Human Molecular Genetics, 2003, Vol. 12, No. 9 985-994
DOI: 10.1093/hmg/ddg109
© 2003 Oxford University Press
Raised intracellular glucose concentrations reduce aggregation and cell death caused by mutant huntingtin exon 1 by decreasing mTOR phosphorylation and inducing autophagy
1Department of Medical Genetics and 2Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK and 3Taisho Laboratory of Functional Genomics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
Received December 16, 2002; Revised January 28, 2003; Accepted February 13, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntington's disease is caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract. This gain-of-function mutation is associated with huntingtin aggregation and cell death. Autophagy is an important clearance route for mutant huntingtin exon 1. While mammalian target of rapamycin (mTOR) is a key regulator of autophagy, the upstream modifiers of this process are poorly understood. Our previous expression profiling studies in HD cell models observed changes in four genes associated with glucose metabolism, including the GLUT1 glucose transporter. A role for intracellular glucose as a modulator for polyglutamine toxicity was suggested as cell death was reduced by GLUT1 overexpression. Here we show that the protective effect of GLUT1 is associated with decreased huntingtin exon 1 aggregation in cell models. Consistent with this result, we also observed reduced aggregation and enhanced clearance of mutant huntingtin when cells were cultured in raised glucose concentrations (8 g/l). These effects were mimicked by 8 g/l 2-deoxyglucose (2DOG) (transported, phosphorylated but not metabolized further), but not with 8 g/l 3-O-methyl glucose (transported but not metabolized further). Thus, this phenomenon is probably mediated by glucose-6-phosphate. Increased clearance of mutant huntingtin by raised glucose (8 g/l) and 2DOG correlated with increased autophagy and reduced phosphorylation of mTOR, S6K1 and Akt. Thus, raised intracellular glucose/glucose 6-phosphate levels reduce mutant huntingtin toxicity by increasing autophagy via mTOR and possibly Akt. As mTOR and Akt regulate a diversity of crucial cellular processes, our data also suggest a major new set of targets for intracellular glucose signalling.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntington's disease (HD) is a devastating autosomal dominant neurodegenerative disease associated with abnormal movements, cognitive deterioration and psychiatric symptomatology. It is a member of a family of nine known neurodegenerative diseases caused by CAG trinucleotide repeat expansion mutations that are translated into abnormally long polyglutamine tracts. The HD gene encodes a very large protein (>3000 amino acids) called huntingtin and the disease is associated with expansions of more than 37 consecutive glutamines that are found close to its N-terminus. While these diseases share a number of features, including the propensity for the mutant protein to aggregate intracellularly, the role of these inclusions/aggregates is the subject of vigorous debate.
Genetic and transgenic studies are consistent with a model where expanded polyglutamines cause disease by conferring a novel toxic function on the disease proteins (1). Mutant huntingtin is cleaved in vivo and in vitro (2,3) to form N-terminal fragments containing the polyglutamine repeats. It is believed that these fragments are the toxic forms of the protein that are also found in aggregates. Although the exact identity of these fragments is not known, they appear to comprise the first 100–150 residues of huntingtin. Accordingly, we and many other laboratories have modelled HD pathogenesis with exon 1 fragments, which cause toxicity and aggregate in cell models and in vivo (1).
We previously reported that inhibiting or inducing autophagy affected the turnover of mutant huntingtin exon 1 containing the expanded polyglutamines but not green fluorescent protein (GFP) in mammalian cell culture systems, suggesting that mutant huntingtin construct is cleared by autophagy (4). In these experiments autophagy was inhibited with 3-methyl adenine (3MA), dimethyladenosine and bafilomycin A1, which act at different stages of the process and we stimulated autophagy with rapamycin (4). Decreased turnover and increased levels of mutant huntingtin correlated with increased aggregation and cell death and vice versa. Autophagy is a bulk degradation system in which a double membrane structure, an autophagic vacuole/autophagosome, sequesters a portion of cytoplasm along with proteins and organelles to be degraded. These structures subse-quently fuse with the lysosome/vacuole where their contents are degraded (5). Our data are consistent with observations of Kegel et al. (6) and Yamada et al. (7) that mutant huntingtin accumu-lated in autophagosomes and early lysosomes.
Yeast genetic screens have identified many genes involved in autophagy, named APG, AUT and CVT (8). However, there is only limited knowledge about regulation of autophagy in mammalian cells. For instance, autophagy is induced by starvation and formation of autophagic bodies is inhibited by amino acids (9) and insulin (10). However, it is likely that this process is regulated by other upstream metabolic signals. Several protein kinases regulate autophagy in yeast and mammalian cells. The yeast gene TOR (target of rapamycin), and its mammalian orthologue mTOR (also known as RAFT1, RAPT1 or FRAP) encode a phosphatidylinositol kinase-related kinase that regulates autophagy. Inactivation of TOR, which is associated with dephosphorylation in metabolic contexts, induces autophagy (5). The phosphorylation state of mTOR and thus its catalytic activity can be modulated by several factors including amino acid deprivation (11,12). The upstream regulators of mTOR are not clearly identified. While some studies suggest that mTOR is phosphorylated and activated by Akt/PKB (13,14), these conclusions have been disputed (15). mTOR phosphorylates at least two downstream effectors namely S6K1 (also known as p70S6K) and 4E-BP1 (also known as PHAS-1) (16–18). Phosphorylation of S6 ribosomal protein, the target of S6K1 correlates with autophagy in mammalian cells (19,20).
Recently, we performed gene expression profiling in stable inducible PC12 cell lines expressing either mutant or wild-type forms of huntingtin exon 1. These studies observed changes in mRNA levels of a number of genes involved in glucose metabolism, including GLUT1, a protein that transports glucose across cell membranes (21). In order to test if GLUT1 could play a role in polyglutamine pathology, we overexpressed this glucose transporter and showed that it reduced cell death caused by mutant huntingtin exon 1 in neuronal and non-neuronal cell lines. Here we have examined the mechanism for this effect. Overexpression of GLUT1 reduced aggregation of mutant huntingtin exon 1. Consistent with the effects of GLUT1 overexpression leading to increased intracellular glucose levels, elevated glucose levels in the tissue culture medium enhanced mutant huntingtin clearance and reduced cell death. Increased degradation of huntingtin exon 1 in response to raised glucose was not accompanied by changes in expression levels of Cathepsin B or D but was associated with increased autophagy. We report for the first time that raising intracellular glucose levels is a novel signal stimulating autophagy. This effect is particularly interesting, as it acts in the opposite direction to the decreased autophagy seen with amino acid addition or insulin stimulation. Our data suggest that these effects of glucose are mediated at the level of glucose-6 phosphate. Glucose/glucose-6 phosphate reduces phosphorylation of Akt, mTOR and its downstream effector S6K1. As mTOR is a key negative regulator of autophagy and its dephosphorylation stimulates this process in other contexts, our data suggest that glucose is inducing autophagy via this pathway.
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RESULTS |
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Effect of glucose and its analogues on huntingtin aggregation
We used huntingtin exon 1 with 74 polyglutamine repeats tagged to EGFP (Q74) in our studies. In cell models, only the mutant protein with a long polyglutamine stretch forms stable aggregates, while the wild type protein with a short polyglutamine tract (e.g. Q23) does not aggregate. In cell culture, increased aggregation of the mutant protein positively correlates with cell death characterized by apoptotic nuclear morphology. Reduction of aggregates by overexpression of molecular chaperones (22) or by inducing autophagy (4) correlates with reduced cell death.
We previously reported that overexpression of GLUT1 glucose transporter significantly reduced cell death induced by EGFP-Q74 in COS-7 (monkey kidney) and SK-N-SH neuroblastoma cell lines, when compared with EGFP-Q74 co-transfected with empty vector control (21). In order to try to elucidate the mechanism for this effect, we analysed the effect of GLUT1 overexpression on the proportion of EGFP-Q74 cells with aggregates. COS-7 and SK-N-SH cells were transfected with EGFP-Q74 (0.5 µg per six-well dish) and co-transfected with GLUT1 cDNA (1.5 µg per six-well dish), or an equivalent amount of empty vector (pBudCE4, Invitrogen). The proportion of EGFP-expressing cells with aggregates was significantly reduced in the presence of GLUT1 in both COS-7 cells (mean reduction=17%) and SK-N-SH cells (mean reduction=40%), compared with an equivalent amount of empty vector [COS-7, odds ratio (OR)=0.84, 95% CI 0.74–0.95, P=0.007; SK-N-SH, OR=0.57, 95% CI 0.51–0.63, P<0.0001; data from two experiments each performed in triplicate].
Since GLUT1 transports glucose across cell membranes into cells, one would expect that the effects of its overexpression would be mimicked by raised glucose concentrations in the tissue culture medium. We tested the effect of glucose on aggregation of mutant huntingtin exon 1 by transiently transfecting COS-7 cells with mutant huntingtin exon 1 and incubating the cells in 1, 4 and 8 g/l glucose in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Increasing glucose concentrations caused a dose-dependent reduction in the percentages of cells with aggregates and cell death (Fig. 1). This effect plateaued with further increases in the glucose levels (data not shown). We obtained similar reductions of aggregation and cell death with 8 versus 1 g/l glucose if cells were grown in these conditions for 24 h prior to transfection (data not shown). Huntingtin aggregation and cell death were reduced by 8 g/l 2-deoxy glucose (2DOG), compared with 1 g/l glucose (Fig. 1). 2DOG is a transportable glucose analogue that can be phosphorylated to a glucose-6-phosphate analogue, which is not metabolized further. These effects were not seen with 8 g/l 3-O-methyl glucose (3OMG), a transportable and non-metabolizable analogue which is not phosphorylated, and with L-glucose, which is not transported (Fig. 1). These data together suggest that transport and phosphorylation of glucose is essential for the downstream signals that reduce mutant huntingtin exon 1 aggregation and cell death.
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Enhanced clearance of mutant huntingtin exon 1 with increased glucose
We next tested if reduced aggregation of mutant huntingtin exon 1 was due to its enhanced clearance in high glucose. We used inducible PC12 cell lines, stably expressing huntingtin exon 1 protein with 23 or 74 glutamine repeats under the control of a doxycycline (dox)-sensitive promoter (Tet-On), which we have previously characterized (23). We induced expression of huntingtin exon 1 in these cells for 15 h by adding dox to the medium, after which the expression was switched off by removing dox from the medium. We have previously shown that this strategy leads to the clearance of both aggregated and soluble forms of the protein (4). When dox was removed from the medium, cells were then treated with the different glucose doses (1, 4 or 8 g/l), 2DOG and 3OMG for 72 h and the Q23/Q74 protein levels were determined by western blotting. Increased glucose (8 g/l) (Fig. 2Ai and ii) and 2DOG (Fig. 2Ai and ii) enhanced the clearance of mutant protein. However none of the treatments affected the turnover of wild-type protein (Fig. 2B), suggesting that the mutant huntingtin exon 1 is more dependent on autophagy for its clearance than its wild-type counterpart. This is also supported by the finding in the same experiment that mutant, but not wild-type huntingtin exon 1 tagged to HA, accumulates in COS-7 cells treated for 48 h with 3-methyl adenine, an inhibitor of autophagosome formation (Fig. 2C).
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-tubulin or actin).We tested if the effect we observed was due to increased levels of lysosomal cathepsins B and D, as elevated glucose levels increased expression of these proteases in ras-transformed fibroblasts (24). Expression levels of cathepsin B (Fig. 2D) and cathepsin D (Fig. 2E) did not change with the different glucose doses in both COS-7 cells (Fig. 2D and E) and PC12 cells (data not shown).
Increased glucose increases the numbers of autophagic vacuoles
Monodansylcadaverine (MDC) is a fluorescent dye that specifically labels autophagic vacuoles in vivo. MDC incorporation is an indicator of autophagic activity (25–27). Increased glucose (8 g/l) and 2DOG increased the number of MDC-labelled vesicles and overall MDC fluorescence in cells, compared with 3OMG and 1 g/l glucose (Fig. 3). The MDC fluorescence suggested qualitatively similar vesicle shapes and sizes in 1 g/l (Fig. 3A) and 8 g/l (Fig. 3B) glucose samples—this impression was confirmed by electron microscopy (EM) (Fig. 4A and B). There were markedly more MDC-positive vesicles with 2DOG (Fig. 3C) compared with 1 or 8 g/l glucose, suggesting even more autophagic activity with 2DOG than with 8 g/l glucose. EM revealed a marked profusion of vesicles in 2DOG-treated cells (Fig. 4F) compared with 8 g/l glucose (Fig. 4E), but the vesicles (Fig. 4C) were qualitatively similar to those seen with 1 g/l (Fig. 4A) and 8 g/l (Fig. 4B) glucose. The apparently increased autophagic activity of 2DOG compared with 8 g/l glucose correlates with the greater clearance of mutant huntingtin and decreased aggregation with 2DOG versus 8 g/l glucose in Fig. 1. The 3OMG treatment led to less vesicular/punctate MDC staining pattern, and a more diffuse pattern of fluorescence (Fig. 3D). EM of these samples revealed extraordinary large autophagosome-like structures (Fig. 4D) not seen with any of the other treatments—the appearance may be consistent with a block in degradation of the autophagosome content. Figure 4E and F shows low power images of B and C, respectively.
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Reduced phosphorylation of mTOR, S6K1 and Akt
Cells treated with increased glucose (8 g/l) and 2DOG showed reduced levels of phospho mTOR (Ser 2448) relative to total mTOR (Fig. 5Ai and ii). In mammalian cells phosphorylation of ribosomal protein S6, a substrate of S6K1, strongly correlates with autophagy. Increased glucose (8 g/l) resulted in reduced phosphorylation of mTOR (Fig. 5Ai and ii) and its downstream effector S6K1 (Fig. 5B), suggesting that glucose signalling of autophagy was regulated via mTOR. We found reduced levels of phospho Akt (Ser 473) with increased glucose (8 g/l) and 2DOG (Fig. 5C). This correlates well with the reduced mTOR phosphorylation. However, since the role of Akt in the mTOR pathway is a subject of debate (15), care should be taken in interpreting its exact role.
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Our data show that 8 g/l glucose and 2DOG increase autophagy and huntingtin clearance on one hand and decrease mTOR phosphorylation (a marker for mTOR activity) on the other hand. The phosphorylation level of the mTOR substrate S6K1 is decreased by increased glucose, consistent with a reduction in the activity of mTOR. While there is general agreement that a reduction in mTOR activity will increase autophagy (20), we have confirmed that this is the case in our system. We treated cells for 48 h with 0.2 µg/ml rapamycin, a specific inhibitor of mTOR. This regime previously increased mutant huntingtin clearance (4), and we have confirmed that this increases the number of MDC-stained autophagic vesicles (Fig. 6). Thus, the decrease in mTOR activity in 8 g/l glucose and 2DOG is sufficient to account for the increased autophagy and mutant huntingtin clearance we have observed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our data suggest that the protective effect of increased intracellular glucose transport against polyglutamine-mediated cell death is related to enhanced clearance of the toxic protein. This is mediated by increased autophagy, which is associated with decreased phosphorylation of one its key regulators, mTOR. The effect we have observed is analogous to what we described previously using rapamycin, a specific inhibitor of mTOR that also reduced polyglutamine aggregation and cell death by enhancing clearance (4).
Besides its role in energy metabolism, glucose is now increasingly considered as an important signalling molecule. We have discovered that raised intracellular glucose is a novel stimulus for autophagy in mammalian cells, as raised glucose concentrations and 2DOG increase the number of autophagic vacuoles and result in increased clearance and decreased aggregation of mutant huntingtin exon 1. Our data suggest that this process requires glucose transport, as no effect is seen with L-glucose (which is not transported). Furthermore, this effect is probably mediated at the level of glucose 6-phosphate, since it is stimulated by 2DOG (transported and phosphorylated to be a glucose-6 phosphate analogue but not metabolized further) and not 3OMG (transported but not phosphorylated). The greater efficacy of 2DOG compared with 8 g/l glucose may be because greater levels of glucose-6-phosphate or its equivalent would be expected with 2DOG which cannot be metabolized past this point. It is interesting to note that the effects of glucose are opposite to insulin, which inhibits autophagy (10). Glucose also mediates opposite effects to amino acids, which induce autophagy when they are deficient in the culture medium (9).
The effects of glucose on autophagy are probably mediated via mTOR, a known key regulator of this process—both mTOR and S6K1 phosphorylation were reduced with high glucose and the activities of these kinases correlate with their phosphorylation. mTOR negatively regulates autophagy. This is consistent with studies in yeast and mammalian cells, suggesting that mTOR functions in other nutrient-sensing pathways, regulating the cellular response to starvation conditions such as amino acid deprivation (11,12). Inhibition of phosphorylation resulting from inactivation of mTOR by treatment with rapamycin induces autophagy; however, the details of the regulatory mechanism are not understood. It is also not known how these proteins regulate the autophagic process at the molecular level. The upstream pathways regulating mTOR activity are poorly understood—for instance, the steps connecting amino acid starvation to autophagy are not known even though this phenomenon was characterized many years ago. Consequently, the current literature provides no obvious clues as to possible immediate targets of glucose-6-phosphate that may be linked to mTOR pathway.
In conclusion, our data provide functional explanations of how intracellular glucose transport and metabolism can impact on polyglutamine toxicity. Glucose/glucose 6-phosphate has been identified as a novel stimulus for autophagy via mTOR and possibly Akt and this leads to enhanced clearance of the toxic huntingtin exon 1 fragment. As mTOR is involved in a diversity of crucial cellular processes like growth and translation of certain transcripts and Akt is a key regulator of cell growth and survival, our results suggest a host of other important functions may be regulated by the effects of glucose on these kinases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmids used
HD gene exon 1 fragment with 74 polyglutamine repeats (Q74) in pEGFP-C1 (Clontech) and haemeagglutinin-tagged HD gene exon 1 with 23 and 74 polyglutamine repeats in pHM6 vector (Q23/Q74-HA) were described and characterized previously (28). Glut1 (H-K03195M) was from GeneStorm human clones (Invitrogen, The Netherlands). PC12 stable lines expressing exon 1 of HD gene are described in Wyttenbach et al. (23).
Mammalian cell culture and transfection
African green monkey kidney cells (COS-7) were grown in DMEM (Sigma) supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine and 1 mM sodium pyruvate at 37°C, 5% carbon dioxide. The cells were grown in six-well dishes to 60–80% confluency for 24 h and transfected using LipofectAMINE reagent (Invitrogen) using the manufacturer's protocol. The transfection mixture was replaced by either low-glucose (1 g/l) medium, or low-glucose medium supplemented with 4 or 8 g/l glucose, 8 g/l 2DOG, 8 g/l 3OMG or 8 g/l L-Glucose (all from Sigma) after 5 h incubation at 37°C. Transfected cells were analysed by immunofluorescence or immunoblot after 48 h as described previously (4). The PC12 stable cells were maintained at 75 µg/ml hygromycin in standard DMEM (Sigma) with 100 U/ml penicillin/streptomycin, 2 mM L-glutamine (Invitrogen), 10% heat-inactivated horse serum (Invitrogen), 5% Tet-approved FBS (Clontech) and 100 µg/ml G418 (Invitrogen) at 37°C, 10% CO2. The cells were seeded at 1–2x105 per well in six-well dishes and were induced with 1 µg/ml dox (Sigma) for 15 h. Expression of transgenes was switched off by removing dox from the medium, cells were either left in dox-free medium unsupplemented or treated with 1, 4 or 8 g/l glucose, 8 g/l 2DOG or 8 g/l 3OMG for 72 h and the medium with glucose or its analogues changed every 24 h.
Western blot analysis
The primary antibodies used include anti-GFP (8362-1, Clontech), anti-mTOR (2972), anti-phospho-mTOR (Ser 2448) (2971), anti-p70 S6 kinase (9202), anti-phospho-p70 S6 kinase (Thr389) 1A5 (9206), anti Akt (9272) and anti-phospho-Akt (Ser473) (9271) all from Cell Signalling Technology, anti-Cathepsin B (ab7430, Abcam), anti-Cathepsin D (Clone CTD-19, Sigma) and anti-HA antibodies (12CA5, Covance). Blots were probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Bio Rad) at 1:2000 and bands visualized using ECL or ECL Plus detection kit (Amersham). Densitometry analysis was performed using Scion Image Beta 4.02 software.
Quantification of aggregate formation and abnormal cell nuclei
Aggregate formation and nuclear morphology were assessed using a fluorescence microscope. 200 EGFP positive COS-7 cells were selected and the proportion of cells with aggregates was counted. Aggregates are described in Narain et al. (28). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, 3 mg/ml, Sigma) and nuclei were considered abnormal if they showed apoptotic morphology (fragmentation or pyknosis). Pyknotic nuclei are typically <50% diameter of normal nuclei and show increased DAPI intensity. We have demonstrated that these criteria are specific for cell death, as they show a very high correlation with propidium iodide staining in live cells (29). Analysis was performed with observer blinded to identity of slides—slides were coded and the code was broken after completion of the experiment. All experiments were done in triplicate at least twice.
Labelling autophagic vacuoles with monodansylcadaverine
Autophagic vacuoles were labelled with monodansylcadaverine (MDC, Sigma) by incubating cells on coverslips with 0.05 mM MDC for 1 h at 37°C (25). After incubation cells were rinsed three time with 1x PBS and analysed immediately using a fluorescent microscope. Rapamycin (Sigma) treatment was done at 0.2 µg/ml for 48 h before MDC staining.
Transmission electron microscopy
The cells were prepared for ultrastructural analysis as previously described (30).
Statistical analysis
Pooled estimates for the changes in inclusion formation or cell death, resulting from perturbations assessed in multiple experiments were calculated as odds ratios with 95% confidence intervals [(percentage of cells expressing construct with inclusions in perturbation conditions/percentage of cells expressing construct without inclusions in perturbation conditions)/(percentage of cells expressing construct with inclusions in control conditions/percentage of cells expressing construct without inclusions in control conditions)]. Odds ratios and P-values were determined by unconditional logistical regression analysis, using the general log–linear analysis option of SPSS 9 software (SPSS, Chicago, IL, USA). Odds ratios were considered to be the most appropriate summary statistic for reporting multiple independent replicate experiments of this type (21,22), because the percentage of cells with inclusions under specified conditions can vary between experiments on different days, whereas the relative change in the proportion of cells with inclusions induced by an experimental perturbation is expected to be more consistent.
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ACKNOWLEDGEMENTS |
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We are grateful to the Commonwealth Scholarship Commission (B.R.), Medical Research Council programme grants to D.C.R. and Steve Brown, and Paul Luzio and Margaret Robinson (A.S.), Hereditary Disease Foundation (D.C.R.) and Japan Science and Technology Corporation (K.K.) for funding. We are grateful for Wellcome Trust Senior Research Fellowships in Clinical Science (D.C.R.) and Basic Biomedical Science (R.D.). We thank Paul Luzio for valuable comments on the manuscript.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +44 1223762608; Fax: +44 1223331206; Email: dcr1000{at}cus.cam.ac.uk
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