Rapamycin inhibits polyglutamine aggregation independently of autophagy by reducing protein synthesis

King, Matthew A., Hands, Sarah, Hafiz, Farida, Mizushima, Noboru, Tolkovsky, Aviva M. and Wyttenbach, Andreas (2008) Rapamycin inhibits polyglutamine aggregation independently of autophagy by reducing protein synthesis. Molecular Pharmacology, 73 (4), 1052-1063. (doi:10.1124/mol.107.043398).

Abstract

Accumulation of misfolded proteins and protein assemblies is associated with neuronal dysfunction and death in several neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's disease (HD). It is therefore critical to understand the molecular mechanisms of drugs that act on pathways that modulate misfolding and/or aggregation. It is noteworthy that the mammalian target of rapamycin inhibitor rapamycin or its analogs have been proposed as promising therapeutic compounds clearing toxic protein assemblies in these diseases via activation of autophagy. However, using a cellular model of HD, we found that rapamycin significantly decreased aggregation-prone polyglutamine (polyQ) and expanded huntingtin and its inclusion bodies (IB) in both autophagy-proficient and autophagy-deficient cells (by genetic knockout of the atg5 gene in mouse embryonic fibroblasts). This result suggests that rapamycin modulates the levels of misfolded polyQ proteins via pathways other than autophagy. We show that rapamycin reduces the amount of soluble polyQ protein via a modest inhibition of protein synthesis that in turn significantly reduces the formation of insoluble polyQ protein and IB formation. Hence, a modest reduction in huntingtin synthesis by rapamycin may lead to a substantial decrease in the probability of reaching the critical concentration required for a nucleation event and subsequent toxic polyQ aggregation. Thus, in addition to its beneficial effect proposed previously of reducing polyQ aggregation/toxicity via autophagic pathways, rapamycin may alleviate polyQ disease pathology via its effect on global protein synthesis. This finding may have important therapeutic implications.

Previous SectionNext SectionThe polyglutamine/CAG disorders comprise a group of neurodegenerative diseases that are associated with polyglutamine (polyQ) expansion mutations in the respective disease genes that are otherwise unrelated (Cummings and Zoghbi, 2000). Abnormally long polyQ stretches cause proteins to misfold and produce intracellular protein aggregates. It is believed that polyQ aggregation follows a stochastic nucleation-dependent process that initiates oligomerization, amyloid-like fibril formation, and the production of structures called inclusion bodies (IBs) (Perutz and Windle, 2001). Because polyQ misfolding/aggregation is associated with cellular toxicity (Ross and Poirier, 2004), it is crucial to understand the cellular mechanisms that control misfolding/aggregation with a view to the development of drugs that modify these pathways and alleviate disease.

The accumulation of intracellular IBs points to the inability of cells to dispose of mutant polyQ proteins using chaperone-assisted refolding (Muchowski and Wacker, 2005) and proteasome-mediated degradation (Jana and Nukina, 2003). Deciphering the mechanisms of degradation and clearance of polyQ-expanded proteins and how such mechanisms might be targeted using drugs is a major focus of current research. Macroautophagy (here referred to as autophagy) is a process alternative to that of proteasomal degradation by which some long-lived proteins and organelles are cleared (Shintani and Klionsky, 2004). Autophagy may be responsible for clearing polyQ-expanded proteins and their assemblies (Rubinsztein, 2006). Clearance by autophagy occurs by sequestration of the target organelle/protein into double-membrane structures called autophagosomes that fuse with endo/lysosomes and discharge their contents, which are subsequently degraded. The mammalian homolog of Atg8 MAP-LC3 (LC3) is a key mediator of autophagy: after LC3 is C-terminally cleaved (LC3 I), phosphatidylethanolamine is added to the C-terminal glycine by the Atg5/12 complex, generating LC3 II bound to the nascent autophagosomal membrane (Tanida et al., 2004). Because the Atg5/12 complex catalytically activates the lipidation of LC3, trace amounts of Atg5 can support substantial autophagy, whereas Atg5 knockout cells are totally deficient in autophagy (Hosokawa et al., 2006). The hallmark of autophagic activation is the formation of autophagosome puncta containing LC3 II, whereas the biochemical measurement of autophagic activity is expressed as the amount of LC3 II that accumulates in the absence or presence of lysosomal activity.

Autophagy was first implicated in the regulation of IB formation and clearance of aggregate-prone proteins based on the use of chemical activators/inhibitors, including the proautophagic drug rapamycin and knockdown of different autophagic genes (Rubinsztein, 2006). The finding that rapamycin and its analog CCI-779 protect against neurodegeneration in animal models of misfolding diseases (Ravikumar et al., 2004; Berger et al., 2006) opens up immense hopes for treating debilitating diseases such as the polyQ disorders. Rapamycin, a macrolytic lactone produced by Streptomyces hygroscopicus, has immunosuppressive, antimicrobial, and antitumor properties. It binds intracellularly to FK506 binding protein 12 and targets the protein kinase mammalian target of rapamycin (mTOR). Inhibition of phosphorylation of mTOR by rapamycin activates autophagy, and it has been suggested that rapamycin (or analogs) ameliorates neurodegenerative proteinopathies via activation of autophagy (Rubinsztein, 2006). However, mTOR has an impact on various downstream targets not necessarily involved in autophagy, including the control of protein synthesis (Dann and Thomas, 2006; Wullschleger et al., 2006), and because of these effects, it is currently being evaluated in several phase II clinical trials for cancer (Sabatini, 2006). It is therefore unclear whether rapamycin mediates its protective effects solely via autophagy.

To probe the actions of rapamycin on the formation and clearance of expanded polyQ proteins and IBs, we have taken advantage of clonal cell lines of autophagy-proficient (Atg5+/+) and -deficient (Atg5-/-) mouse embryonic fibroblasts (MEFs) that are easily amenable to biochemical and genetic rescue experiments. Using exon 1 of human Htt containing 97 glutamines and fused to enhanced green fluorescent protein (EGFP) (Ex1HttQ97-EGFP) as an aggregation prone model polypeptide, we show that autophagy-deficient cells accumulate insoluble Ex1HttQ97-EGFP more rapidly and form greater numbers of IBs compared with autophagy-proficient cells. Reexpression of Atg5 in Atg5-deficient cells reversed this phenotype. Most strikingly, rapamycin reduced the amount of insoluble Ex1HttQ97-EGFP and IBs to a similar degree in both Atg5+/+ and Atg5-/- cells. The formation of SDS-insoluble polyQ assemblies is a cooperative process that is highly dependent on the accumulation of a critical mass of the protein (Scherzinger et al., 1999; Colby et al., 2006). We suggest that a major effect of rapamycin is the reduction in protein synthesis required for polyQ aggregation and IB formation to occur.

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