Aims

The mechanisms leading to Tau aggregation are still ill-defined, different molecular features have been identified as involved in the aggregation process. The peptide PHF6 (Paired Helical Filament-6, residues 306-VQIVYK-311) is described as a nucleus of Tau aggregation. Different strategies have been proposed as therapeutic approaches in tauopathies, including immunotherapy. However, it remains important to better define the best region(s) of Tau to target , as well as the Tau species (non/phosphorylated, non/soluble or aggregated) and their localization (intra/extra-cellular). In view of this challenge, we have used VHHs (Variable domain of the Heavy- chain of the Heavy-chain-only-antibodies) for their interesting properties and relative ease of generation.

Methods

In partnership with Hybrigenics Company, a synthetic phage display library of VHHs was screened against full-length recTau protein. The epitopes recognized by the selected VHHs, were defined using Nuclear Magnetic Resonance spectroscopy. A VHH targeting an epitope in the microtubule binding domain of tau, corresponding to the PHF6, was selected. Further optimizations of this VHH, using yeast two-hybrid were performed, increasing its intracellular binding capacity and its binding affinity, resulting in a family of VHHs targeting PHF6. These VHHs were next screened for their inhibitory effect towards tau aggregation in vitro, in a cellular model (FRET analysis) as well as in tau transgenic mice.

Results

VHH seem to have the capacity of blocking tau seeding in all cases.

Conclusions

The promising preliminary results of the in vivo open the way for new studies using these VHHs as molecular tools to decipher the best target in Tau immunothérapies.

Abstract Body

Tau has become an unavoidable therapeutic target in Alzheimer's disease and progressive supranuclear palsy. Decreasing the expression of tau is the most commonly used strategy. In addition to anti-sense oligonucleotides, anti-tau immunotherapy is one of the most advanced therapeutic approaches. However, targeting tau remains a challenge. Tau is primarily an intracellular protein. In such paradigm, immunological tools must reach the cytoplasm of the cells to be targeted. However, tau protein is also secreted, and its pro-aggregative species are believed to participate in tau pathology spreading by a prion-like mechanism. In this hypothesis, immunological tools should block extracellular pathological species. From a technological point of view, immunological tools have largely evolved and there is now a very wide panel: monoclonal antibody (immunoglobulin, IgG), Fab fragment, single-chain variable fragment (Fv), VHH single domain antibody (or nanobody), ... Each of them has advantage.

In the laboratory, we work with IgGs that recognise different tau regions or pathological post-translational modifications (phosphorylation or acetylation). We have also developed numerous anti-tau VHHs that can be used as recombinant protein or by viral vectorisation. We use many experimental models of tauopathy (FRET seeding assay, murine models (seeding/spreading)...) for preclinical studies.

In this work, we will discuss the advantages and disadvantages of using a total anti-tau immunoglobulin versus pathological anti-tau. We will also see the opportunity to use VHH-based gene therapy for certain tauopathies considered as orphan disease (FTLD-MAPT).

Altogether, we show that Tau is a promising therapeutic target, but targeted epitope is crucial to avoid side-effects.

Aims

In few tauopathies such as Alzheimer’s disease (AD), tau pathology first affects a specific region before spreading to other cells following specific neural pathways. It has recently been considered that this staging could be linked to a prion-like propagation¹. According to this hypothesis, seed-competent tau species might be transferred to healthy cells and recruit endogenous tau protein leading to the formation of aggregates. Several mechanisms mediating their transfer have been highlighted such as extracellular vesicles (EVs)². In this context, we wondered if EV’s, isolated from human derived interstitial fluids (ISF), might contain tau seeds that should induce tau aggregation in recipient cells before analyzing their seeding capability in vivo.

Methods

ISF are purified from humans brains (AD, Pick’s disease (PiD), progressive supranuclear palsy (PSP) patients and controls) or mice brains (Tg30tau and WT mice). EV’s are then isolated from ISF and characterized using size-exclusion columns before transferred by lipofection to CFP-/YFP-Tau-RD(P301S) HEK293 cells³. FRET signal is measured by flow cytometry. EV’s with FRET-positive signal are injected into the hippocampus of young Tg30tau and WT mice.

Results

FRET assay shows significant FRET-positive cells treated with EV’s from Tg30tau ISF compared to cells treated with WT EV’s. Moreover, EV’s from AD ISF induce significant FRET-positive signal compared to PSP, PiD and controls. Once injected into the brain of Tau30 mice, EV’s with FRET-positive signal mediate the seeding of endogenous human tau.

Conclusions

These results support that EV’s transport seed-competent tau species that participate to the spreading of tau pathology with different process among tauopathies.

Aims

Neuropathologists defined neurodegenerative specific pathways in tauopathies suggesting the existence of vulnerable neurons. The spreading of extracellular tau species play a major role in this progression and the main remaining questions are now around the molecular species responsible for spreading. Although tau can be secreted through unconventional secretion, we focused on extracellular vesicles (EVs) found in human fluids and compare their role in the transport of seed-competent species among tauopathies.

Methods

Material: Viral vectors, cells, murine models, Human-derived materials (CSF, plasma, brain samples, ISF) from AD, PSP, PiD and controls. Methods: IHC, ELISA, EM, NTA, WB, FRET, intracranial injections.

Results

We demonstrated that 1) EVs-containing tau are secreted in many cell and animal models but also in human fluids (ISF, CSF, plasma) and 2) These secreted EVs are capable to transfer tau in neurons. The seed competency of these EVs was then investigated and, among human fluids, EVs derived from ISF are the most toxic one with a progressive loss of seeding effect from the central to the peripheral fluids. Additionally, whereas tau is detected in EVs-ISF from AD, PSP, PiD and controls, only EVs derived from AD’s ISF are highly converters.

Conclusions

To conclude, seeds from central human fluids are secreted in EVs and transmitted to receiving cells to induce tau lesions, especially in AD. Our data also suggested that secreted species or spreading mechanisms are probably different among tauopathies and identify EVs as good extracellular targets for seeds depletion in AD.