Sequestration of the Abeta peptide prevents toxicity and promotes degradation in vivo

Abstract

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta) peptide by using a small engineered binding protein (Z(Abeta3)) that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3) in the brains of Drosophila melanogaster expressing either Abeta(42) or the aggressive familial associated E22G variant of Abeta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3) not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.

Figure 1. Inhibition of neurotoxicity measured as lifespan of transgenic Drosophila.

Each curve represents 100 flies divided equally into groups of 10. Expression of all Aβ peptides and Affibody proteins was under the control of the UAS-GAL4 system. In these experiments, expression was driven throughout the CNS by the elav^c155-GAL4 driver line. Survival assays were performed to quantify the degree of neurodegeneration when each different combination of Aβ peptide and Affibody proteins or Z domain control was expressed in the CNS. (A) Aβ₄₂ e22g median lifespan = 9 (±0.5) days; Aβ₄₂ e22g + Z_Aβ3 = 20 (±0.2) days, p<0.001 versus Aβ₄₂ e22g alone; Aβ₄₂ e22g + (Z_Aβ3)₂ = 31 (±0.8) days, p<0.001 versus Aβ₄₂ e22g alone. (B) Aβ₄₂ median lifespan = 28 (±0.4) days; Aβ₄₂ + Z_Aβ3 = 32 (±0.7) days, p<0.001 versus Aβ₄₂ alone; Aβ₄₂ + (Z_Aβ3)₂ = 40 (±1.2) days, p<0.001 versus Aβ₄₂ alone. (C) Aβ₄₀ median lifespan = 38 (±2); Aβ₄₀ + Z_Aβ3 = 41 (±2) days; Aβ₄₀ + (Z_Aβ3)₂ = 38 (±2) days. (D) Control experiment: lifespan of flies expressing only the Z domain, Z_Aβ3, or (Z_Aβ3)₂ and non-transgenic flies (wild-type). Median lifespan of wild-type flies = 38 (±1.8) days. Complete survival statistics are shown in Table S1.

Figure 2. Rescue of Drosophila eye morphology.

Scanning electron micrographs (SEM) of eyes of flies expressing Aβ₄₂ e22g alone or in combination with the Z domain control or the (Z_Aβ3)₂ Affibody at low and high magnification. A wild-type non-transgenic fly eye is shown for comparison. Scale bar = 100 µm in main pictures and 20 µm in inserts.

Figure 3. Clearance of Aβ from the Drosophila brain.

(A) Electrophoretic (SDS PAGE) analysis of soluble Aβ in fly brain extracts. A clear Aβ immunoreactive band is seen at 8 kDa (consistent with an Aβ dimer [14]) in flies expressing Aβ₄₂ e22g and flies co-expressing Aβ₄₂ e22g and the Z domain. The 8 kDa Aβ immunoreactive band is absent in flies co-expressing Aβ₄₂ e22g and either Z_Aβ3 or (Z_Aβ3)₂. β-actin immunodetection (bottom row) served as a loading control. (B) ELISA analysis of total (soluble and insoluble) Aβ₄₂ e22g concentration in the brains of flies expressing the different Affibody constructs. The levels of Aβ₄₂ e22g measured in the presence of the different Affibody molecules are expressed as a percentage of the concentration in the Aβ₄₂ e22g alone control. Differences between genotypes were analyzed by ANOVA and post hoc t tests. ** p<0.01. (C) Immunohistochemistry and confocal microscopy analysis of Aβ₄₂ e22g aggregates in intact brains from flies expressing Aβ₄₂ e22g alone or in combination with different Affibody constructs. Anti-Aβ immunostaining is shown in red, with a nuclear counterstain (TOTO-3) shown in blue. White boxes in brain images to the left are magnified to the right. Aβ immunoreactive aggregates are observed as puncta and are abundant in the brains of flies expressing Aβ₄₂ e22g alone or in combination with the Z domain. Immunoreactive Aβ deposits are sparse in brains where Z_Aβ3 is co-expressed with Aβ₄₂ e22g and absent in brains where (Z_Aβ3)₂ is co-expressed with Aβ₄₂ e22g. (D) SDS PAGE analysis of Z_Aβ3 and (Z_Aβ3)₂ levels in the presence and absence of Aβ₄₂ e22g. Twelve kDa anti-c-Myc immunoreactive bands (consistent with a disulfide linked Z_Aβ3 dimer) of equal intensity are detected in Z_Aβ3-expressing flies in the presence or absence of Aβ₄₂ e22g. Twelve kDa anti-Affibody immunoreactive bands of equal intensity are also detected for the head-to-tail linked (Z_Aβ3)₂ dimer. (E) Quantitative RT-PCR analysis of Aβ mRNA levels in flies expressing Aβ in combination with different Affibody constructs or the Z domain control. The relative levels of Aβ mRNA detected in flies expressing Aβ₄₂ e22g in combination with Z (white), Z_Aβ3 (red), and (Z_Aβ3)₂ (blue) compared to that detected in flies expressing Aβ₄₂ e22g alone (black) do not differ significantly (n.s., not significant).

Figure 4. Inhibition of Aβ₄₀ amyloid fibril formation.

(A) Structure of the Z_Aβ3 Affibody (blue and cyan) in complex with an Aβ₄₀ hairpin (residues 16 to 40; red) . White spheres represent buried nonpolar side chains (core) of Z_Aβ3. (B–D) Kinetics of Aβ₄₀ amyloid fibril formation monitored by ThT fluorescence using 30 µM Aβ₄₀ with addition of 36 µM Z_Aβ3 at different times (B and D) or using the specified concentrations of Aβ₄₀ and Z_Aβ3 (C). Time traces of three or four independent experiments are shown for each condition in (B) and (D). The average of three experiments is shown in (C) with error bars representing maximum and minimum values. Experiments in (B–D) were repeated with Aβ₄₂ (Figure S3). (E) Transmission electron microscopy (TEM) of fibrils prepared for the Aβ₄₀ fibril dissolution assay. Scale bar = 200 nm. (F, top) Up-field region of the ¹⁵N HSQC NMR spectrum of a fibril dissolution sample at 37°C starting from 300 µM ¹⁵N-Aβ₄₀ in fibrils and (middle) 24 h after addition of 325 µM Z_Aβ3. The Aβ₄₀ backbone resonances appear as Aβ₄₀ dissociates from fibrils and is captured as complex with Z_Aβ3. For reference: the assigned spectrum of Affibody-bound monomeric Aβ₄₀ (bottom) prepared directly from monomeric Aβ₄₀. (G) Kinetics of Aβ₄₀ fibril dissolution. The concentration of bound Aβ₄₀ was calculated from the intensities of the NMR resonances compared to those of an internal ¹⁵N-Z_Aβ3 standard. The experiments were carried out using recombinantly produced Aβ₄₀ with an N-terminal methionine residue.

Figure 5. Dissolution of Aβ oligomers.

(A–D) Oligomer formation (A and B; 100 µM total Aβ₄₂) and oligomer dissolution (C and D; 20 µM total Aβ₄₂) monitored by SEC in the absence or presence of 1.2-fold excess of Z_Aβ3. SEC elution profiles were integrated and normalized (see Figure S6 and Materials and Methods). The fraction of high molecular weight (HMW) aggregates was calculated as the difference between unity and the sum of the monomer and oligomer fractions. (E) Normalized CD spectra (MRE, mean residue elliptictiy) of monomeric Aβ₄₂ (black), oligomers (red), and fibrils (blue). β-sheet secondary structure is identified by a distinct minimum at ∼215 nm in the spectrum. (F,G) TEM images of oligomeric Aβ₄₂ solutions after isolation and at the endpoint of the dissolution experiment. Scale bar = 100 nm. (H) ¹⁵N HSQC NMR spectrum of an Aβ₄₂ oligomer sample, which has dissociated as a result of the sequestering of monomeric Aβ₄₂ by Z_Aβ3 (black). Starting from 11 µM ¹⁵N-Aβ₄₂ in oligomeric form (such as shown in F), this spectrum was recorded 2 days after the addition of 13 µM Z_Aβ3. The fraction of Aβ₄₂ bound to Z_Aβ3 after 5 days of incubation was estimated by NMR to be 92% (±9%). A spectrum of Z_Aβ3:Aβ₄₂ prepared from monomer solutions is shown for reference (green). The experiments were carried out using recombinantly produced Aβ₄₀ or Aβ₄₂ with N-terminal methionines.