[Autophagy 2:2, 132-134, April/May/June 2006]; ©2006 Landes Bioscience
Inositol and IP3 Levels Regulate Autophagy Addenda
Biology and Therapeutic Speculations ABSTRACT
KEY WORDS lithium, inositol monophosphatase, autophagy, huntington's disease, bipolar affective disorders
ACKNOWLEDGEMENTS
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Lithium has been used for decades as a mood-stabilizing drug for the treatment of bipolar affective disorder. Although its mechanism of action is still not fully understood, inositol depletion is one of the most widely accepted hypotheses for its therapeutic action.1 Lithium targets various intracellular enzymes, including glycogen synthase kinase 3β (GSK-3β) and inositol monophosphatase (IMPase)2 (Fig. 1). Neurodegenerative disorders like Alzheimer’s disease (AD) and Parkinson’s disease (PD) are associated with protein aggregates (also called inclusions) in the brain. Intracellular protein aggregation due to misfolding is also the pathological hallmark of nine CAG triplet repeat expansion disorders, including Huntington’s disease (HD).3 HD is caused by a CAG repeat expansion in the huntingtin gene. These expanded trinucleotide repeats encode an abnormally long polyglutamine (polyQ) tract expansion in the huntingtin protein, which then aggregates.4 We previously showed that lithium reduced mutant huntingtin aggregates and toxicity in HD cell5 and Drosophila6 models. The reduced toxicity could be partly explained by GSK-3β inhibitors and genetic manipulations of this pathway activated apoptosis in HD cell models and neurodegeneration in HD transgenic Drosphilia. However, while lithium decreased aggregation, GSK-3β inhibition increased huntingtin aggregates in cells.5 Thus, we speculated that a non-GSK-3β pathway perturbed by lithium may also be contributing to some of its protective effects. We tested if the reduction of mutant huntingtin aggregate levels by lithium was due to enhanced clearance, since huntingtin fragment expression levels correlate with aggregation. Lithium treatment significantly reduced the levels of the soluble and aggregated mutant huntingtin fragments, correlating with decreased toxicity.7 Since previous data showed that such mutant huntingtin fragments are autophagy substrates,8 we tested and confirmed that lithium induced autophagy.7 The ability of lithium to induce autophagy could be accounted for by its IMPase activity.7 A specific GSK-3β inhibitor (SB216763) did not induce autophagy or enhance mutant huntingtin fragment clearance, while a specific IMPase inhibitor (L-690,330)9 mimicked lithium’s effect in enhancing the clearance of these mutant aggregate-prone proteins and decreasing mutant huntingtin aggregates and toxicity. Furthermore, L-690,330 induced autophagy, and its effects on mutant huntingtin and the A53T α-synuclein mutant were lost upon autophagy inhibition. IMPase catalyzes the hydrolysis of inositol monophosphate (IP1) into free inositol required for the phosphoinositol signaling pathway.10 Lithium affects this pathway by inhibiting IMPase, leading to free inositol depletion, which in turn decreases myo-inositol-1, 4,5-triphosphate (IP3) levels1 (Fig. 1). Inositol depletion is a common mechanism for
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Our work in this area is supported by an M.R.C. programme grant and E.U. Framework VI (EUROSCA) (D.C.R.). We are grateful for Gates Cambridge Scholarship (S.S.) and Wellcome Trust Senior Fellowship in Clinical Science (D.C.R.).
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Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=2387
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Received 12/05/05; Accepted 12/06/05
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*Correspondence to: David C. Rubinsztein: Department of Medical Genetics; University of Cambridge; Cambridge Institute for Medical Research; Wellcome Trust/MRC Building; Addenbrooke's Hospital; Hills Road, Cambridge, CB2 2XY, UK; Tel.: +0.1223.762608; Fax: +0.1223.331206; Email:
[email protected]
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Department of Medical Genetics; University of Cambridge; Cambridge Institute for Medical Research; Wellcome Trust/MRC Building; Addenbrooke's Hospital; Cambridge, UK
We recently showed that lithium induces autophagy via inositol monophosphatase (IMPase) inhibition, leading to free inositol depletion and reduced myo-inositol-1,4, 5-triphosphate (IP3) levels. This represents a novel way of regulating mammalian autophagy, independent of the mammalian target of rapamycin (mTOR). Induction of autophagy by lithium led to enhanced clearance of autophagy substrates, like mutant huntingtin fragments and mutant α-synucleins, associated with Huntington’s disease (HD) and some autosomal dominant forms of Parkinson’s disease (PD), respectively. Similar effects were observed with a specific IMPase inhibitor and mood-stabilizing drugs that decrease inositol levels. This may represent a new therapeutic strategy for upregulating autophagy in the treatment of neurodegenerative disorders, where the mutant protein is an autophagy substrate. In this Addendum, we review these findings, and some of the speculative possibilities they raise.
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Sovan Sarkar David C. Rubinsztein*
SC
Addendum to:
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Lithium Induces Autophagy by Inhibiting Inositol Monophosphatase
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S. Sarkar, R.A. Floto, Z. Berger, S. Imarisio, A. Cordenier, M. Pasco, L.J. Cook, and D.C. Rubinsztein J Cell Biol 2005; 170:1101-11
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Lithium, Inositol and Autophagy
Figure 1. Lithium’s targets and the pathways regulating autophagy. Lithium affects the Wnt and phosphoinositol signaling pathways by inhibiting GSK-3β and IMPase, respectively. The autophagy-inducing ability of lithium is GSK-3β-independent, and is attributed to its IMPase-inhibitory effect in the phosphoinositol signaling pathway, lowering inositol (Ins) and IP3 levels. Lithium also inhibits IPPase and inositol uptake through the sodium myo-inositol transporter (SMIT). Decreased inositol or IP3 levels stimulate autophagy, enhancing the clearance of aggregate-prone proteins like mutant huntingtin and α-synucleins (indicated by arrows). A specific IMPase inhibitor (L-690,330) and mood-stabilizing drugs [carbamazepine (CBZ) and valproic acid (VPA)] have similar effects. IP3 is generated by G-protein-coupled receptor-mediated stimulation of phospholipase C (PLC), which hydrolyses PIP2 into IP3 and diacylglycerol (DAG). IP3 can be increased by adding myo-inositol (Ins), and by an inhibitor (PEI) of prolyl oligopeptidase (PO), allowing IP6 conversion into IP3 by alleviating PO’s inhibition on multiple inositol polyphosphate phosphatase (MIPP). Increased inositol or IP3 levels inhibit autophagy, which reverse lithium’s effect. Autophagy is also stimulated by rapamycin (rap) through mTOR inhibition (indicated by arrows) downstream of the phosphatidylinositol 3-kinase (PI3K) pathway. Simultaneous inhibition of mTOR (by rapamycin) and phosphoinositol signaling (by LiCl or L-690,330) pathways have an additive effect on autophagy and the clearance of aggregate-prone proteins, compared to either single pathway.
mood-stabilizing drugs like lithium, carbamazepine (CBZ) and valproic acid (VPA).11 Consistent with a role for inositol depletion in autophagy regulation, CBZ and VPA also enhanced the clearance of aggregate-prone proteins.7 Thus, drugs that deplete intracellular inositol may be therapeutic candidates in HD and related disorders. To confirm the regulation of autophagy by inositol levels, we studied the effect of increased inositol (addition of myo-inositol) or IP3 [using an inhibitor of prolyl oligopeptidase activity called prolyl endopeptidase inhibitor 2 (PEI), which elevates intracellular IP3]11 on lithium (Fig. 1). These compounds reversed the proautophagic properties of lithium by preventing the clearance of mutant aggregate-prone proteins. Furthermore, treatment with myo-inositol or PEI alone, which elevated IP3 levels, inhibited autophagy, suggesting that IP3 levels regulate mammalian autophagy.7 This novel autophagy pathway was independent of the well known mammalian target of rapamycin (mTOR) pathway, which negatively regulates autophagy.12 Rapamycin did not affect IP3 levels and IP3 did not affect rapamycin signaling. Simultaneous pharmacological inhibition of mTOR (by rapamycin) and phosphoinositol signaling (by lithium or L-690,330) pathways resulted in greater www.landesbioscience.com
clearance of mutant aggregate-prone proteins and protection against their aggregation and toxicity, compared to either pathway alone (Fig. 1). Thus, we demonstrated that autophagy can be further enhanced by an additive effect from two independent pathways, compared to maximal stimulation by either pathway alone.7 This novel pharmacological strategy for autophagy induction may help treatment of neurodegenerative diseases like HD, where the toxic proteins are autophagy substrates.8,13,14 Combination therapy with more moderate IMPase and mTOR inhibition may be safer for long-term treatment, if each pathway is not maximally inhibited. It is tempting to speculate whether the therapeutic efficacies of lithium, CBZ and VPA in bipolar affective disorder is mediated by the clearance of very long half-life autophagy substrates that may mediate the disease (this does not mean protein aggregation is a feature of this disease), since these drugs have acute effects on free inositol levels long before mediating clinical effects.15,16 Is it worth considering autophagy inducers like rapamycin for treating mood disorders? Given the high prevalence of bipolar and unipolar affective disorders, their significant morbidity and cost, coupled with a significant proportion of cases that respond poorly to existing therapy, this
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idea may be worth debating. Autophagy induction may also be an effective strategy in certain infectious diseases caused by agents that are autophagy substrates (such as mycobacteria and group A streptococci)17,18—we believe that it may also be worth considering rapamycin and/or lithium or other inositol-lowering agents in these contexts. References 1. Berridge MJ, Downes CP, Hanley MR. Neural and developmental actions of lithium: A unifying hypothesis. Cell 1989; 59:411-9. 2. Rowe MK, Chuang DM. Lithium neuroprotection: Molecular mechanisms and clinical implications. Expert Rev Mol Med 2004; 6:1-18. 3. Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med 2004; 10:S10-7. 4. Rubinsztein DC. Lessons from animal models of Huntington’s disease. Trends Genet 2002; 18:202-9. 5. Carmichael J, Sugars KL, Bao YP, Rubinsztein DC. Glycogen synthase kinase-3 β inhibitors prevent cellular polyglutamine toxicity caused by the Huntington’s disease mutation. J Biol Chem 2002; 277:33791-8. 6. Berger Z, Ttofi EK, Michel CH, Pasco MY, Tenant S, Rubinsztein DC, O’Kane CJ. Lithium rescues toxicity of aggregate-prone proteins in Drosophila by perturbing Wnt pathway. Hum Mol Genet 2005; 14:3003-11. 7. Sarkar S, Floto RA, Berger Z, Imarisio S, Cordenier A, Pasco M, Cook LJ, Rubinsztein DC. Lithium induces autophagy by inhibiting inositol monophosphatase. J Cell Biol 2005; 170:1101-11. 8. Ravikumar B, Duden R, Rubinsztein DC. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum Mol Genet 2002; 11:1107-17. 9. Atack JR, Cook SM, Watt AP, Fletcher SR, Ragan CI. In vitro and in vivo inhibition of inositol monophosphatase by the bisphosphonate L-690,330. J Neurochem 1993; 60:652-8. 10. Maeda T, Eisenberg Jr F. Purification, structure, and catalytic properties of L-myo-inositol-1-phosphate synthase from rat testis. J Biol Chem 1980; 255:8458-64. 11. Williams RS, Cheng L, Mudge AW, Harwood AJ. A common mechanism of action for three mood-stabilizing drugs. Nature 2002; 417:292-5. 12. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000; 290:1717-21. 13. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O’Kane CJ, Rubinsztein DC. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 2004; 36:585-95. 14. Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC. α-Synuclein is degraded by both autophagy and the proteasome. J Biol Chem 2003; 278:25009-13. 15. Moore GJ, Bebchuk JM, Parrish JK, Faulk MW, Arfken CL, Strahl-Bevacqua J, Manji HK. Temporal dissociation between lithium-induced changes in frontal lobe myo-inositol and clinical response in manic-depressive illness. Am J Psychiatry 1999; 156:1902-8. 16. Gould TD, Chen G, Manji HK. Mood stabilizer psychopharmacology. Clin Neurosci Res 2002; 2:193-212. 17. Ogawa M, Yoshimori T, Suzuki T, Sagara H, Mizushima N, Sasakawa C. Escape of intracellular Shigella from autophagy. Science 2005; 307:727-31. 18. Nakagawa I, Amano A, Mizushima N, Yamamoto A, Yamaguchi H, Kamimoto T, Nara A, Funao J, Nakata M, Tsuda K, Hamada S, Yoshimori T. Autophagy defends cells against invading group A Streptococcus. Science 2004; 306:1037-40.
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