Trehalose, a Novel mTOR-independent Autophagy Enhancer, Accelerates the Clearance of Mutant Huntingtin and α-Synuclein
2006; Elsevier BV; Volume: 282; Issue: 8 Linguagem: Inglês
10.1074/jbc.m609532200
ISSN1083-351X
AutoresSovan Sarkar, J. Eric Davies, Zebo Huang, Alan Tunnacliffe, David C. Rubinsztein,
Tópico(s)Parkinson's Disease Mechanisms and Treatments
ResumoTrehalose, a disaccharide present in many non-mammalian species, protects cells against various environmental stresses. Whereas some of the protective effects may be explained by its chemical chaperone properties, its actions are largely unknown. Here we report a novel function of trehalose as an mTOR-independent autophagy activator. Trehalose-induced autophagy enhanced the clearance of autophagy substrates like mutant huntingtin and the A30P and A53T mutants of α-synuclein, associated with Huntington disease (HD) and Parkinson disease (PD), respectively. Furthermore, trehalose and mTOR inhibition by rapamycin together exerted an additive effect on the clearance of these aggregate-prone proteins because of increased autophagic activity. By inducing autophagy, we showed that trehalose also protects cells against subsequent pro-apoptotic insults via the mitochondrial pathway. The dual protective properties of trehalose (as an inducer of autophagy and chemical chaperone) and the combinatorial strategy with rapamycin may be relevant to the treatment of HD and related diseases, where the mutant proteins are autophagy substrates. Trehalose, a disaccharide present in many non-mammalian species, protects cells against various environmental stresses. Whereas some of the protective effects may be explained by its chemical chaperone properties, its actions are largely unknown. Here we report a novel function of trehalose as an mTOR-independent autophagy activator. Trehalose-induced autophagy enhanced the clearance of autophagy substrates like mutant huntingtin and the A30P and A53T mutants of α-synuclein, associated with Huntington disease (HD) and Parkinson disease (PD), respectively. Furthermore, trehalose and mTOR inhibition by rapamycin together exerted an additive effect on the clearance of these aggregate-prone proteins because of increased autophagic activity. By inducing autophagy, we showed that trehalose also protects cells against subsequent pro-apoptotic insults via the mitochondrial pathway. The dual protective properties of trehalose (as an inducer of autophagy and chemical chaperone) and the combinatorial strategy with rapamycin may be relevant to the treatment of HD and related diseases, where the mutant proteins are autophagy substrates. Trehalose is a non-reducing disaccharide found in many organisms, including bacteria, yeast, fungi, insects, invertebrates, and plants. It is the natural hemolymph sugar of invertebrates. It functions to protect the integrity of cells against various environmental stresses like heat, cold, desiccation, dehydration, and oxidation by preventing protein denaturation (1Chen Q. Haddad G.G. J. Exp. Biol. 2004; 207: 3125-3129Crossref PubMed Scopus (207) Google Scholar). Many of the stress-protecting properties of trehalose were discovered in yeast (2Kandror O. Bretschneider N. Kreydin E. Cavalieri D. Goldberg A.L. Mol. Cell. 2004; 13: 771-781Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar); however, it also has beneficial effects in mammals where it is not endogenously synthesized. For instance, it may be a valuable tool for cryopreservation of cells (1Chen Q. Haddad G.G. J. Exp. Biol. 2004; 207: 3125-3129Crossref PubMed Scopus (207) Google Scholar, 3Wolkers W.F. Walker N.J. Tablin F. Crowe J.H. Cryobiology. 2001; 42: 79-87Crossref PubMed Scopus (301) Google Scholar). It is not clear how trehalose mediates many of its protective effects, but some may be via its ability to act as chemical chaperone and influence protein folding through direct protein-trehalose interactions (4Welch W.J. Brown C.R. Cell Stress Chaperones. 1996; 1: 109-115Crossref PubMed Scopus (433) Google Scholar). Trehalose inhibits amyloid formation of insulin in vitro (5Arora A. Ha C. Park C.B. FEBS Lett. 2004; 564: 121-125Crossref PubMed Scopus (205) Google Scholar) and prevents aggregation of β-amyloid associated with Alzheimer disease (6Liu R. Barkhordarian H. Emadi S. Park C.B. Sierks M.R. Neurobiol Dis. 2005; 20: 74-81Crossref PubMed Scopus (284) Google Scholar). Recently, trehalose was shown to inhibit polyglutamine (polyQ) 3The abbreviations used are: polyQ, polyglutamine; Baf, Bafilomycin A1; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; EGFP-HDQ74, EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats; Glu, glucose; HD, Huntington disease; LC3, microtubule-associated protein 1 light chain 3; Lact, lactacystin; 3-MA, 3-methyladenine; mTOR, mammalian target of rapamycin; MEF, mouse embryonic fibroblasts; PD, Parkinson disease; p70S6K, ribosomal S6 protein kinase; Raf, raffinose; Rap, rapamycin; S6P, ribosomal S6 protein; Sor, sorbitol; suc, sucrose; α-syn, α-synuclein; Tre, trehalose; HA, hemagglutinin; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein. -mediated protein aggregation in vitro, reduce mutant huntingtin aggregates and toxicity in cell models and alleviate polyQ-induced pathology in the R6/2 mouse model of Huntington disease (HD) (7Tanaka M. Machida Y. Niu S. Ikeda T. Jana N.R. Doi H. Kurosawa M. Nekooki M. Nukina N. Nat. Med. 2004; 10: 148-154Crossref PubMed Scopus (646) Google Scholar). This protective effect was suggested to be caused by trehalose binding to expanded polyQ and stabilizing the partially unfolded mutant protein. HD is an autosomal-dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion, which results in an abnormally long polyQ tract in the N terminus of the huntingtin protein. Asymptomatic individuals have 35 or fewer CAG repeats, whereas HD is caused by 36 or more repeats. HD and related polyQ expansion diseases are associated with the formation of intraneuronal inclusions (also known as aggregates) by the mutant proteins containing the expanded polyQ tracts. The toxicity of mutant huntingtin is thought to be exposed after it is cleaved to form N-terminal fragments consisting of the first 100-150 residues containing the expanded polyQ tract, which are also the toxic species found in aggregates. Thus, HD pathogenesis is frequently modeled with exon 1 fragments containing expanded polyQ repeats, which cause aggregate formation and toxicity in cell models and in vivo (8Rubinsztein D.C. Trends Genet. 2002; 18: 202-209Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). The clearance of aggregate-prone proteins like mutant huntingtin fragments, other polyQ mutations, mutant α-synucleins and tau is strongly dependent on macroautophagy, generally referred to as autophagy (9Berger Z. Ravikumar B. Menzies F.M. Oroz L.G. Underwood B.R. Pangalos M.N. Schmitt I. Wullner U. Evert B.O. O′Kane C.J. Rubinsztein D.C. Hum. Mol. Genet. 2006; 15: 433-442Crossref PubMed Scopus (562) Google Scholar, 10Ravikumar B. Duden R. Rubinsztein D.C. Hum. Mol. Genet. 2002; 11: 1107-1117Crossref PubMed Scopus (938) Google Scholar, 11Ravikumar B. Vacher C. Berger Z. Davies J.E. Luo S. Oroz L.G. Scaravilli F. Easton D.F. Duden R. O′Kane C.J. Rubinsztein D.C. Nat. Genet. 2004; 36: 585-595Crossref PubMed Scopus (1990) Google Scholar, 12Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar, 13Sarkar S. Floto R.A. Berger Z. Imarisio S. Cordenier A. Pasco M. Cook L.J. Rubinsztein D.C. J. Cell Biol. 2005; 170: 1101-1111Crossref PubMed Scopus (815) Google Scholar). Autophagy is a process that allows bulk degradation of cytoplasmic contents. It involves the formation of double membrane structures called autophagosomes, which fuse with lysosomes to form autolysosomes where their contents are degraded (14Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2988) Google Scholar). Induction of autophagy reduces the levels of mutant huntingtin and protects against its toxicity in cells and in transgenic Drosophila and mouse models of HD (10Ravikumar B. Duden R. Rubinsztein D.C. Hum. Mol. Genet. 2002; 11: 1107-1117Crossref PubMed Scopus (938) Google Scholar, 11Ravikumar B. Vacher C. Berger Z. Davies J.E. Luo S. Oroz L.G. Scaravilli F. Easton D.F. Duden R. O′Kane C.J. Rubinsztein D.C. Nat. Genet. 2004; 36: 585-595Crossref PubMed Scopus (1990) Google Scholar, 13Sarkar S. Floto R.A. Berger Z. Imarisio S. Cordenier A. Pasco M. Cook L.J. Rubinsztein D.C. J. Cell Biol. 2005; 170: 1101-1111Crossref PubMed Scopus (815) Google Scholar). The apparent clearance of huntingtin aggregates by autophagy is likely to be a consequence of removal of aggregate precursors (soluble and oligomeric species), rather than big inclusions, which do not appear to be membrane-bound and are also much larger than typical autophagosomes. Currently, the only suitable pharmacological strategy for upregulating autophagy in mammalian brains is to use rapamycin, or its analogues, that inhibit the mammalian target of rapamycin (mTOR), a negative regulator of autophagy (14Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2988) Google Scholar). Parkinson disease (PD) is another condition associated with aggregate formation. The intraneuronal Lewy body aggregates seen in PD have the protein α-synuclein as a major component. The A53T and A30P point mutations in α-synuclein cause autosomal dominant forms of PD (15Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6667) Google Scholar, 16Kruger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3321) Google Scholar). The A53T and A30P α-synuclein mutants are substrates of autophagy, and the clearance of these mutant forms is retarded when autophagy is inhibited (12Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar). While these forms of α-synuclein aggregate in vivo, we do not observe overt aggregation in our cell lines (12Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar). Furthermore, unlike wild-type α-synuclein, these mutant forms are not cleared by the chaperone-mediated autophagy pathway (17Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1572) Google Scholar), which is distinct from macroautophagy (which we call autophagy in this report). Hence, we have used these mutations as model autophagy substrates. Here we identify a novel role for trehalose as an autophagy inducer. Trehalose, in aqueous solutions, leads to enhanced clearance of aggregate-prone proteins like mutant huntingtin and α-synuclein, and protects cells from subsequent pro-apoptotic insults. Plasmid Constructs—HD gene exon 1 fragment with 74 polyQ repeats in pEGFP-C1 (Clontech) (EGFP-HDQ74) and HA-HDQ74 constructs were characterized previously (18Narain Y. Wyttenbach A. Rankin J. Furlong R.A. Rubinsztein D.C. J. Med. Genet. 1999; 36: 739-746Crossref PubMed Scopus (127) Google Scholar). EGFP-LC3, Myc-LC3-HA (from T. Yoshimori), EGFP-Atg5 (from N. Mizushima), and EGFP-Bax (from R. Youle) constructs were obtained as kind gifts. PABPN1 A17 (from E. Wahle) was subcloned into pHM6 (Roche Applied Science) to generate the HA-PABPN1 A17 construct. Reagents—Compounds used were 100 mm d-(+)-trehalose dihydrate, 100 mm sucrose, 100 mm d-(+)-raffinose pentahydrate, 100 mm d-sorbitol, 0.2 μm rapamycin, 200 nm bafilomycin A1, 10 mm 3-methyladenine, 10 μm lactacystin, 3 μm staurosporine, 200 μg/ml Congo Red (all from Sigma-Aldrich), and 100 mm d-(+)-glucose (BDH). Mammalian Cell Culture and Transfection—African green monkey kidney cells (COS-7), human neuroblastoma cells (SK-N-SH), human cervical carcinoma cells (HeLa), stable HeLa cells expressing EGFP-LC3 (19Bampton E.T.W. Goemans C.G. Niranjan D. Mizushima N. Tolkovsky A.M. Autophagy. 2005; 1: 23-36Crossref PubMed Scopus (314) Google Scholar) (kind gift from A. M. Tolkovsky), and wild-type Atg5 (Atg5+/+) and Atg5-deficient (Atg5-/-) mouse embryonic fibroblasts (20Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar) (MEFs) (kind gift from N. Mizushima) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, and 2 mm l-glutamine (Sigma) at 37 °C, 5% CO2. Inducible PC12 stable cell lines expressing EGFP-HDQ74 or EGFP-HDQ23 (10Ravikumar B. Duden R. Rubinsztein D.C. Hum. Mol. Genet. 2002; 11: 1107-1117Crossref PubMed Scopus (938) Google Scholar, 21Wyttenbach A. Swartz J. Kita H. Thykjaer T. Carmichael J. Bradley J. Brown R. Maxwell M. Schapira A. Orntoft T.F. Kato K. Rubinsztein D.C. Hum. Mol. Genet. 2001; 10: 1829-1845Crossref PubMed Google Scholar), and HA-tagged A30P or A53T α-synuclein mutants (12Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar), previously characterized, were maintained at 75 μg/ml hygromycin B (Calbiochem) in Dulbecco’s modified Eagle’s medium with 10% horse serum, 5% fetal bovine serum, 100 units/ml penicillin/streptomycin, 2 mm l-glutamine, and 100 μg/ml G418 (Invitrogen) at 37 °C, 10% CO2. T-REx 293 (Invitrogen), derived from human embryonal kidney cell line HEK 293, was maintained in Dulbecco’s modified Eagle’s medium with 10% horse serum, 5% fetal bovine serum, 100 units/ml penicillin/streptomycin, 2 mm l-glutamine, and 5 μg/ml Blasticidin S (Invitrogen) at 37 °C, 5% CO2. QA1/12/9A and A21/12/2B (Tre) are stable transfectant cell lines produced from T-REx 293, which were maintained in similar medium and conditions as T-REx 293 along with the addition of 500 μg/ml G418 (Geneticin) and 250 μg/ml Zeocin (Invitrogen). Cells were transfected with the constructs for 4 h using Lipofectamine or Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol, fixed with 4% paraformaldehyde (Sigma) after 48 h (EGFP-HDQ74) or 2 h (EGFP-LC3) post-transfection, and mounted in Citifluor (Citifluor Ltd.) containing 4′,6-diamidino-2-phenylindole (DAPI; 3 μg/ml; Sigma-Aldrich). Quantification of Aggregate Formation and Cell Death—Approximately 200 EGFP-positive cells were counted for the proportion of cells with EGFP-HDQ74 aggregates, as described previously. Only EGFP-positive cells were counted so that we count only the transfected cells. If an EGFP-positive cell has one or many aggregates, the aggregate score is one. If an EGFP-positive cell does not have any aggregate, the aggregate score is zero. Nuclei were stained with DAPI and those showing apoptotic morphology were considered abnormal. These criteria are specific for cell death, which highly correlate with propidium iodide staining in live cells (22Wyttenbach A. Sauvageot O. Carmichael J. Diaz-Latoud C. Arrigo A.P. Rubinsztein D.C. Hum. Mol. Genet. 2002; 11: 1137-1151Crossref PubMed Scopus (425) Google Scholar). Similar assessment for cell death was done after apoptotic insults with staurosporine or Bax. Experiments were done in triplicate at least twice. Quantification of Cells with EGFP-LC3 Vesicles—Similar analyses in triplicate were done for counting the proportion of EGFP-positive cells with EGFP-LC3 vesicles. Approximately 100 EGFP-positive cells were counted for the proportions of EGFP-positive cells with >5 LC3-positive vesicles. We considered an EGFP-positive cell as having a score of zero if there were 5 or fewer vesicles (as cells have basal levels of autophagy) and cells scored one if they had >5 LC3-positive vesicles (13Sarkar S. Floto R.A. Berger Z. Imarisio S. Cordenier A. Pasco M. Cook L.J. Rubinsztein D.C. J. Cell Biol. 2005; 170: 1101-1111Crossref PubMed Scopus (815) Google Scholar). Clearance of Mutant Huntingtin and α-Synucleins—Stable inducible PC12 cell lines expressing EGFP-HDQ74, EGFP-HDQ23, or α-synuclein mutants (A30P or A53T) were induced with 1 μg/ml doxycycline (Sigma) for 8 and 48 h, respectively (10Ravikumar B. Duden R. Rubinsztein D.C. Hum. Mol. Genet. 2002; 11: 1107-1117Crossref PubMed Scopus (938) Google Scholar, 12Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar). Transgene expression was switched off by removing doxycycline from medium. Cells were treated with or without compounds for time points as indicated in experiments. Compounds were replenished every 24 h for EGFP-HDQ74 clearance. Clearance of soluble huntingtin or α-synuclein mutants was measured by immunoblotting with antibody against EGFP or HA, respectively. Western Blot Analysis—Cell pellets were lysed on ice in Laemmli buffer (62.5 mm Tris-HCl, pH 6.8, 5% β-mercaptoethanol, 10% glycerol, and 0.01% bromphenol blue) for 30 min in presence of protease inhibitors (Roche Applied Science). Primary antibodies include anti-EGFP (8362-1, Clontech), anti-HA (12CA5, Covance), anti-complex IV subunit IV (A-21348, Molecular Probes), anti-cytochrome c (4272), anti-caspase 3 (9665), anti-mTOR (2972), anti-phospho-mTOR (Ser2448) (2971), anti-p70 S6 kinase (9202), anti-phospho-p70 S6 kinase (Thr389) (9206), anti-4E-BP1 (9452), anti-phospho-4E-BP1 (Thr37/46) (9459) (all from Cell Signaling Technology), anti-LC3 (kind gift from T. Yoshimori), anti-Beclin-1 (3738, Cell Signaling), anti-Atg7 (600-401-487, Rockland), anti-Atg12 (36-6400, Zymed Laboratories), anti-c-Myc (C3956, Sigma), anti-actin (A2066, Sigma), and anti-tubulin (Clone DM 1A, Sigma). Blots were probed with anti-mouse or anti-rabbit IgG-horseradish peroxidase and visualized using ECL or ECL Plus detection kit (Amersham Biosciences). Generation of Stable Human Cell Line Synthesizing Intracellular Trehalose—T-REx 293 (Invitrogen), derived from the human embryonal kidney cell line HEK 293, expresses TetR, which represses transcription from promoters containing the tet operator unless tetracycline or derivatives are present in the medium. QA1/12/9A and A21/12/2B (Tre) are stable transfectant cell lines produced from T-REx 293, which contains a tetracycline-inducible form of the trehalose-6-phosphate synthase gene (otsA) of Escherichia coli, together with constitutively expressed otsB (trehalose-6-phosphate phosphatase gene). Intracellular trehalose concentration in QA1/12/9A (Tre) cell line, 24 h after tetracycline induction, is ∼20 mm as determined by gas chromatography (23de Castro A. Garcia Tunnacliffe A. FEBS Lett. 2000; 487: 199-202Crossref PubMed Scopus (79) Google Scholar). Immunocytochemistry—Cells were fixed with 4% paraformaldehyde in 0.1 m PBS, pH 7.6, immunolabeled with antibody against active caspase 3 (G7481, Promega) or HA (12CA5, Covance) and fluorophore-conjugated secondary antibody (Alexa Fluor 488 (green) goat anti-rabbit or Alexa Fluor 594 (red) goat anti-mouse (Molecular Probes, Cambridge Bioscience), respectively), and mounted in Citifluor containing DAPI (3 μg/ml; Sigma-Aldrich) to visualize nuclei. Approximately 200 cells were scored for abnormal apoptotic nuclei (cell death) and active caspase 3 (bright, fluorescent immunolabeling using the active caspase 3 antibody). Microscopy—Transfected cells were analyzed on a Nikon Eclipse E600 fluorescence microscope (plan-apo 60×/1.4 oil immersion lens at room temperature) (Nikon, Inc.). Images of EGFP-LC3 HeLa stable or COS-7 cells were acquired on a Zeiss LSM510 META confocal microscope (63 × 1.4 NA plan-apochromat oil immersion or fluar 40×/1.3 oil lens, respectively) at room temperature using Zeiss LSM510 v3.2 software (Carl Zeiss, Inc.), and Adobe Photoshop 6.0 (Adobe Systems, Inc.) was used for subsequent image processing. Statistical Analysis—Aggregate formation, cell death, or EGFP-LC3 vesicles were expressed as percentages from triplicate samples, and the error bars denote S.E. p values were determined by unconditional logistical regression analysis, using the general log-linear analysis option of SPSS 9 software (SPSS, Chicago). Densitometry analysis on the immunoblots was done by Scion Image Beta 4.02 software (Scion Corporation) from three independent experiments (n = 3). Significance for the clearance of mutant proteins was determined by factorial ANOVA test using STATVIEW software, version 4.53 (Abacus Concepts), where the control condition was set to 100%. The y-axis values are shown in percentage (%), and the error bars denote S.E. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, nonsignificant. Trehalose Reduces polyQ-mediated Aggregation and Cell Death and Enhances the Clearance of Soluble Mutant Huntingtin—We confirmed that trehalose reduced aggregation and cell death caused by EGFP-tagged huntingtin exon 1 with 74 polyQ repeats (EGFP-HDQ74) in COS-7 (non-neuronal) and SK-N-SH (neuronal precursor) cells (Fig. 1, A and B and supplemental Fig. S1A). This effect of 100 mm trehalose was not caused by osmotic stress and was not a general property of the disaccharides, as no such effect was seen with 100 mm sucrose (a disaccharide), raffinose (a trisaccharide), or sorbitol (a sugar alcohol) (Fig. 1, A and B). We tested if the reduced aggregation of the huntingtin construct was partly because of enhanced clearance leading to lower levels (10Ravikumar B. Duden R. Rubinsztein D.C. Hum. Mol. Genet. 2002; 11: 1107-1117Crossref PubMed Scopus (938) Google Scholar, 18Narain Y. Wyttenbach A. Rankin J. Furlong R.A. Rubinsztein D.C. J. Med. Genet. 1999; 36: 739-746Crossref PubMed Scopus (127) Google Scholar), using a stable doxycycline-inducible PC12 cell line expressing EGFP-HDQ74, where transgene expression is first induced by adding doxycycline and then switched off by removing doxycycline from the medium. If the transgene expression level is followed at various times after switching off expression after an initial induction period, one can assess if specific agents alter the clearance of the transgene product, as the amount of transgene product decays when synthesis is stopped (10Ravikumar B. Duden R. Rubinsztein D.C. Hum. Mol. Genet. 2002; 11: 1107-1117Crossref PubMed Scopus (938) Google Scholar). Trehalose significantly reduced EGFP-HDQ74 aggregates at 48 and 72 h and enhanced the clearance of soluble EGFP-HDQ74 and insoluble mutant huntingtin (that gets retarded in the stacking gel) at 120 h (Fig. 1, C and D and supplemental Fig. S1B). Sucrose, raffinose, and sorbitol did not have any effects on the clearance of EGFP-HDQ74 when used at similar concentrations (Fig. 1E). The enhanced clearance of EGFP-HDQ74 is not simply a chaperone effect, as no clearance was observed with Congo Red at doses that do reduce EGFP-HDQ74 aggregation and toxicity (supplemental Fig. S1, C and D). However, trehalose did not influence the clearance of wild-type huntingtin exon 1 (EGFP-HDQ23) (Fig. 1F and supplemental Fig. S1E). Trehalose Enhances the Clearance of α-Synuclein Mutants—We also assessed the clearance of A53T and A30P α-synuclein mutants using stable doxycycline-inducible PC12 cell lines, using similar switch-on/off paradigms to EGFP-HDQ74, as these are autophagy substrates (12Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar). Trehalose significantly enhanced the clearance of A30P and A53T mutants of α-synuclein at 24h (Fig. 2, A and B). However, it had no significant effect on the clearance of wild-type α-synuclein at 24 h (Fig. 2C). This is entirely consistent with previous observations that wild-type α-synuclein clearance is not obviously retarded when autophagy is blocked, in contrast to the mutants (12Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar, 17Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1572) Google Scholar). Trehalose Reduces Mutant huntingtin Aggregates by Autophagic Route—We tested if the enhanced clearance of mutant huntingtin mediated by trehalose was by autophagy or the proteasomal route, using inhibitors of autophagy (3-methyl adenine, 3-MA) and the proteasome (lactacystin). Both these inhibitors increased EGFP-HDQ74 aggregates and toxicity in COS-7 cells (Fig. 3A and supplemental Fig. S2, A and B), consistent with our previous observations that this protein is cleared both by autophagy and proteasome (10Ravikumar B. Duden R. Rubinsztein D.C. Hum. Mol. Genet. 2002; 11: 1107-1117Crossref PubMed Scopus (938) Google Scholar, 12Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1154) Google Scholar). When autophagy was inhibited by 3-MA, trehalose could not further reduce EGFP-HDQ74 aggregates (Fig. 3A and supplemental Fig. S2A). However, cells treated with the proteasome inhibitor lactacystin and trehalose had significantly reduced EGFP-HDQ74 aggregates, compared with cells treated with lactacystin alone (Fig. 3A and supplemental Fig. S2A). When 3-MA was used together with lactacystin, the beneficial effect of trehalose on aggregation was lost and inclusions were significantly increased (Fig. 3A and supplemental Fig. S2A). These data suggest that trehalose enhanced clearance of EGFP-HDQ74 through the autophagic route. We further confirmed these data by comparing mutant huntingtin aggregation in autophagy-competent MEFs (Atg5+/+) or matched MEFs lacking the essential autophagy gene Atg5 (Atg5-/-) (20Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1161) Google Scholar). In untreated Atg5-/- cells, the Atg5/autophagy deficiency dramatically increased mutant huntingtin aggregation and toxicity compared with untreated Atg5+/+ cells, as this mutant protein is an autophagy substrate (Fig. 3, B and C). When these cells were treated with trehalose, the mutant huntingtin aggregation and toxicity were significantly reduced in Atg5+/+ cells, but not in Atg5/autophagy-deficient (Atg5-/-) cells, thus confirming that the ability of trehalose to induce autophagy is a major factor behind its ability to reduce mutant huntingtin aggregation in cells (Fig. 3, B and C). Trehalose Induces Autophagy—We first assessed the effect of trehalose on autophagy in the Atg5+/+ and Atg5-/- MEFs by measuring the levels of microtubule-associated protein 1 light chain 3 (LC3). Endogenous LC3 is processed post-translationally into LC3-I, which is cytosolic. LC3-I is converted to LC3-II, which associates with autophagosome membranes (24Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar). LC3-II levels relative to actin/tubulin correlate with autophagosome number per cell (24Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar, 25Mizushima N. Int. J. Biochem. Cell Biol. 2004; 36: 2491-2502Crossref PubMed Scopus (812) Google Scholar). As we are interested in autophagosome number per cell, we have not quantified LC3-II versus LC3-I, as some LC3-II can be converted back to LC3-I (26Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Trehalose significantly increased LC3-II levels in the autophagy-competent Atg5+/+ cells, but not in the autophagy-deficient Atg5-/- cells (Fig. 3D). LC3-II levels were significantly increased also in COS-7 cells treated with trehalose for 24 h (Fig. 4A). HeLa cells stably expressing LC3 fused to EGFP (EGFP-LC3) (19Bampton E.T.W. Goemans C.G. Niranjan D. Mizushima N. Tolkovsky A.M. Autophagy. 2005; 1: 23-36Crossref PubMed Scopus (314) Google Scholar) treated for 24 h with trehalose had significantly higher EGFP-LC3-II levels (Fig. 4B) compared with untreated cells. The increase in LC3-II by trehalose is similar to what has been observed previously when autophagy is induced (24Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5468) Google Scholar). In this cell line, 100 mm trehalose (the concentration used in our experiments) induced autophagy (supplemental Fig. S3A). Accumulation of LC3-II can occur because of increased autophagosome formation, but also if there is impaired autophagosome-lysosome fusion. We assayed LC3-II in the presence of bafilomycin A1, which blocks autophagosome-lysosome fusion (27Yamamoto A. Tagawa Y. Yoshimori T. Moriyama Y. Masaki R. Tashiro Y. Cell Struct. Funct. 1998; 23: 33-42Crossref PubMed Scopus (1077) Google Scholar). Bafilomycin A1 resulted in the expected increase in EGFP-LC3-II in stable HeLa cells (Fig. 4C). The dose of bafilomycin A1 used is saturating for LC3-II levels in this assay (data not shown). Further blockage of autophagosome-lysosome fusion via a bafilomycin A1-independent mechanism, using the dynein inhibitor erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) (28Ekstrom P. Kanje M. J. Neurochem. 1984; 43: 1342-1345Crossref PubMed Scopus (21) Google Scholar), along with this dose of bafilomycin A1, results in no increase in LC3-II compared with bafilomycin A1 alone (data
Referência(s)