We found that the formation of amyloid -like huntingtin fibrils in vitro and in vivo critically depends on polyglutamine repeat length, protein concentration, and time. Furthermore, huntingtin aggregation can be seeded by preformed fibrils, suggesting that fibrillogenesis in HD, as in Alzheimer’s disease, is caused by nucleation-dependent polymerization.43 Our findings that the assembly of huntingtin aggregates requires the formation of a nucleus and is time- and protein concentration-dependent may account for the late onset and progressive phenotype in HD. Although fibril formation occurs within hours in the in vitro system, it may take years in neuronal cells of HD patients.

The concentration Inhibitors,research,lifescience,medical of mutant huntingtin in medium spiny neurons, which are affected most in HD, is unknown, but it is likely to be much lower than that used in the in vitro aggregation assays. Thus, we Inhibitors,research,lifescience,medical suggest that the lag time during which huntingtin dinners, trimers,

and oligomers are formed in vivo is significantly elongated in HD patients. For the identification of huntingtin aggregation inhibitors, we have developed a rapid and sensitive filter retardation assay, which is suitable for the highthroughput screening of drugs that prevent aggregate formation.45 Inhibitors,research,lifescience,medical This assay is based on the finding that HD exon 1 aggregates are insoluble in sodium dodecyl sulfate (SDS) and are retained on a cellulose acetate filter, whereas monomelic forms of the HD exon 1 protein do not bind to this filter membrane. The captured aggregates Inhibitors,research,lifescience,medical are then detected by simple immunoblot analysis using a specific anti-huntingtin antibody. Using the filter retardation assay, we first tested a number of known inhibitors of β-amyloid, PrPscr, and microtubule fibril formation for their effect on huntingtin aggregation in vitro.46 We found that Congo red, thioflavine S, chrysamine G, and Direct fast yellow inhibited HD exon 1 protein aggregation in a dose-dependent manner, whereas other potential inhibitors of β-amyloid Inhibitors,research,lifescience,medical formation, such as thioflavine T, gossypol, melatonin, and rifampicin, had little or no effect on huntingtin aggregation. The results obtained in vitro

were confirmed in cell culture model systems. Furthermore, we found that the monoclonal antibody 1C2, which specifically recognizes the elongated polyglutamine stretch in huntingtin, and the heat almost shock proteins Hsp70 and Hsp40 are potent inhibitors of huntingtin aggregation.46, 47 Interestingly, the addition of heat shock proteins to in vitro aggregation reactions shifts the self-association pathway of huntingtin from fibrillar to amorphous aggregation. This suggests that in vivo chaperones may have the potential to transform toxic fibrillar aggregates into nontoxic aggregates, which can then be Daporinad cost degraded by the ubiquitin/proteasome system. Thus, small molecules that activate a heat shock response in neurons may be effective in delaying the onset and progression of HD.