Potent Tau Aggregation Inhibitor D-Peptides Selected against Tau-Repeat 2 Using Mirror Image Phage Display

Abstract

Alzheimer's disease and other Tauopathies are associated with neurofibrillary tangles composed of Tau protein, as well as toxic Tau oligomers. Therefore, inhibitors of pathological Tau aggregation are potentially useful candidates for future therapies targeting Tauopathies. Two hexapeptides within Tau, designated PHF6* (275-VQIINK-280) and PHF6 (306-VQIVYK-311), are known to promote Tau aggregation. Recently, the PHF6* segment has been described as the more potent driver of Tau aggregation. We therefore employed mirror-image phage display with a large peptide library to identify PHF6* fibril binding peptides consisting of D-enantiomeric amino acids. The suitability of D-enantiomeric peptides for in vivo applications, which are protease stable and less immunogenic than L-peptides, has already been demonstrated. The identified D-enantiomeric peptide MMD3 and its retro-inverso form, designated MMD3rev, inhibited in vitro fibrillization of the PHF6* peptide, the repeat domain of Tau as well as full-length Tau. Dynamic light scattering, pelleting assays and atomic force microscopy demonstrated that MMD3 prevents the formation of tau β-sheet-rich fibrils by diverting Tau into large amorphous aggregates. NMR data suggest that the D-enantiomeric peptides bound to Tau monomers with rather low affinity, but ELISA (enzyme-linked immunosorbent assay) data demonstrated binding to PHF6* and full length Tau fibrils. In addition, molecular insight into the binding mode of MMD3 to PHF6* fibrils were gained by in silico modelling. The identified PHF6*-targeting peptides were able to penetrate cells. The study establishes PHF6* fibril binding peptides consisting of D-enantiomeric amino acids as potential molecules for therapeutic and diagnostic applications in AD research.

Keywords: Alzheimer's disease; D-peptides; phage display; tau aggregation inhibitors; therapy.

Figure 1

Scheme for selection of PHF6*‐binding peptides using mirror image phage display. (A) The largest human Tau isoform (Tau 2 N4R) in the central nervous systems contains four microtubule binding repeats. PHF6*, consisting of amino acids 275 to 280, is located at the beginning of repeat two. (B) The D‐enantiomeric form of PHF6* was synthesized. After fibrillization, the D‐enantiomeric fibrils were used for phage display. An L‐peptide, binding to the D‐enantiomeric PHF6* fibrils, was selected and the D‐enantiomeric version of the selected L‐peptide was synthesized, which will bind to the L‐enantiomeric form of the target, regular PHF6* fibrils.

Figure 2

PHF6 fibrillizes spontaneously by incubation at room temperature. The assay was performed using 50 μM PHF6 in NaPi buffer with 10 μM ThT (gray column). NaPi and 10 μM ThT without addition of PHF6 was used as control (red column). Peptides MMD3 or MMD3rev were added in concentrations of 500 μM to 50 μM PHF6 samples (green column and blue column, respectively). Fluorescence was measured at 490 nm in relative units (mean +/− standard deviations of results, three replicates per run). Samples treated with D‐peptides do not show a decreasing ThT signal, indicating no inhibition of PHF6 aggregation. (B) PHF6* fibrillization was performed by incubating 100 μM PHF6* in NaPi buffer with 10 μM ThT at room temperature (gray column). NaPi and 10 μM ThT without addition of PHF6 was used as control (red column). Peptides MMD3 or MMD3rev were added in concentrations of 1000 μM to 100 μM PHF6* samples (green column and blue column respectively). Fluorescence was measured at 490 nm in relative units (mean +/− standard deviations of results, three replicates per run). Samples treated with D‐peptides show a decreasing ThT signal, indicating an inhibition of PHF6* aggregation.

Figure 3

ELISA was performed with FAM‐labeled versions of MMD3 and MMD3rev. The plate was coated with Tau fibrils in a 10 μg/mL concentration or with PHF6* fibrils at a concentration of 50 μg/mL. As negative control, only coating buffer was added to the wells. The peptides MMD3‐FAM and MMD3rev‐FAM were added in an increasing concentration; the bound peptides were detected with anti‐FAM antibodies. After adding the TMB substrate, the absorption at 450 nm, which represents the binding affinities, was measured. We observed that the absorption signal increased with increasing the concentration of MMD3 or MMD3rev, which indicates that MMD3 and MMD3rev bound to Tau fibrils as well as PHF6* fibrils.

Figure 4

MMD3 has low affinity for binding to monomeric hTau40. (A) Superposition of 2D ¹H‐¹⁵N SOFAST‐HMQC of hTau40 in the absence and presence of MMD3 (hTau40 : MMD3 molar ratio of 1 : 30). (B) MMD3‐induced CSPs in hTau40 at hTau40 : MMD3 mole ratios of 1 : 3 (green), 1 : 10 (blue), and 1 : 30 (red). The dotted black line corresponds to a threshold of 0.0019 ppm, which is two standard deviations above the average CSP value for the mole ratio 1 : 10. (C) NMR signal intensity ratios I/I₀ from 2D ¹H‐¹⁵N SOFAST‐HMQC of hTau40 for hTau40 : MMD3 mole ratios of 1 : 3 (green), 1 : 10 (blue), and 1 : 30 (red). The dotted black line corresponds to a threshold of 0.94, which is two standard deviations below the average I/I₀ value for the mole ratio 1 : 10.

Figure 5

Changes in the size of Tau aggregates in the presence of MMD3 and MMD3rev using dynamic light scattering (DLS) and pelleting assay. Tau^RDΔK (10 μM) was incubated with 2.5 μM of heparin 16000 (H16 K) and 100 μM of peptides. (A) Hydrodynamic size of Tau^RDΔK monomer is <10 nm in diameter (red curve). Tau^RDΔK aggregated in the presence of heparin 16000 (H16 K) formed aggregates in the size of 15–100 nm in diameter (blue curve). Tau^RDΔK in the presence of both peptides (MMD3 and MMD3rev) forms aggregates with the size ranging from 3000–4000 nm in diameter (green and black curves). Peptides alone in the presence of heparin did not form larger aggregates, see Figure S3. (B) Pelleting assay: Aggregated Tau^RDΔK centrifuged samples resolved on SDS gels showing supernatant and pellet fractions (lanes 1, 2; S, P). The majority of the high molecular weight aggregated Tau is present in the pellet fractions of Tau^RDΔK treated with peptides MMD3 and MMD3rev (lanes 4, 6). (C) Quantification of the Tau pellet and supernatant fractions from western blot gels shows differences between the Tau pellets fractions treated with/without peptides (bars 2, 4, 6).

Figure 6

Tau forms amorphous aggregates in the presence of Tau peptides. AFM height and amplitude images reveal that Tau^RDΔK (with heparin 16000 (H16 K)) aggregates into fine structured filaments with the height of ∼15 nm (image A, B). In presence of peptides MMD3 (images C, D) and MMD3rev (images E, F), Tau aggregates into amorphous aggregates without attaining definite structure.

Figure 7

Interaction of inhibitory peptides with the Tau polymorph 1 and 2. Binding mode of (A) W‐MINK, (B) MMD3, and (C) MMD3rev to the Tau fibril polymorph 1. The peptides are shown in color presentation and key interacting residues described in the text are labelled. Major sites of interference with interface A and B are marked by red and yellow circles, respectively. Binding mode of (D) W‐MINK, (E) MMD3, and (F) MMD3rev to the Tau fibril polymorph 2. The peptides are shown in color presentation and key interacting residues described in the text are labelled. Major sites of interference with interface C are marked by blue circles.

Figure 8

Uptake of fluorescently labelled D‐peptides analyzed by confocal microscopy and flow cytometry. At different concentrations (25, 50 and 100 μM) of fluorescently labelled peptides (MMD3 and MMD3 reverse) were incubated with N2a cells expressing Tau^RDΔK for 48 h. The uptake of A647 peptides were analyzed by confocal microscopy (A, B) and by flow cytometry (C). As shown in the representative images the peptides are predominantly localized in the cytoplasm. A1, B1: DAPI shown as green; A2, B2: A647‐MMD3, A647‐MMD3 reverse; A3, B3: merged images. (C) The uptake of A647 labelled peptides were monitored by flow cytometry. As shown in the representative flow cytometry data, almost 100 % of the cells take up both the peptides. MMD3 reverse peptides show concentration dependent increase in intensity the fluorescence.