Research ArticleAlzheimer’s Disease

Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer’s disease

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Science Translational Medicine  25 May 2016:
Vol. 8, Issue 340, pp. 340ra72
DOI: 10.1126/scitranslmed.aaf1059
  • Fig. 1. Aβ expression protects against S. Typhimurium meningitis in genetically modified AD mouse models.

    Transgenic (5XFAD) mice expressing human Aβ and mice lacking murine APP (APP-KO) were compared to genetically unmodified littermates [wild type (WT)] for resistance to S. Typhimurium meningitis. One-month-old mice received single ipsilateral intracranial injections of S. Typhimurium, and clinical progression was followed to moribundity. (A to C) Performance of 5XFAD (n = 12) mice compared to WT (n = 11) are shown after infection for survival (P = 0.009) (A), clinical score (P < 0.0001) (B), and percent weight loss (P = 0.0008) (C). (D) S. Typhimurium load 24 hours after infection in 5XFAD (n = 4) and WT (n = 4) mouse brain hemisphere homogenates shown as mean CFU ± SEM (*P = 0.03 and **P = 0.04). (E) APP-KO mice (n = 15) show a trend (P = 0.104) toward reduced survival compared to WT (n = 15) littermates after infection. (F) No mortality was observed among control sham-infected WT (n = 6) or 5XFAD (n = 6) mice injected with heat-killed S. Typhimurium. Statistical significance was calculated by log-rank (Mantel-Cox) test for survival (A, E, and F), linear regression for clinical score and weight (B and C), and statistical means compared by t test for brain bacterial loads (D). For survival and clinical analysis (A to C), data were pooled from three independent experiments.

  • Fig. 2. Aβ expression in nematodes and cultured cells increases host resistance to infection by Candida.

    Aβ-mediated protection against C. albicans (Candida) was characterized in C. elegans and cultured host cell monolayer mycosis models. Experimental nematodes included control (Cont.) non-Aβ expressing (CL2122) and transgenic (Tg) human Aβ-expressing (GMC101) strains. Host cell lines included control nontransformed (H4-N and CHO-N) and transformed Aβ-overexpressing (H4-Aβ40, H4-Aβ42, and CHO-CAB) cells. (A) Survival curves for CL2122 (n = 61) and GMC101 (n = 57) nematodes after infection with Candida (P < 0.00001). (B) Viability of nontransformed and transformed host cell monolayers after 28-hour incubation with Candida. Host cell viability was followed by prelabeling host cell monolayers with BrdU and then comparing wells for an anti-BrdU signal. Signal of infected wells shown as percentage of uninfected control wells (*P = 0.002, **P = 0.001, and ***P = 0.004). (C) Candida adherence to host cells. Fluorescence micrograph of Calcofluor white–stained Candida adhering to control H4-N or transformed H4-Aβ42 host cell monolayers after 2 hours of co-incubation in preconditioned culture medium. (D) Quantitative analysis of Candida host cell colonization. Adhering Candida were detected using an immunochemical luminescence assay with anti-Candida antibodies (*P = 0.003, **P = 0.001, and ***P = 0.004). Well comparisons use arbitrary luminescence units (AU). (E) Phase-contrast micrographs of agglutinated Candida after overnight incubation with H4-N or H4-Aβ42 host cells. (F) Quantitative analysis of Candida agglutination. Wells were compared for yeast aggregate surface area using image analysis software (*P = 0.007, **P = 0.002, and ***P = 0.009). Bars in (B), (D), and (F) are means of six replicate wells ± SEM. Statistical significance was calculated by log-rank (Mantel-Cox) test for nematode survival (A) and statistical mean comparisons by t test (B, D, and F). Micrographs (C and E) are representative of data from three replicate experiments and multiple discrete image fields (table S1A).

  • Fig. 3. Aβ’s protective actions in cell culture are mediated by adhesion inhibition and agglutination activities against Candida.

    C. albicans adhesion to abiotic surfaces and agglutination in the bulk phase were characterized in the presence of cell-derived or synthetic Aβ. After 36 hours of conditioning, host cell–free culture medium was collected from control nontransformed (H4-N or CHO-N) or transformed Aβ-overexpressing (H4-Aβ40, H4-Aβ42, or CHO-CAB) cultured cells. Aβ-immunodepleted (ID α-Aβ) and control immunodepleted [ID IgG (immunoglobulin G)] media were prepared by incubation with immobilized anti-Aβ or nonspecific antibodies. Experimental synthetic peptides included Aβ (Aβ40 and Aβ42), AMP positive control (LL-37), and negative control scrambled Aβ42 (scAβ42). (A and B) Comparison of ID α-Aβ and ID IgG medium’s adhesion inhibition (*P = 0.009, **P = 0.001, and ***P = 0.004) and agglutination (*P = 0.001, **P = 0.0005, and ***P = 0.004) activities. (C and D) Comparison of anti-Candida activities of serially diluted conditioned medium and synthetic peptides. (E and F) Activities of synthetic Aβ42 monomer, soluble oligomeric ADDLs, and protofibril preparations. (G and H) Conditioned culture medium adhesion inhibition (*P = 0.003 and **P < 0.0003) and agglutinating (*P < 0.02 and **P < 0.003) source activities alone (Neat) or in the presence of soluble yeast wall carbohydrates (+Glucan or +Mannan). (I) Synthetic monomeric Aβ42 and cell-generated peptide from H4-Aβ42 cells were compared for Candida binding using an Aβ/Candida binding ELISA. (J) Untreated, immunodepleted, or glucan (Glu)- or mannan (Man)–spiked H4-Aβ42 conditioned media were incubated with intact immobilized yeast cells in an Aβ/Candida binding ELISA assay (*P = 0.006, **P = 0.008, and ***P < 0.004). Synthetic peptide incubations (C to F and I) were performed in H4-Aβ42 conditioned culture medium pretreated to remove cell-derived Aβ by α-Aβ immunodepletion. Symbols and bars for (A) to (J) are statistical means of six replicate wells ± SEM. Statistical significance was by t test.

  • Fig. 4. β-Amyloid fibrils propagate from yeast surfaces and capture Candida in H4-Aβ42 medium.

    Early-stage C. albicans aggregates harvested from H4-Aβ42 conditioned medium were probed with α–Aβ-Au nanoparticles and analyzed by TEM. (A) Yeast agglutination is mediated by fibrillar structures. The micrograph shows fibrils binding cells within yeast aggregates and linking C. albicans clusters. (B) Fibrillar structures extending from yeast cell surfaces. (C and D) α–Aβ-Au nanoparticle labeling of short fibrillar structures extending from C. albicans surfaces and long fibrils running between yeast clumps. (E) Absorption experiment showing ablated α–Aβ-Au binding of fibrils extending from yeast in the presence of soluble synthetic Aβ peptide. Data are consistent with specific α–Aβ-Au labeling of β-amyloid fibrils. Micrographs are representative of data from three replicate experiments and multiple discrete image fields (table S1A).

  • Fig. 5. Candida cells are entrapped by Aβ in H4-Aβ42 culture medium.

    After overnight incubation with H4-Aβ42 medium, yeast (C. albicans) aggregates were harvested and probed for β-amyloid markers. (A and B) Visible yeast aggregates (VIS), yeast aggregates stained with green fluorescent Thioflavin S (ThS FLU), yeast aggregates probed with red fluorescent anti-Aβ (α-Aβ FLU) antibodies, and superimposed images (VIS/FLU overlay). Yeast aggregates generated with the control synthetic LL-37 peptide (A) are negative for Thioflavin S–enhanced fluorescence. (B) Yellow denotes colocalization of anti-Aβ and Thioflavin S signals. Colocalization of these signals is the hallmark of Aβ. (C) SEM analysis revealed fibrous material in H4-Aβ42 yeast aggregates that is absent from control C. albicans pellets prepared by centrifugation in H4-N medium. (D) H4-Aβ42 yeast aggregates incubated with immunogold nanoparticles coated with anti-Aβ antibodies (α–Aβ-Au) and analyzed by TEM. The first and second panels show labeling of fibrous material by α–Aβ-Au. The third panel shows inhibition of α–Aβ-Au nanoparticle binding by soluble synthetic Aβ peptide (α–Aβ-Au + Aβ peptide), consistent with specific labeling of β-amyloid. Micrographs are representative of data from two or more replicate experiments and multiple discrete image fields (table S1A).

  • Fig. 6. Intestinal infection with Candida induces Aβ fibrillization in transgenic GMC101 nematode gut.

    Aβ42-expressing GMC101 C. elegans were infected with C. albicans (Candida) and probed for anti-Aβ immunoreactivity and β-amyloid markers using TEM and confocal fluorescence microscopy (CFM). (A) Micrograph shows positive labeling of yeast cell surface in GMC101 worm gut by immunogold nanoparticles coated with anti-Aβ antibodies (α–Aβ-Au) after Candida ingestion. (B to D) Visible (VIS) and fluorescence signals from freeze-fracture nematode sections with advanced Candida infections. (B) Comparison of uninfected and infected worms. (C and D) Thioflavin S and anti-Aβ staining for gut yeast aggregates. Signals include anti-Candida immunoreactivity (α-Candida), Thioflavin S–enhanced fluorescence (ThS), anti-Aβ immunoreactivity (α-Aβ), and superimposed (Overlay) signals. Yellow denotes signal colocalization. Uninfected and infected CL2122 nematode controls were negative for anti-Aβ immunoreactivity and enhanced Thioflavin S fluorescence (figs. S2 and S8). Micrographs are representative of data from three or more replicate experiments and multiple discrete image fields (table S1B).

  • Fig. 7. Infection-induced β-amyloid deposits colocalize with invading S. Typhimurium cells in 5XFAD mouse brain.

    Four-week-old WT mice or transgenic 5XFAD animals expressing high levels of human Aβ were injected intracerebrally with viable S. Typhimurium bacteria. Mice were also injected with heat-treated S. Typhimurium cell debris as a negative control for the injection procedure. (A and B) Mouse brain sections were prepared 24 (A) or 48 hours (B) after infection. Signals shown include visible (VIS), anti-Salmonella immunoreactivity (α-Salmonella), enhanced Thioflavin S fluorescence (ThS) or anti-Aβ immunoreactivity (α-Aβ), and superimposed (Overlay) signals. Panels are representative images of multiple images captured as Z-sections using CFM. Yellow denotes signal colocalization (Z-series projections showing β-amyloid surrounding and entrapping bacterial colonies in a rotating three-dimensional section of 5XFAD mouse brain are also included in video S1). Micrographs are representative of data from three replicate experiments and multiple discrete image fields (table S1C).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/340/340ra72/DC1

    Materials and Methods

    Fig. S1. Aβ deposition and inflammation in 5XFAD mice before infection and criteria used for assessing clinical performance after infection.

    Fig. S2. Aβ42 localizes to gut and muscle in GMC101 nematodes.

    Fig. S3. Aβ expression protects GMC101 nematodes and CHO-CAB cells against S. Typhimurium.

    Fig. S4. Confirmation of increased Candida resistance among transformed host cells using three independent assays.

    Fig. S5. Transformed cell lines generate Aβ oligomers at levels found in the soluble fraction of human brain.

    Fig. S6. Transformed H4-Aβ40 and CHO-CAB host cells resist Candida colonization and agglutinate yeast.

    Fig. S7. Birefringence of Congo red–stained yeast aggregates from H4-Aβ42 medium.

    Fig. S8. Anti-Aβ antibodies do not label CL2122 tissues or yeast.

    Fig. S9. β-Amyloid colocalizes with S. Typhimurium cells in 5XFAD brain.

    Fig. S10. Model for antimicrobial activities of soluble Aβ oligomers.

    Table S1. Figure micrographs are representative of data from multiple repeat experiments and image fields.

    Video S1. Z-section projection of 5XFAD mouse brain showing β-amyloid entrapment of S. Typhimurium cells.

  • Supplementary Material for:

    Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer's disease

    Deepak Kumar Vijaya Kumar, Se Hoon Choi, Kevin J. Washicosky, William A. Eimer, Stephanie Tucker, Jessica Ghofrani, Aaron Lefkowitz, Gawain McColl, Lee E. Goldstein, Rudolph E. Tanzi,* Robert D. Moir*

    *Corresponding author. Email: moir{at}helix.mgh.harvard.edu (R.D.M.); tanzi{at}helix.mgh.harvard.edu (R.E.T.)

    Published 25 May 2016, Sci. Transl. Med. 8, 340ra72 (2016)
    DOI: 10.1126/scitranslmed.aaf1059

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Aβ deposition and inflammation in 5XFAD mice before infection and criteria used for assessing clinical performance after infection.
    • Fig. S2. Aβ42 localizes to gut and muscle in GMC101 nematodes.
    • Fig. S3. Aβ expression protects GMC101 nematodes and CHO-CAB cells against S. Typhimurium.
    • Fig. S4. Confirmation of increased Candida resistance among transformed host cells using three independent assays.
    • Fig. S5. Transformed cell lines generate Aβ oligomers at levels found in the soluble fraction of human brain.
    • Fig. S6. Transformed H4-Aβ40 and CHO-CAB host cells resist Candida colonization and agglutinate yeast.
    • Fig. S7. Birefringence of Congo red–stained yeast aggregates from H4-Aβ42 medium.
    • Fig. S8. Anti-Aβ antibodies do not label CL2122 tissues or yeast.
    • Fig. S9. β-Amyloid colocalizes with S. Typhimurium cells in 5XFAD brain.
    • Fig. S10. Model for antimicrobial activities of soluble Aβ oligomers.
    • Table S1. Figure micrographs are representative of data from multiple repeat experiments and image fields.
    • Legend for video S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Video S1 (.mov format). Z-section projection of 5XFAD mouse brain showing β-amyloid entrapment of S. Typhimurium cells.

    [Download Video S1]

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