Abstract
Intracellular accumulation of polyglutamine (polyQ)-expanded Huntingtin (Htt) protein is a hallmark of Huntington's disease (HD). This study evaluated whether activation of Sirt1 by the anti-cancer agent, β-lapachone (β-lap), induces autophagy in human neuroblastoma SH-SY5Y cells, thereby reducing intracellular levels of polyQ aggregates and their concomitant cytotoxicity. Treatment of cells with β-lap markedly diminished the cytotoxicity induced by forced expression of Htt exon 1 containing a pathogenic polyQ stretch fused to green fluorescent protein (HttEx1(97Q)-GFP). β-lap increased autophagy in SH-SY5Y cells, as evidenced by the increased formation of LC3-II and autolysosomes. Furthermore, β-lap reduced HttEx1(97Q)-GFP aggregation, which was significantly prevented by co-incubation with 3-methyladenine, an inhibitor of autophagy. β-lap increased Sirt1 activity, as shown by the increased deacetylation of the Sirt1 substrates, PARP-1 and Atg5, and the nuclear translocation of FOXO1. Both the induction of autophagy and attenuation of HttEx1(97Q)-GFP aggregation by β-lap were significantly prevented by co-incubation with sirtinol, a general sirtuin inhibitor or by co-transfection with shRNA against Sirt1. The pro-autophagic actions of β-lap were further investigated in a transgenic Caenorhabditis elegans (C. elegans) line that expressed Q67 fused to cyanine fluorescent protein (Q67). Notably, β-lap reduced the number of Q67 puncta and restored Q67-induced defects in motility, which were largely prevented by pre-treatment with RNAi against sir-2.1, the C. elegans orthologue of Sirt1. Collectively, these data suggest that β-lap induces autophagy through activation of Sirt1, which in turn leads to a reduction in polyQ aggregation and cellular toxicity. Thus, β-lap provides a novel therapeutic opportunity for the treatment of HD.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenine / analogs & derivatives
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Adenine / pharmacology
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Animals
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Animals, Genetically Modified / genetics
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Animals, Genetically Modified / growth & development
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Animals, Genetically Modified / metabolism
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Apoptosis / drug effects
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Autophagy / drug effects*
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Blotting, Western
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Caenorhabditis elegans / genetics
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Caenorhabditis elegans / growth & development
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Caenorhabditis elegans / metabolism
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Cell Movement / drug effects
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Cell Proliferation / drug effects
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Humans
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Huntingtin Protein
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Immunoenzyme Techniques
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Immunoprecipitation
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Microscopy, Fluorescence
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Naphthoquinones / pharmacology*
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism*
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Neuroblastoma / drug therapy
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Neuroblastoma / genetics
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Neuroblastoma / pathology*
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Peptides / chemistry
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Peptides / genetics
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Peptides / pharmacology*
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RNA, Messenger / genetics
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RNA, Small Interfering / genetics
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Real-Time Polymerase Chain Reaction
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Reverse Transcriptase Inhibitors / pharmacology
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Reverse Transcriptase Polymerase Chain Reaction
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Sirtuin 1 / antagonists & inhibitors
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Sirtuin 1 / genetics
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Sirtuin 1 / metabolism*
Substances
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HTT protein, human
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Huntingtin Protein
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Naphthoquinones
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Nerve Tissue Proteins
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Peptides
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RNA, Messenger
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RNA, Small Interfering
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Reverse Transcriptase Inhibitors
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Green Fluorescent Proteins
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polyglutamine
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3-methyladenine
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beta-lapachone
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SIRT1 protein, human
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Sirtuin 1
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Adenine
Grants and funding
This work was supported by a grant from the National Research Foundation of Korea (2009-0085747), a grant from the Global Research Laboratory Program (M6-0605-00-0001) funded by the Korean government (MEST), and a grant from the "Systems biology infrastructure establishment grant" provided by Gwangju Institute of Science and Technology (GIST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.