Protofibril Binding Peptides Recognize and Inhibit Huntingtin Amyloid Formation in vitro and in vivo

In Huntingtons disease, polyglutamine expansion in huntingtin exon 1 (Httex1) results in stepwise misfolding, amyloid formation, and neuronal death. Here we used mRNA display directed evolution to generate peptide ligands targeting Httex1 protofibrils, an early, toxic misfolding intermediate. Two distinct peptide families bind protofibrils, one tryptophan rich and the other glutamine rich, resulting in two predominant peptides HD1 (W-rich) and HD8 (Q-rich). Both peptides bind with high affinity and specificity to the misfolded polyQ structure present in protofibrils, a toxic component that is not recognized by existing huntingtin-directed antibodies. Homo- and heterodimers of HD1 and HD8 bind protofibrils with antibody-like affinity, and potently inhibit aggregation of recombinant and cellular Httex1. The HD8-1 heterodimer can be used like an antibody for immunocytochemistry to identify Httex1 aggregates in transfected cells and in the retina of a Huntingtons disease mouse model system (R6/1). Peptide binding to both in vitro and in vivo Httex1 validates in vitro generated Httex1 protofibrils share the same structural features as Httex1 amyloid from cellular and mammalian disease model systems. Further, HD8-1 protofibril recognition enables direct detection of a pathogenic form of Httex1 (misfolded polyQ) as a disease biomarker. Finally, HD1, HD8, and HD8-1 binding and aggregation inhibition defines the protofibril sites that mediate fibril growth. Overall, our observations support developing protofibril-directed ligands as novel, selective, diagnostics and therapeutics for Huntingtons disease. Significance StatementHuntingtin protein polyQ misfolding plays a central role in Huntingtons disease pathogenesis. We used mRNA display to discover a new class of huntingtin-directed ligands protofibril-binding peptides. Unlike presently available antibodies, these peptides selectively recognize misfolded polyQ region, enable using toxic huntingtin aggregates as disease biomarkers, and block huntingtin aggregation in vitro and in vivo. Protofibril binding peptides may thus assist analysis of huntingtin pathology and provide a starting point for designing protein misfolding inhibitors as Huntingtons disease therapeutics.