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Novel targets for Huntington's disease in an mTOR-independent autophagy pathway

Abstract

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the Gi signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.

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Figure 1: Identification of autophagy-inducing drugs.
Figure 2: I1R agonists act through cAMP on Rap2B and PLC-ε to modulate autophagy.
Figure 3: The effects of L-type Ca2+ channel agonist on autophagy and mutant huntingtin aggregation are abrogated by calpain inhibition.
Figure 4: Calpain inhibition increases aggregate-prone protein clearance.
Figure 5: Calpain inhibition induces autophagy.
Figure 6: Calpain cleaves G, creating a link between the cAMP and Ca2+-calpain pathways.
Figure 7: Regulation of autophagy by calpain is mTOR independent.
Figure 8: L-type Ca2+ channel antagonists, I1R agonists, cAMP antagonists and calpain inhibitors rescue Huntington's disease phenotypes in zebrafish.

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Acknowledgements

We thank T. Yoshimori (Osaka University) for LC3 antibody, myc-LC3 and EGFP-LC3 constructs, N. Mizushima (Tokyo Medical and Dental University) for wild-type and knockout Atg5 MEFs, wild-type Atg5 and K130R Atg5 constructs, G. Jackson (University of California, Los Angeles) for the gmrQ120 flies, K.L. Guan (University of Michigan, Ann Arbor) for rheb construct, A. Wells (University of Pittsburgh) for constitutive active S50E m-calpain construct, J. Lomasney (Northwestern University, Chicago) for wild-type PLC-ε construct, J. de Gunzburg (Institut Curie, Paris) for dominant-negative Rap2B construct, R.F. Irvine (University of Cambridge) for cytosolic IP3 kinase A construct, M. Mizuguchi (Toyama Medical and Pharmaceutical University) for human LC3B construct, J.P. Luzio (University of Cambridge) for GFP-lgp20 construct, R. Tsien (University of California, San Diego) for mCherry construct, A.M. Tolkovsky (University of Cambridge) for EGFP-LC3 HeLa stable cells, N.P. Dantuma (Karolinska Institute, Stockholm) for UbG76V-GFP HeLa stable cells, M. Mahaut-Smith for use of the spectrophotometer, J.N. Skepper for EM, A. Roach for critical comments and A. Cordenier for technical assistance. We are grateful for the Gates Cambridge Scholarship and Hughes Hall Research Fellowship (S. Sarkar), Medical Research Council Studentships (A.W., E.K.T.), Biotechnology and Biological Sciences Research Council Career Development Award (C.J.O.'K.), Eli Lilly Pergolide Fellowship (S. Saiki), Academy of Medical Sciences–Medical Research Council Clinical Scientist Fellowship (R.A.F.), Wellcome Trust Senior Fellowship in Clinical Science (D.C.R.), Knowledge Transfer Partnership grant (Department of Trade and Industry), an MRC Programme Grant, an MRC Link Grant, European Union Framework VI (EUROSCA) and the National Institute for Health Research Biomedical Research Centre at Addenbrooke's Hospital for funding.

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A.W., S. Sarkar, P.C., E.K.T., S. Saiki, F.H.S., L.J., D.P. and R.A.F. performed experiments. All authors participated in the design and analysis of various experiments. A.W., S. Sarkar and D.C.R. wrote the paper.

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Correspondence to David C Rubinsztein.

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D.P. and A.F. are employees of Summit plc and have share options in this company. A.F. and P.G. are shareholders in Summit plc. So.S., A.W., D.C.R., E.K.T., C.J.O'K. and R.A.F. are inventors on patents relating to the use of autophagy activation in various diseases.

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Williams, A., Sarkar, S., Cuddon, P. et al. Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nat Chem Biol 4, 295–305 (2008). https://doi.org/10.1038/nchembio.79

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