Aggregation-Prone Region Mapping of Full-Length Huntingtin Links Non-PolyQ Domains to Human Huntington’s Disease Pathology

Introduction: Mutations in exon 1 of the huntingtin (HTT) protein promote its aggregation and accumulation in the brain and periphery, leading to neuronal death and Huntington’s disease (HD). Although HTT has over 3,000 amino acids, exon 1 comprises less than 3% of its length. Proteolysis outside exon 1 yield toxic fragments, but their role in HD remains poorly defined. Objective: To investigate the aggregation potential and pathological relevance of non-exon 1 HTT fragments. Methods: We employed in-silico tools (Aggrescan, Amylpred2, AggreProt, PeptideCutter), in-vitro assays (proteomics, Thioflavin-T fluorescence, TEM, dot blot), and human post-mortem brain analyses (western blotting, TEM, proteomics). Results: In-silico prediction identified 110 aggregation-prone regions, and eight aggregation-prone peptides (AgPPs) were synthesized for validation. These peptides rapidly formed fibrils and oligomers. In-silico, trypsin produced the highest number of AgPPs, and in-vitro digestion of HTT confirmed their generation and aggregation. Western blotting of human HD brains showed elevated trypsin levels. Mass spectrometry identified 103 non-exon 1 HTT fragments in human HD brain aggregates; one, tP1, matched a predicted trypsin product and aggregated in vitro. Notably, non-exon 1 fragments were more abundant in Stage I than Stage III brain aggregates, suggesting early pathological involvement. In iPSC-derived neurons from HD patients, tP1 aggregates induced neurodegeneration mechanisms similarly to exon 1 aggregates. Conclusions: These findings demonstrate that non-exon 1 HTT fragments aggregate and accumulate in the HD brain, implicating them in disease pathology. This expands the current understanding of HTT aggregation beyond exon 1 and suggests a novel avenue for therapeutic exploration.