Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease

ARTICLES Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease Brinda Ravikumar1,6, Coralie Vacher1,6, Zdenek Berger1,2, Janet E Davies1, Shouqing Luo1, Lourdes G Oroz1, Francesco Scaravilli3, Douglas F Easton4, Rainer Duden5, Cahir J O’Kane2 & David C Rubinsztein1 Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. Here we show that mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, as the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of Huntington disease, and inhibition of autophagy has the converse effects. Furthermore, rapamycin protects against neurodegeneration in a fly model of Huntington disease, and the rapamycin analog CCI-779 improved performance on four different behavioral tasks and decreased aggregate formation in a mouse model of Huntington disease. Our data provide proof-ofprinciple for the potential of inducing autophagy to treat Huntington disease. Huntington disease is an autosomal dominant neurodegenerative condition caused by a (CAG)n expansion mutation (n > 35), which is translated into an abnormally long polyglutamine tract at the N terminus of huntingtin. There is no effective treatment for Huntington disease. Huntington disease and related diseases associated with polyglutamine expansions (spinocerebellar ataxia types 1, 2, 3, 6, 7 and 17; spinobulbar muscular atrophy and dentatorubral-pallidoluysian atrophy) are probably caused by gain-of-function mechanisms. The mutant proteins causing these diseases accumulate in intraneuronal aggregates (also called inclusions)1. Some propose that aggregates are deleterious, but others argue that aggregates are protective, which accounts for the partial discordance between the presence of aggregates in neuronal subtypes and the specific brain regions affected in Huntington disease2–4. No protective mechanisms for aggregates have been reported. Polyglutamine-expanded proteins interact with various targets, including several transcription factors or cofactors, leading to dysregulation of certain transcriptional pathways. Polyglutamine diseases may be ‘transcriptionopathies’, but the transcriptional pathways affected may differ in specific diseases5–7. Mutant huntingtin is cleaved to form N-terminal fragments comprising the first 100–150 residues containing the polyglutamine repeats, which are believed to be the toxic species found in aggregates1. Accordingly, the pathogenesis of Huntington disease is frequently modeled with exon 1 fragments, which cause toxicity and aggregate in cell models and in vivo. The turnover of mutant huntingtin fragments and other aggregateprone proteins is impaired by inhibitors of the autophagy-lysosome pathway in cell lines8,9. To test whether induction of autophagy protected against the toxicity of mutant huntingtin, we previously treated cells with rapamycin, a specific inhibitor of mTOR10. mTOR is a kinase that regulates important cellular processes10 (Supplementary Fig. 1 online), and its activity inhibits autophagy in cells from yeast to human10. Treatment with rapamycin for 15 h, starting 9 h after transfection with mutant huntingtin exon 1, reduced aggregate formation and cell death, consistent with the induction of autophagy. But cells had many more aggregates 33 h after transfection than 9 h after transfection with the same constructs, and treatment with rapamycin for 15 h starting at 33 h after transfection did not decrease aggregation or cell death. Thus, we concluded that the ability of rapamycin to inhibit mTOR activity might be impaired after prolonged huntingtin expression or increased aggregate formation8. Therefore, early treatment with rapamycin may attenuate Huntington disease in vivo, as rapamycin (and its analogs) are lipophilic, show good blood-brain barrier penetration11–14 and are designed for long-term use in humans11. 1Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2XY, UK. 2Department of Genetics, Cambridge University, Cambridge CB2 3EH, UK. 3Division of Neuropathology, Institute of Neurology, University College London, London, UK.4 Cancer Research U.K., Genetic Epidemiology Unit, Department of Public Health, University of Cambridge, Cambridge, UK. 5Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2XY, UK. 6These authors contributed equally to this work. Correspondence should be addressed to D.C.R. (). Published online 16 May 2004; doi:10.1038/ng1362 NATURE GENETICS VOLUME 36 | NUMBER 6 | JUNE 2004 585 ARTICLES RESULTS mTOR is sequestered into huntingtin aggregates We speculated that the ability of rapamycin to inhibit mTOR activity might become impaired after prolonged huntingtin expression if aggregates sequester and inactivate mTOR. mTOR was diffusely distributed in untransfected cells, in cells expressing wild-type protein (Gln23; Fig. 1a) and in cells expressing mutant protein (Gln74) without aggregates but colocalized with both cytoplasmic and nuclear aggregates in >98% of mutant cells with aggregates (Fig. 1a and Supplementary Fig. 2 online). This degree of mTOR colocalization is much greater than observed with most other proteins previously analyzed15. We never observed mTOR aggregation in the absence of huntingtin aggregates (data not shown). Sequestration of mTOR was not an artefact of protein overexpression or aggregation, as we found no colocalization of certain upstream mTOR regulators or unrelated proteins (Akt, Pdk1, Pten) with mutant huntingtin aggregates (data not shown). mTOR was also sequestered in huntingtin aggregates in brains of transgenic mice expressing N-terminal mutant huntingtin fragments16 (Fig. 1b) and of individuals with Huntington disease (Fig. 1c), but not in brains of controls. a c b d f 586 e g Western blots of whole-cell lysates showed that a substantial amount of mTOR (normally a 289-kDa protein) was present in the form of an abnormally slowly migrating, high-molecular-mass product in the stacking gel (characteristic of aggregates) in mutant but not wild-type cells (Fig. 1d,e). The presence of mutant huntingtin in the stacking gel is frequently considered to be indicative of its insolubility17. We also detected this high-molecular-mass product in the stacking gel using antibodies to phosphorylated mTOR (Fig. 1e) and to green fluorescent protein (GFP) directed to the huntingtin (...truncated)