Identification of structural determinants on tau protein essential for its pathological function: novel therapeutic target for tau immunotherapy in Alzheimer's disease

Abstract

Introduction: Pathologically modified tau protein is the main feature of Alzheimer's disease (AD) and related tauopathies. Therefore, immunotherapies that target mis-disordered tau represent a promising avenue for the disease-modifying treatment of AD. In this report, we present our discovery of (1) a novel target for tau immunotherapy; (2) monoclonal antibody DC8E8, which neutralizes this target; and (3) the results of efficacy studies of DC8E8 in a murine model of tauopathy.

Methods: In vitro tau oligomerisation assays were used for the selection of antibodies. The therapeutic efficacy of DC8E8 was evaluated in transgenic mice. The structure of the DC8E8 epitope was determined by X-ray crystallography.

Results: Screening of a panel of monoclonal antibodies for their inhibitory activity in an in vitro pathological tau-tau interaction assay yielded DC8E8, which reduced the amount of oligomeric tau by 84%. DC8E8 recognised all developmental stages of tau pathology in AD human brains, including pretangles and intra- and extracellular tangles. Treatment with DC8E8 in a mouse AD model expressing mis-disordered human tau significantly reduced the amount of insoluble oligomerised tau and the number of early and mature neurofibrillary tangles in the transgenic mouse brains. By using a panel of tau-derived peptides in a competitive enzyme-linked immunosorbent assay, we identified the tau domain essential for pathological tau-tau interaction, which is targeted by DC8E8. The antibody was capable of binding to four highly homologous and yet independent binding regions on tau, each of which is a separate epitope. The X-ray structure of the DC8E8 Fab apo form, solved at 3.0 Å, suggested that the four DC8E8 epitopes form protruding structures on the tau molecule. Finally, by kinetic measurements with surface plasmon resonance, we determined that antibody DC8E8 is highly discriminatory between pathological and physiological tau.

Conclusions: We have discovered defined determinants on mis-disordered truncated tau protein which are responsible for tau oligomerisation leading to neurofibrillary degeneration. Antibody DC8E8 reactive with these determinants is able to inhibit tau-tau interaction in vitro and in vivo. DC8E8 is able to discriminate between the healthy and diseased tau proteome, making its epitopes suitable targets, and DC8E8 a suitable candidate molecule, for AD immunotherapy.

Figures

Figure 1

Monoclonal antibody DC8E8 inhibits pathological tau–tau interaction. (A) Inhibition of pathological tau–tau interaction by DC8E8. The amount of oligomerised tau (297-391/4R) was measured by thioflavin T fluorescence in the absence and in the presence of the monoclonal antibody (mAb) DC8E8 at the time points 1, 4 and 20 hours. Inhibitory activity of DC8E8 was statistically significant for the indicated time points when analysed using a nonparametric t-test (P < 0.0001). (B) Analysis of the inhibitory potential of DC8E8 showing prevention of the formation of tau dimers, trimers and oligomers by mis-disordered truncated tau by immunoblotting using horseradish peroxidase–conjugated mAb DC25. The positions of the molecular weight markers are indicated on the right.

Figure 2

Monoclonal antibody DC8E8 recognises developmental stages of Alzheimer’s disease tau neurodegeneration and Alzheimer’s disease–specific insoluble tau complexes. DC8E8 recognises preclinical Alzheimer’s disease (AD) (A), clinically incipient AD (B) and the fully developed final stage of AD (C). Hippocampal staining shows that DC8E8 detects early pretangle stages (D), intracellular neurofibrillary tangles (E) and extracellular neurofibrillary tangles (F). Tool bar: A-C 100 µm; D-F 10 µm. (G) DC8E8 recognizes soluble tau (lane 1) and sarkosyl-insoluble tau proteins (lane 2) in the material extracted from Braak stage VI AD brain tissue (allocortex tissue including hippocampus, entorhinal and temporal cortex). Western blot shows that DC8E8 detects AD-specific tau species with low and high molecular weights. For soluble tau (lane 1), 15 μg of total protein was loaded per lane. Sarkosyl-insoluble tau fraction (lane 2) was 50-fold enriched by solubilisation in a small volume of SDS-PAGE loading buffer (for details, see the Methods section).

Figure 3

DC8E8 immunotherapy significantly reduces levels of insoluble tau oligomers in brains of transgenic mice. Tau transgenic mice were immunized with DC8E8 and with an irrelevant antibody, DC51, and brain tissue of the animals was fractionated into soluble and sarcosyl-insoluble tau fractions. (A) and (B) Amount of total soluble tau is unchanged after DC8E8 treatment compared with treatment with a mock antibody, DC51. (C) and (D) Amount of sarcosyl-insoluble tau oligomers in transgenic mouse brain is significantly diminished after treatment with DC8E8 (n = 3, **P < 0.01). All Western blots were stained with pan-tau monoclonal antibody DC25, and lanes 1 to 6 contain samples from individual animals used in the experiments. Signal quantification was performed using AIDA software.

Figure 4

DC8E8 significantly reduces the number of neurofibrillary tangles in transgenic mice. Transgenic mice were treated with mock antibody DC51 (A) and (D) and with DC8E8 (B) and (E). Neurofibrillary tangles (NFTs) were visualised with AT8 staining (A) and (B) and with pS214 antibody (D) and (E). Transgenic mice treated with therapeutic antibody DC8E8 showed significantly less tau pathology than mice treated with mock antibody DC51 (C) and (F). * - P < 0.05; *** - P < 0.001; boxes represent 75 percentiles, middle bars represent medians and outer horizontal bars represents data range.

Figure 5

Mapping of domain on tau protein recognised by DC8E8. (A) For the epitope mapping of DC8E8 monoclonal antibody, we used full-length three- and four-repeat tau isoforms with two N-terminal inserts (1 and 2), tau deletion mutants (3–8) and tau-derived synthetic peptides (9–12). All deletion mutants that contained the microtubule-binding repeat (MTBR) region (3–6) were recognized by DC8E8; tau deletion mutants lacking the MTBR region were not recognized (8 and 9). Importantly, DC8E8 recognized each of synthetic peptides derived from individual MTBRs (9–12). The result is that DC8E8 recognised four binding sites (epitopes) located in the MTBR region of the tau protein, and each epitope is separately located within one MTBR. (B) To narrow down the DC8E8 minimal epitope, homologous peptides derived from the tau protein repeat region (MTBR1–4) were analysed in competitive enzyme-linked immunosorbent assays. Tau peptides containing at least six amino acids of the DC8E8 recognition sequence HXPGGG were capable of competing with mis-disordered tau (amino acids 151–391) for binding to antibody DC8E8. Tau peptides containing five amino acids of the DC8E8 recognition sequence did not compete with mis-disordered tau for binding to antibody DC8E8. (C) Schema of epitopes on tau protein molecule recognised by DC8E8. The DC8E8 monoclonal antibody is capable of binding four separate binding regions, with each region forming one individual epitope. These four epitopes are separately located within the first, second, third and fourth MTBR of protein tau. Note: All listed molecules are numbered in reference to the longest human tau isoform (2N4R) sequence.

Figure 6

The flexibility of the binding site allows DC8E8 to adapt to four homologous, albeit not identical, structural determinants in the tau microtubule-binding repeats. Two independently refined X-ray structures of DC8E8 antigen-binding fragment (A) and (B) (stereoview) show the flexibility of the antigen-binding site. The surface of the antibody is shown in grey. The backbones of complementarity determining regions (CDRs) are represented as tubes, with the diameter and colour reflecting their averaged atomic displacement parameters, that is, flexibility. The flexibility is expressed as a colour scale ranging from blue to red, corresponding to B-factors 30 to 150 Å2. The CDRs L1 and H3 exhibit higher B-factors than the remaining parts of the model. The pronounced flexibility of these CDRs is essential to allowing DC8E8 to bind each of four slightly different epitopes within the microtubule-binding repeats (MTBRs). (C) Superposition of both independently refined DC8E8 molecules shows that the core of the binding pockets is invariant (molecule A shown as grey solid, molecule B as blue mesh). The CDR loops (italic letters) create a 7- to 9-Å-deep pocket with surface dimensions 18 × 14 Å (red axes). The shape of this pocket necessitates that the minimal DC8E8 epitope HXPGGG adopts a fold protruding into this space to bind in the DC8E8 combining site.

Figure 7

DC8E8 discriminates between pathological and physiological ‘healthy’ tau. (A) Affinity comparison of DC8E8–four-repeat tau protein complex formation. The monoclonal antibody DC8E8 exhibits preferential affinity to the mis-disordered truncated tau 151-391/4R. Surface plasmon resonance (SPR) revealed that mis-disordered truncated tau protein is recognised by DC8E8 nearly seven times stronger than the full-length tau protein isoform. Extrapolation of kinetic SPR sensorgrams of DC8E8 interaction with mis-disordered truncated (B) and full-length (C) four-repeat tau proteins revealed that mis-disordered truncated tau is recognised by DC8E8 with kon = 2.9 × 106 M−1 s−1 and koff = 0.04 s−1, whereas full-length tau exhibits kon = 4.4 × 105 M−1 s−1 and koff = 0.04 s−1. (D) DC8E8 affinity comparison of three-repeat truncated tau and its full-length counterpart. Similarly to the four-repeat tau, extrapolation of kinetic SPR sensorgrams of DC8E8 interaction with truncated (E) and full-length (F) three-repeat tau proteins revealed that truncated tau is recognised by DC8E8 with kon = 1.5 × 105 M−1 s−1 and koff = 0.08 s−1, whereas full-length tau exhibits kon = 2.8 × 104 M−1 s−1 and koff = 0.4 s−1. These results show that three-repeat mis-disordered truncated tau protein is recognised by DC8E8 with 25 times higher affinity than full-length three-repeat tau protein. Black curves represent experimental data, and red curves were fitted by evaluation software for kinetic parameter calculations.