Correspondence to David C. Rubinsztein: dcr1000{at}hermes.cam.ac.uk
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Introduction |
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Autophagy also can help cells clear the toxic, long-lived, aggregate-prone proteins, like mutant huntingtin and A53T, and A30P mutants of -synuclein (Ravikumar et al., 2002; Webb et al., 2003), which cause neurodegenerative disorders, such as Huntington's disease (HD) (Rubinsztein, 2002) and forms of Parkinson's disease (PD) (Polymeropoulos et al., 1997; Kruger et al., 1998), respectively. Induction of autophagy reduced the levels of mutant huntingtin and protected against its toxicity in cells (Ravikumar et al., 2002), and in transgenic Drosophila and mouse models of HD (Ravikumar et al., 2004). The only suitable pharmacologic strategy for up-regulating autophagy in mammalian brains is to use rapamycin, or its analogs, that inhibit the mammalian target of rapamycin (mTOR), a negative regulator of autophagy.
HD is an autosomal-dominant neurodegenerative disorder that is caused by a CAG trinucleotide repeat expansion in the huntingtin gene (Huntington's Disease Collaborative Research Group, 1993), which results in an expansion of the polyglutamine tract in the NH2 terminus of the huntingtin protein. Asymptomatic individuals have 35 CAG repeats, whereas HD is caused by
36 repeats (Rubinsztein et al., 1996). Mutant huntingtin accumulates in intraneuronal aggregates (also called inclusions). Huntingtin is cleaved to form NH2-terminal fragments that consist of the first 100150 residues containing the expanded polyglutamine tract, which are believed to be the toxic species found in the aggregates. Thus, HD pathogenesis frequently is modeled with exon 1 fragments that contain expanded polyglutamine repeats, which cause aggregate formation and toxicity in cell models and in vivo (Rubinsztein, 2002).
PD is another condition that is associated with aggregate formation. The intraneuronal Lewy body aggregates that characterize PD have the protein -synuclein as a major component. The A53T and A30P point mutations in
-synuclein cause autosomal dominant forms of Parkinson's disease (Polymeropoulos et al., 1997; Kruger et al., 1998). Unlike wild-type
-synuclein, the clearance of these mutant forms is retarded when autophagy is inhibited (Webb et al., 2003; Cuervo et al., 2004). Although these forms of
-synuclein aggregate in vivo, we do not observe overt aggregation in our cell lines (Webb et al., 2003). Furthermore, unlike wild-type
-synuclein, these mutant forms are not cleared by the chaperone-mediated autophagy pathway (Cuervo et al., 2004), which is distinct from macroautophagy (called "autophagy" in this paper). Hence, we have used these mutations as model autophagy substrates.
We showed previously that lithium is protective in HD cell models, because it reduced mutant huntingtin aggregates and cell death (Carmichael et al., 2002). Lithium is used as a mood-stabilizing treatment of bipolar disorder, which is characterized by recurrent fluctuations in mood (Manji and Lenox, 1998). Although lithium has been used for decades for bipolar and unipolar affective disorders, the mechanism that underlies its therapeutic action is not understood fully. Here we describe a novel role for lithium as an inducer of autophagy. Lithium facilitated the clearance of known autophagy substrates, like mutant huntingtin and A53T, and A30P mutants of -synuclein. Lithium induced autophagy by inhibiting inositol monophosphatase (IMPase), which led to reduced free inositol and myo-inositol-1,4,5-triphosphate (IP3) levels. This represents a novel pathway for regulation of mammalian autophagy.
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Results |
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Furthermore, we assessed clearance of A53T and A30P -synuclein mutants. After induction of stable inducible PC12 cell lines (Webb et al., 2003) and subsequent removal of doxycycline, lithium treatment for 24 h significantly enhanced the clearance of A53T (Fig. 2 A) and A30P (Fig. 2 B)
-synuclein mutants; no effect was observed with NaCl (Fig. 2 C).
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If lithium facilitates clearance of EGFP-HDQ74 and mutant -synucleins through autophagy, then it should be blocked by the autophagy inhibitor, 3-methyladenine (3-MA). 3-MA increased EGFP-HDQ74 aggregate formation in COS-7 cells (Ravikumar et al., 2002) (Fig. 2 G; Fig. S2 C), and delayed clearance of A53T
-synuclein (Webb et al., 2003) (Fig. 2 H). Furthermore, 3-MA completely abolished the effect of lithium in reducing EGFP-HDQ74 aggregation (Fig. 2 G; Fig. S2 C), or promoting A53T
-synuclein clearance (Fig. 2 H). Thus, lithium induces autophagy and enhances the clearance of known autophagy substrates, such as EGFP-HDQ74 and mutant
-synucleins.
Lithium enhances clearance of mutant proteins by inositol monophosphatase inhibition
Lithium inhibits a number of enzymes, including glycogen synthase kinase-3ß (GSK-3ß) and IMPase (Gould et al., 2002; Coyle and Duman, 2003). To test if either of these enzymes was involved in autophagy regulation, we used specific inhibitors of GSK-3ß (SB216763) (Coghlan et al., 2000) and IMPase (L-690,330) (Atack et al., 1993) (Fig. S3; available at http://www.jcb.org/cgi/content/full/jcb.200504035/DC1). L-690,330 reduced aggregation and cell death that were caused by EGFP-HDQ74 in COS-7 and SK-N-SH cells, whereas SB216763 increased EGFP-HDQ74 aggregates but reduced cell death, which was consistent with our earlier findings (Carmichael et al., 2002) (Fig. 3, A and B). The idea that IMPase was involved in autophagy regulation was supported further because L-690,330 facilitated clearance of soluble EGFP-HDQ74 (Fig. 3 C) and the mutant -synucleins (Fig. 3 D), whereas SB216763 had no effect on clearance of soluble EGFP-HDQ74 (Fig. 3 E) and seemed to slow clearance of mutant
-synucleins in stable PC12 cell lines (Fig. 3 F). Because SB216763, if anything, slows clearance of these substrates, the enhanced clearance of mutant huntingtin and
-synucleins that is mediated by lithium cannot be due to the drug inhibiting GSK-3ß.
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Mood-stabilizing drugs facilitate clearance of mutant proteins
Inositol depletion is a common mechanism for mood stabilizing drugs like lithium, carbamazepine (CBZ), and valproic acid (VPA) (Williams et al., 2002). Consistent with a role for inositol depletion in autophagy regulation, CBZ significantly reduced EGFP-HDQ74 aggregates and attenuated polyglutamine toxicity in COS-7 cells (Fig. 4 I), and enhanced clearance of A30P -synuclein (Fig. 4 J). Similar results were seen with VPA (unpublished data). Thus, drugs that can deplete intracellular inositol may be of therapeutic value in HD and related neurodegenerative diseases.
The effect of lithium on clearance of mutant proteins is regulated by IP3 levels
To determine the importance of inositol levels in autophagy regulation, we tested if the effect of lithium on autophagy could be overcome by the addition of extracellular inositol in the form of myo-inositol (Williams et al., 2002). We also used an inhibitor of prolyl oligopeptidase activity, called prolyl endopeptidase inhibitor 2 (PEI), which elevates intracellular IP3 and abolishes some other effects of lithium (Williams et al., 1999) (Fig. S4 B). Lithium may deplete inositol by inhibiting IMPase and by decreasing the transport of myo-inositol into cells (Lubrich and van Calker, 1999). Accordingly, we pretreated cells with myo-inositol and PEI before adding lithium. As predicted, myo-inositol and PEI pretreatment raised IP3 levels in COS-7 cells that were treated with lithium, compared with lithium treatment alone (Fig. 5 A). Myo-inositol and PEI significantly reversed the protective effect of lithium on EGFP-HDQ74induced aggregation and cell death in COS-7 cells (Fig. 5 B). (The failure of myo-inositol and PEI to reverse completely the lithium effect on EGFP-HDQ74 aggregation at 48 h [Fig. 5 B]despite their effects on IP3 levels at 5 min [Fig. 5 A]can be explained by the different timings of these observations.) These compounds also inhibited the effect of lithium on the clearance of soluble EGFP-HDQ74 (Fig. 5 C) and A53T -synuclein (Fig. 5 D) in stable PC12 cell lines.
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Lithium and IMPase inhibitor induce autophagy independently of mTOR inhibition
We were interested if this novel means of pharmacologic regulation of autophagy was independent of the known pathway that is regulated negatively by mTOR. The activity of mTOR, a protein kinase, can be inferred by the levels of phosphorylation of its substrates, ribosomal S6 protein kinase (S6K1, also known as p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) at Thr389 and Thr37/46, respectively (Fig. S5 A; available at http://www.jcb.org/cgi/content/full/jcb.200504035/DC1), and the phosphorylation of the ribosomal S6 protein (S6P), a substrate of S6K1 (Schmelzle and Hall, 2000). Whereas rapamycin (a specific mTOR inhibitor) reduced phosphorylation of S6K1, S6P, and 4E-BP1 in COS-7 cells as expected, lithium or L-690,330 did not reduce their phosphorylation (Fig. 6, AD; Fig. S5 B), which suggests that their effects are independent of mTOR inhibition.
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The effect of IP3 on autophagy is unlikely to be a downstream consequence of mTOR inhibition, because rapamycin had no effect on IP3 levels (Fig. 5 E). Furthermore, neither myo-inositol nor PEI abolished the protective effect of rapamycin on polyglutamine toxicity in COS-7 cells (Fig. 7 A), and the increased clearance of soluble EGFP-HDQ74 and A53T -synuclein in stable PC12 cell lines (Fig. 7, B and C), in contrast to what we observed in the context of lithium. Therefore, rapamycin and intracellular inositol seem to regulate autophagy independently.
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Discussion |
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Lithium is a major therapy for affective disorders (Manji and Lenox, 1998). It takes 34 wk to stabilize mood, although brain inositol levels in patients who have bipolar disorder are reduced by lithium much sooner after treatment is initiated (Moore et al., 1999). Because autophagy modulates the half-lives of many long-lived proteins (Klionsky and Emr, 2000; Ravikumar et al., 2002, 2004; Webb et al., 2003), it is tempting to speculate that part of the therapeutic efficacy of lithium and related drugs (e.g., CBZ and valproate) may be mediated by clearance of very long half-life autophagy substrates that may contribute to the disease. Steady-state levels of some long half-life proteins could take weeks (many multiples of the half-life) to change in a biologically significant way after the half-life is shortened. This is a plausible explanation for why mood-stabilizing drugs have acute effects on free inositol levels long before they begin to mediate clinical effects. The different lag times that are seen with various mood-stabilizing drugs may be related to their pharmacokineticsthe fact that these drugs may deplete intracellular inositol by different mechanisms (Gould et al., 2002; Ju et al., 2004; Shaltiel et al., 2004) and/or effects on other target enzymes that are unrelated to autophagy. This speculative model does not mean necessarily that abnormal protein degradation is a feature of affective disorders. It is tempting to speculate whether trials of rapamycin or related autophagy-inducing drugs may be worthwhile for mood disorders, given the lack of understanding of their biology and the enormous patient need.
Although the inositol depletion hypothesis may be one of the most widely accepted hypotheses for lithium's actions in bipolar affective disorder (Silverstone et al., 2005), it remains controversial. In rodents, lithium (and valproate) reduces myo-inositol levels and increases IP1 concentrations in the brain (Silverstone et al., 2005). Although the studies in humans have been more difficult to interpret, this may be due, in part, to technical limitations of magnetic resonance spectroscopy methods in humans, and sample size and clinical heterogeneity issues (for review see Silverstone et al., 2005). Nevertheless, the meta-analysis of data from manic and depressed studies generally is consistent with the inositol depletion hypothesis of lithium action in these disorders (Silverstone et al., 2005). It is clearly outside the realm of this study to resolve the controversy of the inositol depletion hypothesis for lithium. Note also that lithium can reduce inositol levels by two mechanisms. The most commonly considered is by inhibition on inositol monophosphatase. However, lithium also inhibits inositol uptake into cells (Lubrich and van Calker, 1999). We performed most studies using 10 mM lithium, following the precedent of Williams et al. (1999). Although this is higher than the desired concentration in humans, we were able to show that the autophagy effects at this dose were mediated specifically by inositol depletion, because they were attenuated by myo-inositol and prolyl endopeptidase inhibition. We previously showed that 2.5 mM lithium reduced polyglutamine aggregation and toxicity in cell models (Carmichael et al., 2002). Furthermore, 1 mM lithium (approximating the therapeutic levels in humans [Camus et al., 2003]) induces autophagy (Fig. S2, A and B). Even if lithium turns out to be not very effective at inducing autophagy in humans, similar effects may be achieved with other drugs that reduce inositol levels, like valproate or CBZ (Williams et al., 2002) (Fig. 4, I and J; not depicted).
These issues do not detract from our fundamental cell biologic elucidation of a novel mTOR-independent autophagy regulatory pathway. This may be of potential value in neurodegenerative diseases that are caused by aggregate-prone proteins, such as HD (Ravikumar et al., 2004). Some benefit was reported with lithium in a mouse model of HD (Wood and Morton, 2003). Combination therapy with more moderate IMPase and mTOR inhibition may be safer for long-term treatment than using doses of either inhibitor that result in more severe perturbation of a single pathway.
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Materials and methods |
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Mammalian cell culture and transfection
African green monkey kidney cells (COS-7) and human neuroblastoma cells (SK-N-SH) were maintained in DME (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich), 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine (Sigma-Aldrich) at 37°C, 5% CO2. Cells were plated in six-well dishes at a density of 105 cells per well for 24 h, and transfected with pEGFP-HDQ74 using LipofectAMINE reagent for COS-7 cells and LipofectAMINE PLUS reagent for SK-N-SH according to the manufacturer's protocol (Invitrogen). Transfection mixture was replaced after 4 h incubation at 37°C by various compounds, such as 10 mM LiCl, 0.2 µM rapamycin, 10 mM 3-MA, 10 µM lactacystin, 50 µM CBZ, 1 mM myo-inositol (all from Sigma-Aldrich), 10 mM NaCl (BDH), 10 µM SB216763 (Tocris), 100 µM L-690,330 (Tocris), or 24 µM prolyl endopeptidase inhibitor 2 (Z-PP-CHO, Calbiochem). Transfected cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) after 48 h and mounted in DAPI (3 mg/ml; Sigma-Aldrich) over coverslips on glass slides and analyzed for aggregation and cell death. For immunoblotting, cells were plated at a density of 3 x 105 cells per well. HeLa cells stably expressing UbG76V-GFP reporter (gift from N.P. Dantuma, Karolinska Institutet, Stockholm, Sweden) were grown in similar media used for growing COS-7 cells, which is supplemented with 0.5 mg/ml G418.
Inducible PC12 stable cell lines expressing EGFP-tagged exon 1 of HD gene (EGFP-HDQ74) (Wyttenbach et al., 2001) and HA-tagged A53T and -synuclein mutants (Webb et al., 2003), were maintained at 75 µg/ml hygromycin B (Calbiochem) in standard DME with 10% horse serum (Sigma-Aldrich), 5% FBS, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, and 100 µg/ml G418 (GIBCO BRL) at 37°C, 10% CO2.
Quantification of aggregate formation and cell death
200 EGFP-positive cells were counted for proportion of cells with aggregates, as described previously (Narain et al., 1999). Nuclei were stained with DAPI; those that showed apoptotic morphology (fragmentation or pyknosis) were considered abnormal. These criteria are specific for cell death, and correlate highly with propidium iodide staining in live cells (Wyttenbach et al., 2002). Analysis was performed using a Nikon Eclipse E600 fluorescence microscope (plan-apo 60x/1.4 oil immersion lens at room temperature); the observer was 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. Images were acquired with a Nikon Digital Camera DXM1200 using Nikon Eclipse E600 fluorescence microscope (40x plan-fluor 40x/0.75 lens or plan-apo 60x/1.4 oil immersion lens at room temperature). Acquisition software was Nikon ACT-1 version 2.12; Adobe Photoshop 6.0 (Adobe Systems, Inc.) was used for subsequent image processing.
Clearance of mutant huntingtin and -synucleins
Stable inducible PC12 cell lines expressing EGFP-HDQ74, or A53T or A30P -synuclein mutants, were plated at 3 x 105 per well in six-well dishes and induced with 1 µg/ml doxycycline (Sigma-Aldrich) for 8 h and 48 h, respectively. Expression of transgenes was switched off by removing doxycycline from the medium (Ravikumar et al., 2002; Webb et al., 2003); cells were treated with or without various compounds for 120 h for EGFP-HDQ74 clearance and 24 h for mutant
-synuclein clearance. For additive effect of LiCl or L-690,330 with rapamycin, treatment was done for 72 h for EGFP-HDQ74 clearance and 8 h for mutant
-synuclein clearance. The medium with various compounds was changed every 24 h. Cells were fixed and mounted in DAPI, or processed for immunoblotting analysis with EGFP for soluble EGFP-HDQ74 clearance or HA for mutant
-synuclein clearance.
Western blot analysis
Cell pellets were lysed on ice in Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 5% ß-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue) for 30 min in the presence of protease inhibitors (Roche Diagnostics). Samples were subjected to SDS-PAGE, and proteins were transferred to nitrocellulose membrane (GE Healthcare). Primary antibodies used include anti-EGFP (83621, CLONTECH Laboratories, Inc.), anti-HA (12CA5, Covance), anti-mTOR (2972), antiphospho-mTOR (Ser2448) (2971), anti-p70 S6 kinase (9202), antiphospho-p70 S6 kinase (Thr389) (9206), anti4E-BP1 (9452), antiphospho-4E-BP1 (Thr37/46) (9459), anti-S6 ribosomal protein (2212), and antiphospho-S6 ribosomal protein (Ser235/236) (2211), all from Cell Signaling Technology; anti-complex IV subunit IV (A-21348, Molecular Probes), anti-LC3 (gift from T. Yoshimori), anti-actin (A2066, Sigma-Aldrich), and anti-tubulin (clone DM 1A, Sigma-Aldrich). Blots were probed with antimouse or antirabbit IgG-HRP (GE Healthcare) and visualized using ECL or ECL Plus detection kit (GE Healthcare).
Immunocytochemistry
COS-7 cells were fixed with 4% paraformaldehyde. Primary antibodies included anti-LC3 (gift from T. Yoshimori, National Institute of Genetics, Japan) and antiphospho-S6 ribosomal protein (Ser235/236) (2211, Cell Signaling Technology). Standard fluorescence method was used for detection; secondary antibodies used were goat antirabbit Alexa 488 Green and Alexa 594 Red (Cambridge Biosciences). Images were acquired on a Zeiss LSM510 META confocal microscope (63x 1.4NA plan-apochromat oil immersion) at room temperature using Zeiss LSM510 v3.2 software; Adobe Photoshop 6.0 (Adobe Systems, Inc.) was used for subsequent image processing.
Inositol phosphate measurements
IP1-2 were assayed as described previously (Harnett and Harnett, 1993). COS-7 cells were labeled with myo-[3H]inositol (1 µCi/106 cells) for 24 h, stimulated, and then subjected to chloroform/methanol (1:2) extraction followed by Bligh-Dyer phase separation. Levels of IP1-2 were determined by liquid scintillation counting of fractions eluted following Dowex (formate form) ion exchange chromatography of aliquots of the aqueous phase. Results were calculated as a percentage of total incorporated radioactivity. Levels of inositol-1,4,5-trisphosphate [IP3(1,4,5)] were measured in perchloric acid extracted samples (107 cells/aliquot) using the [3H] Biotrak Assay System (GE Healthcare) according to the manufacturer's instructions. Data are presented as mean ± SD of triplicate measurements and are representative of at least three separate experiments.
Statistical analysis
Pooled estimates for the changes in aggregate formation or cell death, resulting from perturbations assessed in multiple experiments, were calculated as odds ratios with 95% confidence intervals. We have used this method frequently in the past to allow analysis of data from multiple independent experiments (Wyttenbach et al., 2001, 2002; Carmichael et al., 2002). Odds ratios and P values were determined by unconditional logistical regression analysis, using the general log-linear analysis option of SPSS 9 software. Densitometry analysis was done by Scion Image Beta 4.02 software on immunoblots from three independent experiments. Significance for clearance of mutant proteins was determined by factorial ANOVA test using STATVIEW software, version 4.53 (Abacus Concepts) and the error bar denotes standard error of the mean.
Online supplemental material
Fig. S1 shows lithium facilitated clearance of mutant huntingtin aggregates, an effect not seen with NaCl. Fig. S2 shows lithium-induced autophagy to facilitate clearance of mutant proteins. Fig. S3 shows a schematic diagram of some molecular targets of lithium. Fig. S4 shows aspects of the inositol cycle pertinent to lithium. Fig. S5 shows the mTOR pathway in relation to autophagy. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200504035/DC1.
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Acknowledgments |
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We are grateful for Gates Cambridge Scholarship (S. Sarkar), Wellcome Trust Senior Fellowship in Clinical Science (D.C. Rubinsztein), Academy of Medical Sciences (A.M.S.)/Medical Research Council (M.R.C.) Clinical Scientist Fellowship (R.A. Floto), a Prize Studentship (Z. Berger), and Overseas Research Students Award (Z. Berger). This work was supported by an M.R.C. program grant and by E.U. Framework VI (EUROSCA).
Submitted: 6 April 2005
Accepted: 19 August 2005
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