Development of oligomer-specific antibodies against tau protein and testing of therapeutic potential in a cell model of tau pathology

Tau, a microtubule associated protein, forms abnormal aggregates in many neurodegenerative diseases such as Alzheimer disease (AD). There is an urgent need for disease-modifying therapies of AD and related tauopathies. Inhibiting the aggregation of tau and the accumulation of neurofibrillary tangles (NFTs) could be helpful in combating tau pathology. Recent studies show that tau induced toxicity is mainly due to the presence of oligomers of tau rather than the monomers and fibrillar aggregates (Kaniyappan et al., 2017, Flach et al., 2012, Lasagna-Reeves et al., 2010). To combat the toxicity of tau oligomers we developed antibodies against the purified low-n tau oligomers (dimers to hexamers) of TauRDΔK, the strongly aggregating repeat domain of tau. Monoclonal antibodies were tested by various biochemical and biophysical methods for their specificity to bind to the toxic oligomers. Some antibodies show specificity to aggregates of tau while others detect all forms of tau. Antibodies 2B10 and 6H1, described as representative examples, bind to tau oligomers with high specificity as judged by dotblot, dynamic light scattering (DLS) and immunofluorescence analysis. As these antibodies are dependent on tau conformations, they appear non-specific in denaturing methods like western blotting. 2B10 and 6H1 antibodies are able to inhibit the tau aggregation up to ~90% in vitro (TauRDΔK, hTauP301L), as judged by the Thioflavin S fluorescence assay which is sensitive to ß-structure. In the presence of antibodies tau protein forms only up to low-n oligomers as judged by light scattering and atomic force microscopy (AFM). The choice of the pH of the column elution buffer of the antibodies plays a key role in determining the activity of the antibodies, as antibodies eluted at low pH have a higher activity compared to the same antibodies eluted at high pH. The ability of antibodies to inhibit the aggregation of tau was tested in an N2a cell model of tau pathology which expresses the pro-aggregant tau repeat domain TauRDΔK. Antibodies were added to the extracellular medium, without or with protein transfection reagent (Xfect) which stimulates cellular uptake. In this assay, 2B10 antibody failed to inhibit tau aggregation (ThS signal) and failed to prevent aggregation induced apoptosis (Annexin V signal). By contrast, in the split-luciferase complementation assay the antibody 2B10, applied extracellularly, was able to prevent the dimerization/oligomerization of tau. Surprisingly this antibody has only a relatively low affinity to tau but is still very active in inhibiting tau aggregation in vitro. Antibodies added extracellularly were taken up by the cells and sorted into lysosomes. Their inhibitory effect can be explained by the fact that the internalized antibody recruits the toxic tau protein or oligomers to the lysosomes for degradation. In summary, a subset of antibodies raised against the purified low-n oligomers of TauRDΔK are able to inhibit tau aggregation both in vitro and in a cell model of tau pathology

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

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

A novel D-amino acid peptide with therapeutic potential (ISAD1) inhibits aggregation of neurotoxic disease-relevant mutant Tau and prevents Tau toxicity in vitro

Background: Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death. Methods: We aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively. Results: While ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive β-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK. Conclusions: ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity. Keywords: Alzheimer’s disease; D-amino acid peptides; Phage display; Tau aggregation inhibitors; Therap