Abstract
Aim:
Huntingtin protein (Htt) was a neuropathological hallmark in human Huntington's Disease. The study aimed to investigate whether the macroautophagy regulator, Beclin1, was involved in the degradation of Htt.
Methods:
PC12 cells and primary cultured brain neurons of rats were examined. pDC316 adenovirus shuttle plasmid was used to mediate the expression of wild-type Htt-18Q-552 or mutant Htt-100Q-552 in PC12 cells. The expression of the autophagy-related proteins LC3 II and Beclin1, as well as the lysosome-associated enzymes Cathepsin B and L was evaluated using Western blotting. The locations of Beclin1 and Htt were observed with immunofluorescence and confocal microscope.
Results:
Htt552 expression increased the expression of LC3 II, Beclin1, cathepsin B and L in autophagy/lysosomal degradation pathway. Treatment with the autophagy inhibitor 3-MA or the proteasome inhibitors lactacystin and MG-132 increased Htt552 levels in PC12 cells infected with Ad-Htt-18Q-552 or Ad-Htt-100Q-552. The proteasome inhibitor caused a higher accumulation of Htt552-18Q than Htt552-100Q, and the autophagy inhibitor resulted in a higher accumulation of Htt552-100Q than Htt552-18Q. Similar results were observed in primary cultured neurons infected with adenovirus. In Htt552-expressing cells, Beclin1 was redistributed from the nucleus to the cytoplasm. Htt siRNA prevented Beclin1 redistribution in starvation conditions. Blockade of Beclin1 nuclear export by leptomycin B or Beclin1 deficiency caused by RNA interference induced the formation of mHtt552 aggregates.
Conclusion:
Beclin1 regulates the accumulation of Htt via macroautophagy.
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References
Kim YJ, Yi Y, Sapp E, Wang Y, Cuiffo B, Kegel KB, et al. Caspase 3-cleaved N-terminal fragments of wild-type and mutant Htt are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis. Proc Natl Acad Sci USA 2001; 98: 12784–9.
Qin ZH, Gu ZL, Lin F . The advancement of molecular pathology of Huntington's disease. Chin Pham Bull 2004; 20: 378–82.
Gauthier LR, Charrin BC, Borrell-Pages M, Dompierre JP, Rangone H, Cordelieres FP, et al. Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules. Cell 2004; 118: 127–38.
Rigamonti D, Bauer JH, De-Fraja C, Conti L, Sipione S, Sciorati C, et al. Wild-type huntingtin protects from apoptosis upstream of caspase-3. J Neurosci 2000; 20: 3705–13.
Nasir J, Floresco SB, O'Kusky JR, Diewert VM, Richman JM, Zeisler J, et al. Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell 1995; 81: 811–23.
Duyao MP, Auerbach AB, Ryan A, Persichetti F, Barnes GT, McNeil SM, et al. Inactivation of the mouse Huntington's disease gene homolog Hdh. Science 1995; 269: 407–10.
Zeitlin S, Liu JP, Chapman DL, Papaioannou VE, Efstratiadis A . Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue. Nat Genet 1995; 11: 155–63.
Sapp E, Schwarz C, Chase K, Bhide PG, Young AB, Penney J, et al. Huntingtin localization in brains of normal and Huntington's disease patients. Ann Neurol 1997; 42: 604–12.
Kegel KB, Kim M, Sapp E, McIntyre C, Castano JG, Aronin N, DiFiglia M . Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation, and autophagy. J Neurosci 2000; 20: 7268–78.
Ravikumar B, Duden R, Rubinsztein DC . Aggregate-proneproteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum Mol Genet 2002; 11: 1107–17.
Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 2004; 36: 585–95.
Sarkar S, Davies JE, Huang Z, Tunnacliffe A, Rubinsztein DC . Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein. J Biol Chem 2007; 282: 5641–52.
Qin ZH, Wang Y, Kegel KB, Kazantsev A, Apostol BL, Thompson LM, et al. Autophagy regulates the processing of amino terminal huntingtin fragments. Hum Mol Genet 2003; 12: 3231–44.
Heng MY, Duong DK, Albin RL, Tallaksen-Greene SJ, Hunter JM, Lesort MJ, et al. Early autophagic response in a novel knock-in model of Huntington disease. Hum Mol Genet 2010; 19: 3702–20.
Wellington CL, Ellerby LM, Gutekunst CA, Rogers D, Warby S, Graham RK, et al. Caspase cleavage of mutant Htt precedes neurodegeneration in Huntington's disease. J Neurosci 2002; 22: 7862–72.
Zhai W, Jeong H, Cui LB, Krainc D, Tjian R . In vitro analysis of Htt-mediated transcriptional repression reveals multiple transcription factor targets. Cell 2005; 123: 1241–53.
Wang J, Wang CE, Orr A, Tydlacka S, Li SH, Li XJ . Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice. J Cell Biol 2008; 180: 1177–89.
Bergamini E, Cavallini G, Donati A, Gori Z . The role of macroautophagy in the ageing process, anti-ageing intervention and age-associated diseases. Int J Biochem Cell B 2004; 36: 2392–404.
Heiser V, Scherzinger E, Boeddrich A, Nordhoff E, Lurz R, Schugardt N, et al. Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington's disease therapy. Proc Natl Acad Sci U S A 2000; 97: 6739–44.
Sarkar S, Rubinsztein DC . Huntington's disease: degradation of mutant huntingtin by autophagy. FEBS J 2008; 275: 4263–70.
Chen JJ, Lin F, Qin ZH . The roles of the proteasome pathway in signal transduction and neurodegenerative diseases. Neurosci Bull 2008; 24: 183–94.
Miller RJ, Wilson SM . Neurological disease: UPS stops delivering! Trends Pharmacol Sci 2003 24: 18–23.
Klionsky DJ, Emr SD . Autophagy as a regulated pathway of cellular degradation. Science 2000; 290: 1717–21.
Levine B, Klionsky DJ . Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 2004; 6: 463–77.
Shintani T, Klionsky DJ . Autophagy in health and disease: a double-edged sword. Science 2004; 306: 990–5.
Tassa A, Roux MP, Attaix D, Bechet DM . Class III phosphoinositide 3-kinase-Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes. Biochem J 2003; 376: 577–86.
Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, et al. Induction of autophagy and inhibition of tumorigenesis by Beclin 1. Nature (London) 1999; 402: 672–6.
Shibata M, Lu T, Furuya T, Degterev A, Mizushima N, Yoshimori T, et al. Regulation of intracellular accumulation of mutant Huntingtin by Beclin 1. J Biol Chem 2006; 281: 14474–85.
Saeki K, Yuo A, Okuma E, Yazaki Y, Susin SA, Kroemer G, et al. Bcl-2 down-regulation causes autophagy in a caspase-independent manner in human leukemic HL-60 cells. Cell Death Differ 2000; 7: 1263–9.
Liang XH, Yu J, Brown K, Levine B . Beclin 1 contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function. Cancer Res 2001; 61: 3443–9.
Yamamoto A, Cremona ML, Rothman JE . Autophagy-mediated clearance of Htt aggregates triggered by the insulin-signaling pathway. J Cell Biol 2006; 172: 719–31.
Li X, Wang CE, Huang S, Xu X, Li XJ, Li H, et al. Inhibiting the ubiquitin-proteasome system leads to preferential accumulation of toxic N-terminal mutant huntingtin fragments. Hum Mol Genet 2010; 19: 2445–55.
Kihara A, Kabeya Y, Ohsumi Y, Yoshimori T . Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO Rep 2001; 2: 330–5.
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (No 30600197) and the Natural Science Foundation of Jiangsu Province for College (No 09KJB310014).
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Wu, Jc., Qi, L., Wang, Y. et al. The regulation of N-terminal Huntingtin (Htt552) accumulation by Beclin1. Acta Pharmacol Sin 33, 743–751 (2012). https://doi.org/10.1038/aps.2012.14
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DOI: https://doi.org/10.1038/aps.2012.14
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