Abstract
The accumulation of mutant aggregate-prone proteins is a hallmark of the majority of neurodegenerative disorders, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Autophagy, a cytosolic bulk degradation system, is the major clearance pathway for several aggregate-prone proteins, such as mutant huntingtin. The autophagosome-associated protein LC3-II is a specific marker of autophagic flux within cells, whereas aggregate formation of mutant huntingtin represents a good readout for studying autophagy modulation. Here we describe the method of assessing autophagic flux using LC3-II western blotting and substrate clearance by expressing the N-terminal fragment of huntingtin (htt exon 1) containing an expanded polyglutamine tract in mammalian cells.
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Acknowledgments
We thank the Tau consortium (D.C.R.), Wellcome Trust (Principal Research Fellowship to 095317/Z/11/Z), a Wellcome Trust Strategic Grant to Cambridge Institute for Medical Research (100140/Z/12/Z), and NIHR Biomedical Research Unit in Dementia at Addenbrooke’s Hospital, for funding. Ye Zhu is supported by CSC Cambridge Scholarship from Cambridge Trust and China Scholarship Council.
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Stamatakou, E., Zhu, Y., Rubinsztein, D.C. (2018). Assessing Autophagic Activity and Aggregate Formation of Mutant Huntingtin in Mammalian Cells. In: Precious, S., Rosser, A., Dunnett, S. (eds) Huntington’s Disease. Methods in Molecular Biology, vol 1780. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7825-0_2
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DOI: https://doi.org/10.1007/978-1-4939-7825-0_2
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