Use of a technological platform to screening in vitro and in vivo anti-amyloidogenic drugs able to prevent early neurodegenerative process

Amyloidoses are protein misfolding diseases caused by deposition of fibrillar proteins in target organs. Nowadays, most of them are still incurable and their relevance to public health system is growing, especially as a consequence of population aging. Spinocerebellar ataxia type 3 is a member of this group of pathologies and its causative agent is ataxin-3 (ATX3). This is consists of a globular N-terminal (JD), followed by a flexible tail carrying a poly-glutamine (polyQ) tract. An expanded polyQ tract triggers the aggregation. In this work, I have investigated the capability of tetracycline (Tetra), epigallocatechin-gallate (EGCG), epigallocatechin (EGC), gallic acid (GA) and trifluoroethanol (TFE) to interfere with ATX3 amyloid deposition. Tetra is an antibiotic recently re-evaluated as anti- amyloidogenic compound. EGCG, EGC and GA, which are natural polyphenols, are already known in literature for their anti-amyloidogenic effect; finally, TFE is an osmolyte that stabilizes secondary structure, preferentially α-helix. Data obtained by aggregation assay, spectroscopic analyses (NMR, FTIR) and morphologic characterisation clearly demonstrated Tetra capability of increasing ATX3 aggregates solubility, without a substantial remodelling of the internal structure. Nevertheless, this antibiotic reduced the toxicity of the oligomeric species and ameliorated ataxic C. elegans phenotype. On the contrary, the analysed polyphenols were capable to interfere with ATX3 aggregation but, instead of preventing, they accelerated the aggregation rate redirecting the process towards the formation of soluble, not toxic, off-pathway aggregates. All compounds were also active against the JD in isolation, but only the polyphenols were capable to bind the monomeric form. In particular, they overlapped specific aggregation-prone regions directly involved in the fibrillation. This could explain their capability of redirecting the aggregation pathway and the different mode of action with respect to Tetra. These polyphenols showed a remarkable reduction of ATX3-mediated cytotoxicity and mitigation of ataxic phenotype in C. elegans and E. coli models. However, the compounds displayed a different efficacy, whereby EGCG was the most and GA the least effective. All data strongly support the idea that GA is the minimal functional unit of EGCG. TFE did not show the capability of preventing aggregation; in fact, even at very low concentration it promotes a faster amyloid-like aggregation. Biophysical characterization of its effect on JD aggregation, instead, provided evidence that ATX3 aggregation proceeds along a new identified pathway by which protein misfolding follows protein aggregation. In fact, TFE induces the formation of a native-like state almost indistinguishable from fully native protein, but more aggregation prone.