Multilevel investigation of Tau pathology: from the cytoplasm to the nucleus

Tau protein has been discovered in 1975 from brain tissues and its main function in neurons is to bind and stabilize microtubules. This observation was followed by the identification of Tau as one of the main actors able to induce neuronal toxicity in a group of neurodegenerative disorders named tauopathies. From these discoveries, the scientific community has invested great efforts to elucidate the mechanisms involving Tau and to find a way to prevent its pathological effects. Tau toxicity is due to its displacement from microtubules, progressive aggregation of the protein and spreading in several brain areas causing neuronal dysfunction and death. These are considered the central events leading to neurodegeneration. However, recently it has been demonstrated that Tau is located not only on microtubules or in the cytoplasmic aggregates but also in other subcellular regions, in particular in dendrites and in the nuclear compartment where it exerts functions related to synaptic transmission and genome protection, respectively. In order to investigate the dynamics of Tau from physiological conditions to destabilization and aggregation, we developed a FRET-based biosensor, the Conformational Sensitive Tau (CST), able to determine the conformational changes of Tau during the progression of the pathology. We showed that in physiological conditions, in living cells, Tau binds microtubules with a paperclip conformation. After drug treatments or tauopathy-related mutations, the conformation of Tau opens indicating an impairment of Tau binding to tubulin. Finally, by treating cells with different kinds of Alzheimer’s aggregates, the CST displaced from microtubules and formed FRET-positive intracellular inclusions demonstrating that it is a powerful tool to study also aggregation. The CST employment allowed the characterization of a particular mutation associated to Pick’s disease, Q336H. Remarkably, we found that this mutation induces a closer conformation of Tau and a higher affinity to tubulin, an effect that is opposite to previously discovered mutations. We applied the CST to develop a cell-based aggregation assay to screen compounds impairing Tau pathology. A first screening identified the ERK kinase inhibitor PD-901 as a compound reducing Tau aggregation. Moreover, to test the efficacy of therapeutic compounds in vivo, a transgenic zebrafish expressing the CST is under development to establish a zebrafish-based aggregation platform. The signal of the CST is detectable not only in the cytoplasm but also in the nucleus, however, the FRET analysis in this compartment revealed that nuclear Tau conformation is probably more open and relaxed. We investigated the nuclear function of Tau and we found that the increase in the soluble pool of Tau enhances its translocation into the nucleus and, concomitantly, the nuclear Tau alters the expression of VGluT1, a diseaserelevant gene, indicating that Tau has a role in gene expression modulation. We observed that the increase in VGluT1 expression mediated by Tau causes neuronal hyperexcitability in hippocampal primary neurons, an event typical of the first stages of AD. We found that this Tau function is impaired by the P301L mutation and by pathological aggregation. To identify other possible genes modulated by Tau we performed an RNAseq experiment and we found a global gene expression alteration that strongly resembles the late mild cognitive impairment. The investigation of molecular mechanisms involving nuclear Tau indicates that it competes with HDAC1 for the binding with TRIM28, a nuclear protein involved in heterochromatin formation. This competition causes the delocalization of HDAC1 from the nucleus modifying the chromatin structure and leading to VGluT1 increased levels, suggesting that Tau modulates the gene expression by altering the chromatin condensation. In conclusion, the CST developed in this study allows to follow in real time the pathological process depicting the early and the late stages of aggregation; thus, it is now at the bases of two screening platforms for the drug discovery and validation in reporter cells and in a transgenic model. In addition, this study demonstrated for the first time that Tau in the nuclear compartment modulates the expression of genes probably by altering the chromatin structure and this role seems to be strongly related to mild cognitive impairment stages when Tau is destabilized and partially aggregated.