Exploring the therapeutic potential of Huntingtin native state stabilization through targeting its interacting partners

Huntington's disease (HD) is a devastating neurodegenerative disorder marked by the accumulation of Huntingtin protein (HTT) fragments in HD neuronal cells. These aggregates are predominantly composed of polyglutamine (polyQ)-containing HTT N-terminal fragments (N-HTTs), embedding the exon1-encoded domain (Httex1). However, the mechanism governing the release and aggregation of the N-HTTs into inclusion remains unclear. Elucidating the determinant factors involved in the stability and fragmentation dynamics of HTT is therefore crucial for developing effective therapies, to prevent or slow the disease progression. Our work demonstrates that neither wild-type nor mutant full-length HTT (FL-HTT) with up to 73Q repeats self-assembles into amyloid fibrils. We reveal that proteolytic cleavage of mutant HTT and release of N-HTTs is the rate-limiting step in mutant HTT inclusion formation. By establishing an in vitro model utilizing bacterial proteases, we induce the release of various N-HTTs implicated in HTT aggregation and show that Httex1-like fragments predominantly form amyloid fibrils, while longer N-HTTs tend to self-associate into amorphous structures with higher aggregation kinetics. Similarly, the time-controlled release of Httex1 from FL-HTT in cells by a Tobacco Etch Virus (TEV) system contributes to the formation of inclusions. These models can provide a deeper understanding of N-HTTs release and aggregation dynamics, offering insights into therapeutic approaches aimed at reducing the levels of toxic, aggregation-prone HTT fragments. Furthermore, we explore the interaction between HTT and the huntingtin-associated protein of 40kDa (HAP40). The exceptional stability of the complex, contrasting with the polydisperse nature of the apo-FL-HTT, underscores the potential therapeutic benefits of stabilizing HTT native state conformation. Through in-silico analyses and the development of novel biochemical tools, we identify sequence determinants, including post-translational modifications (PTMs), regulating the HTT-HAP40 interaction, facilitating further studies aimed at disrupting or enhancing the complex formation to assess HTT function and stability in the absence of HAP40. Overall, this research advances our understanding of the molecular and structural determinants of HTT aggregation and stability, paving the way for the development of targeted therapies for HD.