Direct Observation of Secondary Nucleation in Huntingtin Amyloid Formation by High-Speed Atomic Force Microscopy

Amyloid fibril formation is a hallmark of various neurodegenerative diseases such as Huntington's (HD), Alzheimer's, and Parkinson's disease. The protein aggregation process involves slow nucleation events followed by rapid growth and elongation of formed fibrils. Understanding the pathways of amyloid formation is key to development of novel therapeutic agents that can interfere with the pathogenic protein misfolding events. Recent studies of aggregation by polypeptides from Alzheimer's and Huntington's disease have identified the importance of a poorly understood secondary nucleation process that may even be the dominant source of protein aggregate formation. Here, we focus on the polyglutamine-expansion disorder HD and employ mechanistic and structural studies to study different aspects of secondary nucleation in the aggregation of huntingtin Exon 1 (HttEx1). Notably, we apply high-speed atomic force microscopy (HS-AFM) to directly observe the process on the single-particle level and in real time. Our observations show unique features of the amyloid formation dynamics in real time, including secondary nucleation, elongation, and the formation of large bundles of fibrils as a result of nucleated branching. We examine the role of HttEx1 flanking segments during the aggregation process, revealing that the N-terminal Htt^NT segment exhibits a clear primary nucleation-aggregation-enhancing ability; however, it does not seem to induce or affect the secondary nucleation process. The obtained results illuminate the complex aggregation process of HttEx1 and have implications for attempts to inhibit or modulate it for therapeutic purposes.