determined by two-sided Student’s t-test (except in c, which was by one-way ANOVA followed by Tukey).
Extended Data Fig. 4 Expression profile of IFITM3 and other markers in LOAD and age-matched controls.
a, Spearman’s correlation of mRNA expression of human IFITM3 gene with age was analysed in the cortex (n = 158) and hippocampus (n = 123) of normal human brains using the GTEx cohort. b, mRNA expression in samples from non-demented control participants (n = 10) and patients with LOAD (n = 18) of MAP2 (ns), GFAP (**P = 0.0046), and AIF1 (ns) were measured, which were used in Fig. 4c, d. c, Expression profiles of MAP2 (ns), GFAP (****P < 0.0001), and AIF1 (ns) in the temporal cortex of samples from human control participants (n = 76) and patients with LOAD (n = 80) using the Mayo Clinic cohort data. Correlation analyses were carried out and P values were calculated. d, The protein levels of NCT (****P < 0.0001) and PS1-NTF (**P = 0.0042) in human brain membranes (control and LOAD). The samples were analysed by western blotting and quantified (n = 10 and 18, respectively). Signal was normalized to HeLa cell membrane. e, f, IFITM3 SNP genotypes. e, Allelic discrimination plot depicting rs34481144 genotype calls for control (n = 9), LOAD-L (n = 10), and LOAD-H (n = 8) brain samples. The axes show delta Rn values obtained from TaqMan SNP genotyping analysis. Samples without genomic DNA were used as non-template controls (shown as black squares in the left lower quadrant, n = 2). f, Allele frequency of rs34481144 genotype in control (n = 9) and LOAD (n = 18). g, mRNA level of Ifitm3 in four types of eGFP/L10a-expressing mouse hippocampal neurons (GAD2 (glutamate decarboxylase 2), CCK (cholecystokinin), PV (parvalbumin), and CORT (cortistatin) expressing GABAergic neurons) (n = 4 per group, GAD2-PV: ***P = 0.0003, CCK-PV: ****P < 0.0001, CCK-Cort: *P = 0.0363, PV-Cort: **P = 0.0059). h, mRNA levels of IFITM3 in human iPS-cell-derived neurons (n = 4) and human primary astrocytes (n = 3) were measured by qPCR (****P < 0.0001). i, j, Human iPS-cell-derived neurons (i) and human primary astrocytes (j) were stained for IFITM3 with MAP2 (neuronal marker) or S100β (astrocyte marker). DAPI was used for nucleus staining. Scale bars, 200 μm and 500 μm. k, Induction of IFITM3 by IFNα in primary neurons. E16 mouse primary neurons were treated with 100 ng ml−1 of IFNα at DIV12 for 24 h. The protein levels of γ-secretase and IFITM3 were analysed by western blotting (n = 4 per group). β-tubulin III was used as a loading control. l, Effect of IFITM3 induction on γ-secretase activity for Aβ40 (*P = 0.0116) and Aβ42 (*P = 0.0319) activity. Membranes from primary neurons were incubated with the recombinant APP substrate C100-∆ID-FLAG and γ-secretase activity (Aβ cleavage rate) was assayed by human Aβ three-plex MSD kits (n = 12, 10). m, JC8 whole-cell photolabelling. Neuronal membranes were photolabelled with JC8 in the absence or presence of L458 and analysed by anti-PS1-NTF antibody. Photolabelled PS1-NTF protein level was quantified by Odyssey imaging (n = 3, *P = 0.0210). n, Spearman’s correlation between the expression level of IFITM3 and viruses. In the BA-22 region in the MSBB cohort, the expression level of IFITM3 is positively correlated with the expression level of HHV-6B (rho = 0.248, P = 0.044, n = 66). In the BA-36 region, the expression level of IFITM3 is positively correlated with the expression of hepatitis C virus genotype 4 (rho = 0.255, P = 0.033, n = 70). All western blotting images and graphs are representative of two independent experiments (except in b and e, which denote one replicate; in h, data are pooled from two experiments; in k, which denotes three replicates; and in l, data are pooled from four experiments). Data are mean ± s.d. Violin plots represent median (middle line) and interquartile range (outer lines). Two-sided Student’s t-test (except in g, which was by one-way ANOVA followed by Tukey).
Extended Data Fig. 5 Correlation between L505 labelled protein and γ-secretase activity.
a, Structures of JC8, L505, L646, GY4 and L631. b, Western blotting analysis of 11Bt-labelled proteins in the absence or presence of its parent compound, a substrate binding site inhibitor pep11 and imidazole GSM, E2012. Labelled proteins were captured and analysed by western blotting for PS1-NTF (n = 1). c, Pearson’s correlation between γ-secretase activity (Aβ40, Aβ42 cleavage rates in Fig. 4d) and L505-labelled PS1-NTF (in Fig. 5c) in LOAD samples (n = 10). Linear regression analysis was used to calculate R and P values.
Supplementary information
Supplementary Figure
Supplementary Figure 1: Gel Source Data.
Supplementary Table
Supplementary Table 1: List of human brain tissues used in Figure 4.
Supplementary Table
Supplementary Table 2: Spearman’s correlation between the expression level of IFITM3 and Aβ load.
Supplementary Table
Supplementary Table 3: Human IFITM3 SNP Genotypes.
Supplementary Table
Supplementary Table 4: Correlation between IFITM3 and cytokines in a large AD human dataset.
Supplementary Table
Supplementary Table 5: Correlation between IFITM3 and of type I IFN responsive genes in a large AD human dataset.
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Hur, JY., Frost, G.R., Wu, X. et al. The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer’s disease. Nature 586, 735–740 (2020). https://doi.org/10.1038/s41586-020-2681-2
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DOI: https://doi.org/10.1038/s41586-020-2681-2
