P2-047: Inducible cell model of tauopathy expressing full-length tau

Abstract

In the course of our studies to develop drugs that prevent tau protein aggregation and degeneration of neurons in tauopathies, we recently generated several cell models of tauopathy. They are based on the N2a neuroblastoma cell line and express different variants of the repeat domain of tau (tauRD) in an inducible fashion (Tet–on): the wild–type sequence, a pro–aggregation mutant (ΔK280), and an anti–aggregation mutant, containing additional proline residues. The cells expressing K18ΔK280 mutant show robust aggregation of tau. The aggregates are toxic to cells and their removal is beneficial. We also found that fragmentation of tauRD is important for initiation of aggregation and that phosphorylation in the repeat domain cannot be considered as a precursor of PHF–tauRD. To find out which protease(s) are responsible for the fragments generated from K18ΔK280 in N2a Tet–On cells, we used different protease inhibitors against caspases, proteasome, calpain and thrombin. Only the thrombin inhibitor PPACK inhibited the fragmentation of tauRD suggesting the involvement of a thrombin–like activity in tau aggregation in this cell system. We have now obtained a significantly improved N2a cell model generating tau aggregates composed of full length tau molecules. In order to evaluate the influence of the phosphorylation at SP or TP motifs (in the flanking region of the repeats, targets of proline–directed kinases) on aggregation, we generated a new cell model expressing the full–length isoform htau40ΔK280 and observed its aggregation in the N2a cell model. The aggregation was characterized by three methods: 1) on the biochemical level, the presence of aggregates was demonstrated by sarcosyl extraction of the cells and analysis of soluble and insoluble components, 2) fluorescence microscopy using ThS fluorescence which reports on the propensity of the protein to form β–structure, and finally 3) we visualized PHFs by electron microscopy after density gradient enrichment and gold labeling.