Elucidation of the structures and biological activities of huntingtin oligomers

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Huntington's disease (HD)is a fatal, inherited neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin, which leads to its aggregation into amyloid fibrils. Historically, fibrils were thought to directly mediate neurodegeneration in HD and other neurodegenerative diseases such as Alzheimer's disease (AD)and Parkinson's disease (PD). However, recent studies in AD and PD suggest an alternative hypothesis whereby potentially diffusible nanoscale assemblies, such as spherical oligomers, protofibrils and pore-like annular structures, are the structural entities that mediate neurodegeneration. Whether the mutant huntingtin protein forms such structures and shares a similar pathogenic mechanism to AD and PD remains poorly understood. In our preliminary data we demonstrate by ex situ atomic force microscopy (AFM) that a mutant huntingtin fragment with an expanded polyQ repeat forms spherical and annular oligomeric structures reminiscent of those formed by proteins linked to AD and PD. We also show that the molecular chaperones Hsp70 and Hsp40, which are protective in animal models of HD and other neurodegenerative disorders, can attenuate the formation of spherical and annular oligomers. Although our preliminary results are provocative, an important caveat to these experiments is that the AFM was not performed in solution. We now wish to advance our understanding of the neurodegeneration that occurs in HD by elucidating the structures and biological activities of different types of polyQ aggregates using in situ AFM under near physiological conditions. We will first characterize the aggregation of mutant huntingtin fragments on various surface chemistries and under different solution conditions. We will next determine the biological properties of different aggregates by characterizing their effects on the function of nuclear pore complexes in nuclear envelopes by combining in situ AFM with patch clamp analyses. Finally, we will also characterize the effects of molecular chaperones and anti-huntingtin antibodies on the structure and biological activities of mutant huntingtin fragments. Our long-term goal is that a fundamental understanding of the structures of aggregates formed by mutant huntingtin, and how they mediate neurodegeneration, will result in the design of potential therapeutics to suppress their toxicity in vivo.