No abstract

The microtubule (MT)-associated protein Tau is a natively unfolded protein, involved in a number of neurodegenerative disorders, collectively called tauopathies, aggregating in neurofibrillary tangles (NFT). It is an open question how the conversion from a MT bound molecule to an aggregation-prone Tau species occurs and, also, if and how tauopathy-related mutations affect its behavior in the cell. To address these points, we exploited a genetically encoded FRET sensor based on the full length Tau protein, to monitor in real time Tau conformational changes in different conditions in live cells. By studying the FRET signal we found that soluble Tau molecules, detached from MTs, display an unfolded structure. On the contrary, we observed an increased FRET signal generated by Tau monomers bound to MT, indicating that the association with MTs induced a folding of Tau protein, decreasing the distance between its N and C termini. We exploited the FRET sensor to investigate the impact of FTDP-17 mutations and of phosphorylation-site mutations on Tau folding and mobility in live cells. We demonstrated that the FTDP-17 Tau mutations weaken the interaction of Tau with cellular MTs, shifting the equilibrium towards the soluble pool while, conversely, phosphorylation site mutations shift the equilibrium of Tau towards the MT-bound state and a more closed conformation.

Tau plays a central role in a group of neurodegenerative disorders collectively named tauopathies. Despite the wide range of diverse symptoms at the onset and during the progression of the pathology, all tauopathies share two common hallmarks, namely the misfolding and aggregation of Tau protein and progressive synaptic dysfunctions. Tau aggregation correlates with cognitive decline and behavioural impairment. The mechanistic link between Tau misfolding and the synaptic dysfunction is still unknown, but this correlation is well established in the human brain and also in tauopathy mouse models. At the onset of the pathology, Tau undergoes post-translational modifications (PTMs) inducing the detachment from the cytoskeleton and its release in the cytoplasm as a soluble monomer. In this condition, the physiological enrichment in the axon is definitely disrupted, resulting in Tau relocalization in the cell soma and in dendrites. Subsequently, Tau aggregates into toxic oligomers and amyloidogenic forms that disrupt synaptic homeostasis and function, resulting in neuronal degeneration. The involvement of Tau in synaptic transmission alteration in tauopathies has been extensively reviewed. Here, we will focus on non-canonical Tau functions mediating synapse dysfunction.

Tauopathies are neurodegenerative disorders characterized by Tau aggregation. Genetic studies on familial cases allowed for the discovery of mutations in the MAPT gene that increase Tau propensity to detach from microtubules and to form insoluble cytoplasmic Tau aggregates. Recently, the rare mutation Q336H has been identified to be associated with Pick’s disease (PiD) and biochemical analyses demonstrated its ability to increase the microtubules (MTs) polymerization, thus revealing an opposite character compared to other Tau mutations studied so far. Here we investigated the biophysical and molecular properties of TauQ336H in living cells by the employment of the conformational Tau biosensor CST. We found that this mutation alters Tau conformation on microtubules, stabilizes its binding to tubulin, and is associated with a paradoxical lower level of Tau phosphorylation. Moreover, we found that this mutation impacts the cytoskeletal complexity by increasing the tubulin filament length and the number of branches. However, despite these apparently non-pathological traits, we observed the formation of intracellular inclusions confirming that Q336H leads to aggregation. Our results suggest that the Tau aggregation process might be triggered by molecular mechanisms other than Tau destabilization or post-translational modifications which are likely to be detrimental to neuronal function in vivo.

Formation of Tau aggregates is a common pathological feature of tauopathies and their accumulation directly correlates with cytotoxicity and neuronal degeneration. Great efforts have been made to understand Tau aggregation and to find therapeutics halting or reversing the process, however, progress has been slowed due to the lack of a suitable method for monitoring Tau aggregation. We developed a cell-based assay allowing to detect and quantify Tau aggregation in living cells. The system is based on the FRET biosensor CST able to monitor the molecular dynamic of Tau aggregation in different cellular conditions. We probed candidate compounds that could block Tau hyperphosphorylation. In particular, to foster the drug discovery process, we tested kinase inhibitors approved for the treatment of other diseases. We identified the ERK inhibitor PD-901 as a promising therapeutic molecule since it reduces and prevents Tau aggregation. This evidence establishes the CST cell-based aggregation assay as a reliable tool for drug discovery and suggests that PD-901 might be a promising compound to be tested for further preclinical studies on AD.