Anisosmotic Modulation of Mutant Huntingtin Aggregation vis-a-vis HSP70 Induction─Implications for Aging,...

Suboptimal cell hydration is a significant risk factor for age-related deterioration and disease vulnerability. Herein, we use a Huntington disease cell model to evaluate osmolarity-dependent modulation of (1) aggregation of polyQ-expanded mutant Huntingtin-EGFP reporter protein as a readout for structurally dynamic disease proteins versus (2) induction of HSP70 chaperone to report on stress-induced lability of folded proteins. Cell impermeant alkali-metal salts and polyethylene glycols were added to cell media to osmotically dehydrate cells for crowding, whereas water was added to swell cells for macromolecular dispersion. Cell image and biochemical analyses show that addition of sodium chloride and other alkali-metal salts to cell media promoted aggregation of mHTT^Exon1-EGFP protein into forming "inclusion bodies" (IBs) in live cells, while concurrently dampened the induction of HSP70 by heat shock. Conversely, a hypo-osmotic medium tempered the compaction of mHTT^Exon1-EGFP into forming IBs while increasing the induction of HSP70. Cell impermeable PEGs likewise promoted mHTT^Exon1-EGFP aggregation. These observations underscore the importance of an iso-osmotic cell environment for balanced structure and function of disordered versus folded proteome, that deviations from this ideal carry dire consequences on protein homeostasis conducive to disease protein aggregation and stress vulnerability.