Towards deciphering the Nt17 code: How the sequence and conformation of the first 17 amino acids in Huntingtin regulate the aggregation, cellular properties and neurotoxicity of mutant Httex1

The first 17 N-terminal amino acids (the Nt17 domain) flanking the polyQ tract of the Huntingtin protein (Htt) play an important role in modulating its aggregation, life cycle, membrane binding, and toxicity. Therefore, a better understanding of the molecular and structural determinants of the Nt17 code would likely provide important insights and help guide the development of future anti-aggregation and Htt lowering therapeutic strategies. Towards this goal, we sought to elucidate the role of the Nt17 sequence and helical conformation in regulating mutant Httex1 aggregation, morphology, uptake, and neuronal toxicity. To modulate the helical conformation of Nt17, we used a helix and membrane-binding disrupting mutation (M8P) strategy and site-specific introduction of post-translational modifications that are known to enhance (pT3) or disrupt (pS13, pS16, or pS13/pS16) the overall helicity of Nt17. Our in vitro studies show that the Nt17 and polyQ domains synergistically promote Httex1 aggregation, consistent with previous findings. However, we show that the Nt17 sequence, but not its helical conformation, is a key determinant of the morphology and growth of Httex1 fibrils. In cells, we show that the aggregation propensity and the toxic properties of de novo Httex1 were dependent on both the Nt17 sequence and its helical conformation and the synergistic effect of the Nt17 and polyQ domains. Finally, we demonstrate that the uptake of Httex1 into primary striatal neurons is strongly influenced by the helical propensity of Nt17. Phosphorylation (at T3 or S13/S16) or removal of the Nt17 domain increases the uptake and accumulation of Httex1 fibrils into the nucleus and induces neuronal cell death. Altogether our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, cell-to-cell propagation, and neuronal toxicity. These findings have significant implications for targeting the Nt17 domain to develop new disease-modifying therapies for the treatment of Huntington's disease.