Effect of small molecule inhibitors on the aggregation mechanism of Huntingtin Exon1

Greeshama Jain1, Marina Trombetta Lima2, Irina Matlahov1, Amalia Dolga2 and Patrick C.A. van der Wel1

Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands
Molecular Pharmacology - Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands

Huntington’s Disease [HD] is a neurodegenerative disease caused by the expansion of the polyQ domain in the exon 1 region of the huntingtin protein. This expansion of the polyQ domain leads to protein misfolding and the formation of β-sheet-rich fibrillar aggregates. Several studies have indicated that these fibrils can cause cytotoxicity, whereas others argue that certain types of aggregates are non-toxic. In the current study, we probe conformational features of the fibrillar protein and examine how small molecule inhibitors can modulate the pathogenic aggregation process. We perform Thioflavin T (ThT) assays to study the aggregation kinetics of Huntingtin Exon 1 (HttEx1) along with high-resolution imaging technique like transmission electron microscopy to understand the fibril morphology. We used small molecule inhibitors to perturb the aggregation process, seeing effective inhibition of aggregation even at sub stoichiometric ratios of inhibitor relative to the protein. The mechanisms by which the inhibitors modulate aggregation are dissected via kinetic analysis of the ThT aggregation curves, distinguishing, e.g., impacts on de-novo nucleation versus secondary nucleation processes. The mechanistic analysis is combined with measurements of aggregate cytotoxicity performed with cultured neuronal cells. These mechanistic and cytotoxicity studies are combined with morphological and structural studies of the HttEx1 fibrils, based on a combination of electron microscopy and solid-state nuclear magnetic resonance spectroscopy. This integrated multidisciplinary approach provides a novel perspective on the ability of small molecule inhibitors to modulate the misfolding landscape of mutant huntingtin exon 1, with potential implications for future treatment strategies.

Keywords:
Huntingtin Exon1 aggregates, Thioflavin T assays, small molecule inhibitors