Controlling htt protein aggregation to assess toxicity in Caenorhabditis elegans.

Abstract

Aberrant aggregation of proteins into amyloid fibrils is implicated in a wide range of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. Beyond fibrils, a heterogeneous mixture of intermediate aggregates, such as oligomers also form, and these oligomers are potent neurotoxic agents. Because of the transient nature of oligomers and that aggregation reactions result in a complex mixture of aggregate species, it has proven challenging to establish the toxic mechanism of action to specific aggregate species. This is particularly true when expressing aggregating proteins within cells or organisms. Using a mutant huntingtin protein (htt) that readily aggregates, a protocol was developed to introduce well-characterized aggregate species directly to C. elegans to assess toxicity. By precisely controlling aggregation conditions (i.e. temperature, time, and centrifugation), uniform populations of htt aggregates were obtained. These populations were characterized using atomic force microscopy and dynamic light scattering. These aggregates were fed to N2 C. elegans in a dose dependent manner using a 96 well microplate. The impact of this exposure on worm viability was measured using survival and thrashing assays. Both htt oligomers and fibrils were cytotoxic to worms in a dose dependent manner. Having established that these controlled aggregate populations were toxic, this approach can be applied to study how a variety of exogenous factors alters the impact of these aggregates on C. elegans.