Novel repertoire of tau biosensors to monitor pathological tau transformation and seeding activity in living cells

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    Understanding specific tau-tau interactions that play key roles in Alzheimer's disease and tauopathies will enable the elucidation of the toxic tau species involved in the pathogenesis of these diseases and therapeutic development in this area. In this paper, the authors developed a series of NanoBiT complementation-based tau biosensors to monitor tau intramolecular and intermolecular interactions. This paper will be of high interest to a broad target audience including researchers in the field of biophysics, biochemistry, cell biology, neuroscience, neuropathology, and drug discovery as well as physicians.

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Aggregates of the tau protein are a well-known hallmark of several neurodegenerative diseases, collectively referred to as tauopathies, including frontal temporal dementia and Alzheimer’s disease (AD). Monitoring the transformation process of tau from physiological monomers into pathological oligomers or aggregates in a high-throughput, quantitative manner and in a cellular context is still a major challenge in the field. Identifying molecules able to interfere with those processes is of high therapeutic interest. Here, we developed a series of inter- and intramolecular tau biosensors based on the highly sensitive Nanoluciferase (Nluc) binary technology (NanoBiT) able to monitor the pathological conformational change and self-interaction of tau in living cells. Our repertoire of tau biosensors reliably reports i. molecular proximity of physiological full-length tau at microtubules; ii. changes in tau conformation and self-interaction associated with tau phosphorylation, as well as iii. tau interaction induced by seeds of recombinant tau or from mouse brain lysates of a mouse model of tau pathology. By comparing biosensors comprising different tau forms ( i.e . full-length or short fragments, wild-type, or the disease-associated tau(P301L) variant) further insights into the tau transformation process are obtained. Proof-of-concept data for the high-throughput suitability and identification of molecules interfering with the pathological tau transformation processes are presented. This novel repertoire of tau biosensors is aimed to boost the disclosure of molecular mechanisms underlying pathological tau transformation in living cells and to discover new drug candidates for tau-related neurodegenerative diseases.

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  1. eLife assessment

    Understanding specific tau-tau interactions that play key roles in Alzheimer's disease and tauopathies will enable the elucidation of the toxic tau species involved in the pathogenesis of these diseases and therapeutic development in this area. In this paper, the authors developed a series of NanoBiT complementation-based tau biosensors to monitor tau intramolecular and intermolecular interactions. This paper will be of high interest to a broad target audience including researchers in the field of biophysics, biochemistry, cell biology, neuroscience, neuropathology, and drug discovery as well as physicians.

  2. Reviewer #1 (Public Review):

    Pathological conformation and aggregation of tau protein are involved in several neurodegenerative diseases such as tauopathies and Alzheimer's disease. Identifying drug-candidates capable of interfering with pathological transformation of tau remains a challenge for which sensitive and specific assays are needed. This article describes the development and characterization of tau biosensors based on NanoBit technology (nanoluciferase complementation). It is a well-designed and precise study providing very interesting new tools.

    1/ The authors have developed a variety of tau biosensors: some that can be used for basic research to monitor pathological tau transformation and others with properties suitable for drug screening. All biosensors make it possible to evaluate the action of different agents (chemical products, purified proteins, cell or tissue extracts) in a living cell.

    2/ The intermolecular biosensors developed from a shortened version of tau (K18), or the full-length tau, and carrying the P301L mutation possess sensibility and specificity allowing their further development to identify drug-candidates interfering with tau self-interaction.

    3/ The characterization of the tau probes confirmed the physiological and pathological knowledge concerning the tau protein: - proximity of full-length tau when bound to microtubules, - conformational changes of tau during its phosphorylation and - tau-self interaction induced by pathological seeds.

    1/ The suitability of tau biosensors for high-throughput screening needs to be further developed as experiments were only performed in 96-well plates. No scaling up in 384- or 1536-well plates was attempted. Moreover, the transfection of the biosensors could be tedious.

    2/ The models used are basic (HEK-293T cells associated with tau aggregates, Aβ oligomers or mouse brain lysates). The number of drugs tested is also quite low.

    Collectively, the conclusions drawn by the authors are supported by the results. These new biosensors will be easily usable by the scientific community in fundamental research and could also be of interest to pharmaceutical laboratories wishing to carry out screenings of molecules capable of impacting the pathological transformation of tau.

  3. Reviewer #2 (Public Review):

    Cecon et al presented a series of tau biosensors using the NanoBiT complementation system to monitor tau intramolecular and intermolecular interactions. Three major findings shown in the paper are discussed below.

    (1) The authors added two modifications to the existing NanoBiT complementation-based biosensors including K18(P301L) and TauP301L which have the capabilities of monitoring tau-tau interactions in response to phosphorylation and seeding. It is important to first have a thorough characterization of the biosensors such as the basal comparative signals among the different isoforms/mutations (the data in the paper are mostly normalized) and how these signals correspond to their functional units such as whether they are monomers, oligomers or fibrils as confirmed by other biochemistry assays e.g. ThS staining. The interpretation on the functional effect of these biosensors in response to stimulation such as addition of seeds have to be discussed. For example, K18(P301L) biosensor is responding to both mK18 and aggK18 as well as aggTau but not mTau or oAB. It appears that the biosensor is unable to differentiate monomeric and aggregated species of K18 tau. Also, beta-amyloid oligomers have been shown to seed tau aggregation, but this is not the case shown by the study which warrants some discussion. A more thorough characterization of the luciferase biosensors would be essential before moving into other assays and high-throughput screening as it is important to know exactly what kind of tau species are being targeted.

    (2) The authors added colchicine, a MT destabilizing drug, to the luciferase biosensor systems and showed that phosphorylation of WT tau takes place when it is still bound to MTs, as colchicine prevented its phosphorylation and suggested that tau species comprising of K18 and full-length WT tau might represent an interesting new therapeutic target, as K18 tau and tau with P301L mutation renders full-length WT tau responsive to seeding. It is an interesting concept to study how tau aggregation changes with respect to MT destabilization. However, it is worth noting that treatment with chemical compounds may cause many other effects that need to be well controlled/eliminated before reaching a conclusion. The authors showed that treatment with colchicine reduces luciferase signals of the tau biosensors and suggested that the luciferase signals arise from MT bound tau which is interesting. While colchicine is a well-known MT destabilization drug, it is still important to test if colchicine itself is perturbing tau-tau interaction as other studies have shown that colchicine might promote tau aggregation and cause cognitive dysfunction. From a different perspective, one might consider that MT destabilization may result in more tau in the cytosols due to their detachment from MTs and hence resulting in enhanced tau-tau interactions which would be reflected by an increased in biosensor signals. Furthermore, if tau proteins are already interacting when they are on the MTs, a disruption in MTs may not disrupt tau-tau interactions and might lead to enhanced tau-tau interactions. However, this is not the case shown in this study and perhaps a discussion on this interpretation would help to clarify some questions. The luciferase signal for tau on MTs might be due to tau being near one another when they are residing on MTs which acts as a scaffold to hold them together and not exactly due to tau-tau interactions. Hence, upon MT destabilization, the tau proteins lost the scaffolds that hold them together and hence results in a reduction in the luciferase signals. In terms of the therapeutic targeting of K18-WT tau complex, it is important to note that K18 has increased the responsiveness of WT tau to seeding by 2-fold as compared to the 107-fold change upon seeding of K18-K18 tau biosensor. Although significant, it is a very small change as compared to the signal obtained from K18 biosensors.

    (3) Finally, the authors conducted a proof-of-concept study to illustrate the potential of the luciferase biosensor to be used in high-throughput screening drug discovery. The authors used tau seeds (Tg brain lysates), and not small molecules, to show the increase in luciferase signals with Z-factors of >0.5, which indicates excellent assay condition. The authors then further showed that known compounds reduced tau aggregation in Tg brain lysates and reduced luciferase signals of the biosensors. High throughput screening capability typically refers to the perturbation of biosensors or tau-tau interactions directly by drug compounds. From the experimental setup, it seems like the authors will be using luciferase biosensor in the presence of Tg brain lysates (together as a system) to screen for drug candidates, instead of using the biosensor directly to screen for compounds that have a direct effect in perturbing the biosensor. In this case, the Z-factor should be calculated for positive-control compounds that are applied to the biosensor+Tg lysates system. The IC50 of the compounds tested in this system should be determined and compared with the known IC50 values of these compounds in the available literature. It appears that the compounds are only exhibiting good inhibition at very high concentrations, suggesting the need to test and eliminate any non-specific effect such as compound aggregation at a very high concentration.

  4. Reviewer #3 (Public Review):

    The paper by Cecon et al. presents a novel biosensor approach designed to study aspects of Tau aggregation that employ the luciferase-based NanoLuc Binary Technology (NanoBiT). The last decade has seen a rise in the number and variety of Tau biosensor systems, each with its own strengths and weaknesses to study various aspects of Tau aggregation. So far, these have proven to be extremely useful tools for the detection of proteopathic Tau molecules from different origins, by virtue of their capacity to induce easily detectable aggregation of the "endogenous" reporter Tau proteins in the intracellular environment, enabling for example to interrogate the structural features that render the protein pathogenic; in addition, they have been employed for screening of therapeutic candidates that can inhibit or slow down the aggregation process. As regards the study of the aggregation process itself, such systems encounter important limitations in that the modifications done to the protein likely impact reaction rates (both intramolecular and intermolecular interactions) and the aggregation mechanism itself. Additionally, the majority of them rely on overexpression systems, further altering the dynamics of physiological interactions. This paper implements a recently developed and commercially available technology based on nano-luciferase complementation, which has been used to study transient protein-protein interactions but not yet for Tau, and reports on its utility to study both inter- and intra-molecular interactions of Tau in live-cells and seeding activity of exogenously added Tau.

    The field of Alzheimer's will benefit greatly from cellular models that enable faithful replication of aggregation mechanisms that occur intracellularly involving Tau. The elucidation of high-resolution molecular structures of Tau fibrils from cryo-electron microscopy and the realisation that fibrils from different tauopathies display characteristic folds point to altered cellular states that drive the intrinsically-disordered protein (IDP) Tau to adopt specific conformations that spur pathological aggregation processes. The aggregate burden is known now to correlate well with disease progression. Tau has otherwise been described as a highly soluble protein, yet under certain circumstances it adopts a misfolded conformation that in the proximity of other monomers can template further misfolding and spur aggregation. Several biosensor systems have been developed that detect proteopathic Tau with high sensitivity, most notably those that consist of cell lines expressing intracellular FRET pairs. These have been invaluable to the field and have served to demonstrate that seeding activity strongly correlates with disease aggressiveness in Alzheimer's patients (see Dujardin et al. Nat Med 2021), among other important contributions. There are however major limitations in using these models to study aggregation mechanisms in a cellular context in that they rely on significant structural modifications to the protein that alter the aggregation energy landscape, among other artefactual concerns (e.g., protein overexpression).
    This paper sets out to showcase the applicability of the NanoBiT technology on the strength of the considerably smaller size of the fusion proteins. which comprise one large BiT fragment of 17.6 kDa and a small complementation peptide of only 11 amino acids, compared to for instance the popular Tau RD P301S FRET biosensor line that relies on CFP and YFP (both ~27 kDa) Tau-fused constructs as FRET pairs. This is important for interrogating intracellular inter- and intra-molecular interactions as steric effects impact reaction rates and mechanisms. This, coupled with high sensitivity of the bioluminescence signal and amenability for high throughput, comprise the most important advantages of this approach.

    Perhaps the most significant advantage (conceptually) of the NanoBiT technology in this context is the ability to create intramolecular interaction sensors by fusing the fragments to opposite termini. This is especially useful for the N- and C- termini of Tau which are known to be in proximity in certain conformations. The same can be achieved with fluorescence complementation yet with the caveat of introducing larger molecules. Nevertheless, regardless of the smaller dimensions of the fusion protein, the modifications are likely to still alter protein interaction dynamics - this is relevant to both intra- and inter-molecular sensors. While this may not always be a major concern when working with globular proteins, it should be a key consideration when studying Tau aggregation. The energy landscape of intrinsically disordered proteins is highly sensitive to even small structural changes, as exemplified by conformational changes in Tau that render this otherwise highly-soluble protein aggregation-prone. The interaction between the complementary small and large fragments of NanoBiT is reversible and weak (reported as 190 uM), but may still stabilise non-intrinsic conformations. Demonstrating that interaction and aggregation kinetics are not affected significantly compared to the native protein in vitro would be required to support the physiological relevance of the claims related to inter- and intra-molecular interactions.

    An additional concern with the intramolecular sensor is the ability to discriminate whether interactions are indeed intramolecular and not intermolecular, this introduces a confound for instance in the interpretation that a reduction in signal with the WT Tau conformation sensor after treatment with colchicine suggest that microtubules stabilise Tau in a conformation where N- and C- termini of a Tau monomer are in proximity, when this could also well be due to intermolecular interactions, or a combination of both (see the continuous stretch of density of Tau along protofilaments in Kellogg et al. Science 2018). Furthermore, the colocalization data is not of high enough quality to support the claims regarding microtubule interactions, in fact there seems to be stronger colocalization with the intramolecular sensor than with the intermolecular one. Better quality images and co-localization analysis are needed to support these interpretations. The paper thus falls short of providing compelling data to regard this method as a physiologically-relevant approach to study Tau molecular interactions.

    Artefactual problems stemming from the aforementioned alterations are likely not as important for their applicability as sensors, as other Tau biosensors have shown the ability to detect proteopathic forms in a way that reflects the severity of pathology in various contexts, regardless of whether the ensuing aggregates faithfully replicate those encountered in pathology. It would then be of interest to assess how the NanoBiT technology fares compared to alternative cell models in regard to sensitivity. The paper provides a response curve with tissue extracted from a mouse model of tauopathy. The extracts are not purified for tau which makes comparison with other data difficult given that the degree of tauopathy is model and mouse dependent. A more extensive evaluation of the sensing capacity would be needed to establish sensitivity in a meaningful way, for instance with Tau forms for which concentration can be more appropriately estimated, e.g., recombinant Tau and IP-purified extracts from mouse and human tissues, or a direct comparison with other methods.