Research ArticleMOLECULAR NEUROSCIENCE

Selective targeting of primary and secondary nucleation pathways in Aβ42 aggregation using a rational antibody scanning method

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Science Advances  21 Jun 2017:
Vol. 3, no. 6, e1700488
DOI: 10.1126/sciadv.1700488
  • Fig. 1 Schematic representation of the DesAb panel against Aβ42 generated using the antibody scanning method described in this work.

    The five target epitopes, which scan the Aβ42 sequence, are shown as green-framed rectangular boxes, whereas the corresponding designed complementary peptides grafted into the CD3 loop of the single-domain human antibody scaffold are highlighted in green.

  • Fig. 2 Structural and functional characterization of the DesAbs.

    CD spectra (A) and CD thermal denaturation (B) of the DesAbs used in this work. WT, wild type. (B) Denaturation data are reported as fraction of the folded protein (see Materials and Methods). (C) BLI measurements of the binding of the DesAbs to SA sensor chip coated with monomeric biotinylated Aβ42. Each curve was subtracted from a curve of binding of the corresponding DesAb to an uncoated sensor chip; the Kd values of binding to monomeric Aβ42 are reported. Given the proximity of the target peptide of DesAb3–9 to the biosensor surface, the affinity of DesAb3–9 for monomeric Aβ42 was determined with biotin-mediated affinity measurements (fig. S3). n.a., not applicable. (D) Fibril binding experiments of the DesAbs; the Kd values of binding to fibrillar Aβ42 are reported. DesAb3–9, black; DesAb13–19, orange; DesAb18–25, blue; DesAb29–36, green; DesAb36–42, red; DesAb-F (a DesAb that targets α-synuclein), gray. The gray dashed line in (B) indicates the Tm (≈73°C) of the original scaffold as reported in the literature (48).

  • Fig. 3 The antibody scanning method produces antibodies that affect different microscopic steps in Aβ42 aggregation.

    (A) Model of aggregation of Aβ42 showing the primary (red arrow) and the secondary (blue arrow) nucleation of the oligomers and the elongation of the fibrils (black arrow). (B) Solutions containing 2 μM Aβ42 were incubated in the presence of increasing (blue to green) Aβ42 monomer equivalents of the DesAbs (serial dilutions starting from 1 μM DesAb concentration; see fig. S5); each antibody targets a specific epitope within the sequence of Aβ42 (Fig. 1) and inhibits the aggregation of the peptide in a characteristic manner. Continuous lines represent the fits of the data using the integrated rate law for Aβ42 aggregation (see Materials and Methods). (C) Seeded aggregation of Aβ42 in the presence of 10% preformed fibrils with a 0:1 (blue) or 1:1 (green) antibody–to–Aβ42 monomer ratio. a.u., arbitrary units. (D) Bar plot showing the inhibition strength of the DesAbs (which is defined as kAβ42/kAβ42+DesAb) on k+ (black), kn (red), and k2 (blue) rate constants, derived from (B), (C), and fig. S5. The fold change in the presence of the antibodies of each of the rate constants is indicated on the top of the corresponding bar. (E) Relative number of oligomers generated during the aggregation reaction with or without a 1:2 antibody–to–Aβ42 monomer ratio.

  • Fig. 4 Effects of DesAb18–25 and DesAb29–36 in a C. elegans model of Aβ42-mediated toxicity.

    (A) Experimental design for the investigation of the effects of the two selected DesAbs in the C. elegans strain GMC101 (the Aβ42 worm model) compared with strain N2 (the control worm model). The pathological phenotype is induced in the worms by increasing their temperature of incubation from 20° to 24°C, which induces Aβ42 aggregation. A pictorial representation of the populations of monomers (light blue), oligomers formed by primary (blue) and secondary (green) nucleations, and fibrils (maroon) at the different stages (in days) of adulthood of the worms is given to illustrate the aggregation process. (B) Phenotypic fingerprints, which consider speed, body bends per minute (BPM), and fraction not paralyzed or the worms, of Aβ42 worms (C. elegans GMC101; yellow) and control worms (C. elegans N2, WT; gray) treated with empty lipid vesicles and after the administration of DesAb29–36 (green) and DesAb18–25 (blue), screened at day 7 of adulthood. DesAbs were administered starting from a 20 μM concentration (see Materials and Methods) at days 1 and 3 (left) or at day 6 (right). The fingerprints show one representative of three biological replicates that showed similar results. The thickness of the lines represents SEM. The bar plots report the total fitness (see Materials and Methods). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 (relative to untreated worms).

  • Table 1 List of the different DesAbs used in this work.
    AntibodyGrafted sequenceTarget sequence
    DesAb3–9HETLTLR(3)EFRHDSG(9)
    DesAb13–19LSVIKEI(13)HHQKLVF(19)
    DesAb18–25VFVGTEA(18)VFFAEDVG(25)
    DesAb29–36GSMYKATV(29)GAIIGLMV(36)
    DesAb36–42LGIKAEL(36)VGGVVIA(42)
    DesAb-F (38)FQEAVSG(70)VVTGVTA(76)
    (α-synuclein)
    DesAb15–21 (38)FKLSVIT(15)QKLVFFA(21)
  • Table 2 Changes in the microscopic rate constants in Aβ42 aggregation in the presence of the different DesAbs used in this work (elongation, k+; primary nucleation, kn; and secondary nucleation, k2).
    Antibodyk+knk+k2k+knk2
    DesAb3–91.2205.168.7173.858.2
    DesAb13–198.01210.9760.9150.194.8
    DesAb18–253.9723.43.2183.60.8
    DesAb29–361.535.1206.223.4137.4
    DesAb36–429.97.41270.812.9

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/6/e1700488/DC1

    fig. S1. Purified DesAbs used in this study.

    fig. S2. BLI analysis of the interaction of different DesAbs with monomeric α-synuclein.

    fig. S3. Biotin-mediated affinity measurement of DesAb3–9 binding to monomeric Aβ42 and setup of the experimental conditions.

    fig. S4. DesAb binding specificity assessment and interaction of DesAb18–25 and DesAb29–36 with the respective target peptides.

    fig. S5. A DesAb designed to target α-synuclein does not inhibit Aβ42 aggregation.

    fig. S6. Effect of the DesAbs on the global parameters k+kn and k+k2 of Aβ42 aggregation.

    fig. S7. Transduction of the fluorescent protein mCherry into wild-type worms.

    fig. S8. Effects of DesAb18–25 and DesAb29–36 treatments on the C. elegans worms.

    fig. S9. Fingerprints of the Aβ42 worms screened at day 4 of adulthood.

    fig. S10. Effects of DesAb18–25 and DesAb29–36 treatments on wild-type control worms.

    fig. S11. Analysis on the specificity of the treatment with the DesAbs in C. elegans.

    fig. S12. Effects of DesAb18–25 and DesAb29–36 treatments on the aggregation of Aβ42 in C. elegans models.

    fig. S13. Difference between the spectrum of DesAb18–25 and the background.

    movie S1. Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with empty vesicles at days 1 and 3 (AP1, early treatment).

    movie S2. Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with empty vesicles at day 6 (AP2, late treatment).

    movie S3. Representative video clip of the control C. elegans worms N2 at day 7 upon treatment with empty vesicles at day 6 (AP2, late treatment).

    movie S4. Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with DesAb18–25 at days 1 and 3 (AP1, early treatment).

    movie S5. Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with DesAb29–36 at days 1 and 3 (AP1, early treatment).

    movie S6. Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with DesAb18–25 at day 6 (AP2, late treatment).

    movie S7. Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with DesAb29–36 at day 6 (AP2, late treatment).

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Purified DesAbs used in this study.
    • fig. S2. BLI analysis of the interaction of different DesAbs with monomeric α-synuclein.
    • fig. S3. Biotin-mediated affinity measurement of DesAb3–9 binding to monomeric Aβ42 and setup of the experimental conditions.
    • fig. S4. DesAb binding specificity assessment and interaction of DesAb18–25 and DesAb29–36 with the respective target peptides.
    • fig. S5. A DesAb designed to target α-synuclein does not inhibit Aβ42 aggregation.
    • fig. S6. Effect of the DesAbs on the global parameters k+kn and k+k2 of Aβ42 aggregation.
    • fig. S7. Transduction of the fluorescent protein mCherry into wild-type worms.
    • fig. S8. Effects of DesAb18–25 and DesAb29–36 treatments on the C. elegans worms.
    • fig. S9. Fingerprints of the Aβ42 worms screened at day 4 of adulthood.
    • fig. S10. Effects of DesAb18–25 and DesAb29–36 treatments on wild-type control worms.
    • fig. S11. Analysis on the specificity of the treatment with the DesAbs in C. elegans.
    • fig. S12. Effects of DesAb18–25 and DesAb29–36 treatments on the aggregation of Aβ42 in C. elegans models.
    • fig. S13. Difference between the spectrum of DesAb18–25 and the background.

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with empty vesicles at days 1 and 3 (AP1, early treatment).
    • movie S2 (.avi format). Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with empty vesicles at day 6 (AP2, late treatment).
    • movie S3 (.avi format). Representative video clip of the control C. elegans worms N2 at day 7 upon treatment with empty vesicles at day 6 (AP2, late treatment).
    • movie S4 (.avi format). Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with DesAb18–25 at days 1 and 3 (AP1, early treatment).
    • movie S5 (.avi format). Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with DesAb29–36 at days 1 and 3 (AP1, early treatment).
    • movie S6 (.avi format). Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with DesAb18–25 at day 6 (AP2, late treatment).
    • movie S7 (.avi format). Representative video clip of the Aβ42 C. elegans worms GMC101 at day 7 upon treatment with DesAb29–36 at day 6 (AP2, late treatment).

    Download Movies S1 to S7

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