Huntington Exon-1 Aggregation Pathway Revealed by Time-resolved SANS.

Tatiana Perevozchikova (University of Tennessee)

Huntington's disease (HD) is a genetic neurodegenerative disorder, associated with the mutant huntington (Htt) protein, containing an abnormally long stretch of glutamine (polyQ) residues, which upon proteolityc cleavage forms htt-exon1 fragment and aggregates into highly stable and organized beta-sheet structures. Currently, no clear evidence exists to determine if Htt protein aggregates are epiphenomena, if they are beneficial to or pathogenic for neurons. Although the correlation between the length of polyQ repeats, their propensity for aggregation, and disease is undeniable ? the longer the polyQ region, the earlier the onset of HD and its symptoms are more severe ? several cell and HD animal models studies demonstrated an absence of a link between aggregate presence and neuronal toxicity. It was proposed that neuronal toxicity is associated with early stages of protein fibril formation and that mature aggregates actually represent an inert end stage, serving as a rescue mechanism. At present, a detailed understanding of the structures of different intermediate species, formed both on- and off-pathway to Htt fibril formation, is not established. Molecular insights in amyloid research and protein aggregation suffer from fundamental difficulties in stabilizing early intermediate assemblies and characterizing them. To unravel the aggregation pathways of the htt-exon1 fragments containing normal (22Gln) and pathological (42Gln) length of polyglutamine repeats, we performed time-resolved small angle neutron scattering experiments (TR-SANS) combined with ab-initio reconstruction algorithm. Using these approaches the 3-D structures of the earliest htt exon-1 intermediates as well as their structural evolution into protofilbrills and mature fibrillar aggregates are presented for the first time. Complementation of SANS with other biophysical techniques allowed us to find that the length of polyGln repeat within the htt-exon1 fragment does not only affect the kinetics of aggregate formation, but also significantly influence the structural dynamics and mechanism of aggregation. In addition, osmotic stress and the contrast variation methods provided us with an insight into the internal structure of the mature pathological htt fibrils. Put together, these results not only provide the first steps toward the characterization of htt-exon1 aggregation pathway but also demonstrate that SANS is an essential tool for identification of various intermediates associated with amyloid and neurodegenerative diseases.

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