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Tracking mutant huntingtin aggregation kinetics in cells reveals three major populations including an invariant oligomer pool.

J Biol Chem. 2010 May 5;

Authors: Olshina MA, Angley LM, Ramdzan YM, Tang J, Bailey MF, Hill AF, Hatters DM

Huntington's disease is caused by expanded polyglutamine (polyQ) sequences in huntingtin, which procures its aggregation into intracellular inclusion bodies (IBs). Aggregate intermediates such as soluble oligomers are predicted to be toxic to cells yet because of a lack of quantitative methods, the kinetics of aggregation in cells remains poorly understood. We used sedimentation velocity analysis to define and compare the heterogeneity and flux of purified huntingtin with huntingtin expressed in mammalian cells under non-denaturing conditions. Non-pathogenic huntingtin remained as hydrodynamically elongated monomers in vitro and in cells. Purified polyQ-expanded pathogenic huntingtin formed elongated monomers (2.4 S) that evolved into a heterogeneous aggregate population of increasing size over time (100-6,000 S). However in cells, mutant huntingtin formed three major populations: monomers (2.3 S), oligomers (mode s20,w = 140 S) and IBs (mode s20,w = 320,000 S). Strikingly, the oligomer population in proportion of total huntingtin did not change over 3 days and had an unchanging size distribution despite continued monomer conversion to IBs, suggesting oligomers are rate-limiting intermediates to IB formation. We also determined how a chaperone known to modulate huntingtin toxicity, Hsc70 influences in-cell huntingtin partitioning. Hsc70 decreased the pool of 140 S oligomers but increased the overall flux of monomers to IBs, suggesting Hsc70 reduces toxicity by facilitating transfer of oligomers into IBs. Together, our data suggests huntingtin aggregation is streamlined in cells, and is consistent with the 140 S oligomers, which remain invariant over time, as a constant source of toxicity to cells irrespective of total load of insoluble aggregates.

PMID: 20444706 [PubMed - as supplied by publisher]

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