Aggregation of PolyQ Proteins Is Increased upon Yeast Aging and Affected by Sir2 and Hsf1: Novel Quantitative Biochemical and Microscopic Assays

Aviv Cohen,Shoshana Bar-Nun,Liron Ross,Iftach Nachman,Anne C Hart

doi:10.1371/journal.pone.0044785

Abstract

Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in Saccharomyces cerevisiae aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an “aggregation timeline”. This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, SIR2 and HSF1, affect aggregation of the polyQ proteins. In Δsir2 strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.

Highlights

Huntington’s disease (HD) is a progressive neurodegenerative disorder manifested by dysfunction and cell death mainly in the striatal and cortical brain regions [1,2,3]
CAG repeat RNA was recently proposed as an auxiliary toxic agent in polyQ disorders [11], it appears that the deleterious effects in HD are mostly related to the tendency of the mutated huntingtin protein to misfold and aggregate
Quantitative measurements of polyQ proteins aggregation in yeast cells Aggregates of polyQ proteins and their distribution in live cells are usually detected by fluorescence microscopy, owing to a fluorescent tag attached to the polyQ proteins [55;56]

Summary

Introduction

Huntington’s disease (HD) is a progressive neurodegenerative disorder manifested by dysfunction and cell death mainly in the striatal and cortical brain regions [1,2,3]. HD is an autosomal dominant genetic disease, caused by mutations in the first exon of the huntingtin gene, Htt, that increase the number of CAG repeats to generate expanded polyglutamine (polyQ) tracts in the N-terminal region of the huntingtin protein [7]. Among the genetically manipulatable model organisms for studying aggregation and toxicity of polyQ proteins, S. cerevisiae provides unsurpassed tools to decipher disease-associated cellular processes and identify novel therapeutic targets [54]. This unicellular eukaryote neither resembles neurons nor expresses endogenous huntingtin, the relevant cellular pathways appear to be highly conserved between humans and yeast. Sir and Hsf appear to affect aggregation-related processes during aging

Results

Discussion

Materials and Methods