'Scuola Normale Superiore - Edizioni della Normale'
Abstract
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\u2019s 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\u2019s 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