Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease ( HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT ( mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution. For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight ( HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.