The Nt17 domain and its helical conformation regulate the aggregation, cellular properties and neurotoxicity of mutant huntingtin exon 1

Abstract

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.

Nuclear and cytoplasmic huntingtin inclusions exhibit distinct biochemical composition, interactome and ultrastructural properties

“…However, cells expressing Httex1 72Q underwent apoptosis after 96 h. As expected, no inclusions were formed upon overexpression of Httex1 16Q even after 72 h post-transfection (Figure S6). Httex1 39Q inclusions were detected predominantly in the cytoplasm of the HEK cells at all the time points examined (24-72 h), though at lower numbers than in the Httex1 72Q conditions: Httex1 39Q (16%) vs. Httex1 72Q (38%) of transfected cells 41 .…”

“…In this model, nuclear inclusions are also observed in ~ 15% of transfected cells by Httex1 72Q 41 , thus providing an opportunity to investigate and compare the ultrastructural features of cytoplasmic and nuclear Htt inclusions under identical conditions. This model system is widely used to investigate molecular, cellular, and pharmacological modulators of Htt aggregation and inclusion formation 11,12,15,[24][25][26][27][28][29][30][31]42,43 , though very often using Htt constructs with extra non-natural sequences such as Myc, FLAG and GFP tags.…”

“…Despite the formation and abundance of inclusions formed by Httex1 72Q, we did not observe overt toxicity in HEK cells. The initiation of apoptotic events was apparent only after 96 h, as indicated by Caspase 3 activation without loss of plasma membrane integrity 41 .…”

“…Both cytoplasmic and nuclear inclusions have been observed in HD patients' brains and transgenic mouse models of HD [50][51][52] . Therefore, we compared the structural and organizational properties of Httex1 72Q inclusions in the cytoplasm (~85%) and nucleus (~15%) of the transfected HEK cells containing inclusions 41 . Using immunofluorescencebased confocal microscopy, we did not observe significant differences in the size or overall morphology between the Httex1 72Q inclusions in the nucleus and the perinuclear region (Figure 2A).…”

“…Towards this goal, we generated Httex1 39Q and 72Q mutants lacking the entire Nt17 domain (∆Nt17) and compared the structural properties of the inclusions formed by these mutants to those formed by Httex1 39Q and Httex1 72Q. Quantitative confocal microscopy revealed a strong reduction in the number of inclusions (~50% reduction) of cells transfected by Httex1 ∆ Nt17 72Q compared to Httex1 72Q 41 . Surprisingly, inclusions formed by the Httex1 ∆ Nt17 72Q (Figure S7A-B) exhibited an architecture and organization (central core and peripheral shell) similar to those formed by Httex1 72Q (Figure 1D-E and Figure S8A).…”