Lithium is widely used to treat bipolar disorder, but its mechanism of action in this disorder is unknown. Lithium directly inhibits GSK3 (glycogen synthase kinase 3), a critical regulator of multiple signal transduction pathways. Inhibition of GSK3 provides a compelling explanation for many of the known effects of lithium, including effects on early development and insulin signalling/glycogen synthesis. However, lithium also inhibits inositol monophosphatase, several structurally related phosphomonoesterases, phosphoglucomutase and the scaffolding function of β-arrestin-2. It is not known which of these targets is responsible for the behavioural or therapeutic effects of lithium in vivo. The present review discusses basic criteria that can be applied to model systems to validate a proposed direct target of lithium. In this context, we describe a set of simple behaviours in mice that are robustly affected by chronic lithium treatment and are similarly affected by structurally diverse GSK3 inhibitors and by removing one copy of the Gsk3b gene. These observations, from several independent laboratories, support a central role for GSK3 in mediating behavioural responses to lithium.
- bipolar disorder
- glycogen synthase kinase 3 (GSK3)
As has been underlined at the Biochemical Society Focused Meeting on BD (bipolar disorder) at Royal Holloway in April 2009, several plausible targets of lithium have been described and studied, and a strong case can be made for each of these potential targets as a mediator of lithium action within specific experimental contexts. These targets include IMPase (inositol monophosphatase) and structurally related phosphomonoesterases [1,2], PGM (phosphoglucomutase) , GSK3 (glycogen synthase kinase 3) , and the recently described β-arrestin-2–Akt complex . As lithium affects many biological systems, it is likely that each of these potential targets plays an important role in mediating the effects of lithium in specific contexts. However, none has been proved to be a relevant drug target in the therapy of BD.
In addition to its therapeutic actions in BD, lithium alters the function and development of diverse organisms from yeast to vertebrates, and these have provided useful model systems for investigating lithium action. For example, lithium enhances glycogen synthesis, perturbs cell fate decisions in Dictyostelium, sea urchins and vertebrates, stabilizes neuronal growth cones and stimulates mammalian haematopoiesis (reviewed in depth in ). Investigation of lithium action in these model systems has yielded a molecular toolbox for validating putative lithium targets in new biological contexts. In the present brief review, we restrict our attention to targets that are directly inhibited by lithium at therapeutically relevant concentrations and discuss a set of criteria that can be applied to validate a putative target of lithium in diverse biological contexts.
Direct targets of lithium
IMPase and the inositol-depletion hypothesis
The inositol-depletion hypothesis is one of the most compelling and enduring hypotheses to explain lithium action in multiple settings. This hypothesis was enumerated in a seminal paper by Berridge et al. , and will certainly be addressed in greater detail by others in this issue of Biochemical Society Transactions. In brief, the hypothesis holds that inhibition of IMPase by lithium  will limit the regeneration of inositol from IMP, and in settings where the cell is dependent on this source of inositol to synthesize phosphoinositides, lithium should interfere with PtdInsP-dependent signalling pathways. As originally articulated, this would reduce signalling by second messengers generated from signal-induced PtdInsP2 hydrolysis, although it should also interfere with PtdInsP3-dependent pathways (see ). An important feature of this hypothesis is that lithium is an uncompetitive inhibitor of IMPase  and thus the degree of inhibition increases at high substrate concentration.
Phosphomonoesterases related to IMPase
Members of a family of magnesium-dependent phosphatases structurally related to IMPase have also been proposed as biologically relevant lithium targets . These include IPPase (inositol polyphosphate 1-phosphatase), fructose 1,6-bispohosphatase, BPNT (bisphosphate nucleotidase), and the nucleotidase gPAPP (Golgi-resident 3′-phosphoadenosine 5′-phosphate 3′-phosphatase) . Some of these enzymes are significantly more sensitive to lithium than IMPase, and the phenotypes associated with mutations in a few of these genes in metazoan organisms have been described. For example, mutation of the Drosophila ipp gene, which encodes IPPase, phenocopies lithium action in the Drosophila neuromuscular junction, as discussed below. PGM is not a member of this family, but nevertheless also hydrolyses a carbohydrate–phosphomonoester linkage as part of its phosphoryltransferase mechanism, and is similarly magnesium-dependent and lithium-sensitive .
GSK3 is a serine/threonine protein kinase that does not share obvious structural features with other lithium sensitive enzymes . Furthermore, GSK3 is the only protein kinase among more than 70 tested that is inhibited by lithium at therapeutically tolerated concentrations (although several are partially inhibited by lithium at 10 mM ). Lithium competes with magnesium  and most Ki values reported for GSK3 reflect assays carried out at superphysiological magnesium concentrations. Thus the IC50 for lithium inhibition of GSK3 is approx. 1.0 mM or lower at typical intracellular magnesium concentrations [2,11].
GSK3 was first described as an antagonist of glycogen synthase, and insulin activates glycogen synthase in part by Akt/PKB (protein kinase B)-dependent phosphorylation and inhibition of GSK3 . GSK3 also antagonizes Wnt signalling by constitutively phosphorylating β-catenin and promoting its degradation . Thus inhibition of GSK3 by lithium will activate these pathways downstream of GSK3. This downstream activation can explain many of the known effects of lithium on glycogen synthesis, development, circadian rhythm, haematopoiesis and other responses to lithium [2,4].
In addition to direct inhibition of GSK3 by lithium, several modes of indirect inhibition have been described. Lithium enhances the inhibitory N-terminal phosphorylation of GSK3 by increasing Akt activity and by inhibiting the phosphatase that dephosphorylates GSK3 [13,14]. These indirect effects are a consequence of direct GSK3 inhibition, as GSK3 regulates itself through complex feedback loops that involve activation of PP (protein phosphatase) 1 and inhibition of Akt; in addition, in the striatum, lithium disrupts a scaffold of β-arrestin, Akt, PP2A and GSK3, leading to enhanced Akt activity . We propose that GSK3 may play a role in stabilizing this complex, so that inhibition of GSK3 could contribute to the disruption of the β-arrestin–Akt–PP2A–GSK3 complex in vivo, but this has not been tested, and Beaulieu et al.  presented data to show that lithium can disrupt the interaction of β-arrestin and Akt in vitro, in the absence of GSK3.
Criteria for validating a potential direct target of lithium in different biological contexts
With multiple plausible targets and numerous biological effects of lithium, it is essential to establish a set of criteria that can be applied in each new context to validate a given lithium target. These criteria may include the following.
(i) Evidence that a therapeutically relevant concentration of lithium inhibits the target in vitro and in vivo. All of the targets described above are inhibited by lithium in vitro, but the challenge has been to show significant inhibition in vivo. Measurement of enzyme activity or level of product is essential to verify in vivo inhibition directly.
(ii) Pharmacological evidence that structurally distinct inhibitors of the putative target mimic lithium action can provide strong, although not unequivocal, support for a given target. Completely specific enzyme inhibitors are rare, if they exist at all, but it is unlikely that multiple structurally diverse inhibitors will share ‘non-specific’ targets.
(iii) Genetic evidence, for example by gene knockout, RNA interference or expression of dominant-negative constructs, that disruption of gene function mimics lithium action is a powerful approach to validate putative drug targets.
(iv) Reversal of lithium effect by restoring enzyme function or product in the presence of lithium is a valuable, although not infallible, approach to validate that observed effects of lithium are caused by inhibition of a specific target. This is analogous to validating gene-knockout phenotypes by reintroduction of the gene into the mutant background.
Applying the rubric
Cell fate decisions in developing organisms
The ability of lithium to perturb the development of a variety of organisms has been recognized since the late 19th Century . Lithium shifts cell fate of developing Dictyostelium from spore to stalk cell fate , vegetalizes animal blastomeres in sea urchins and dorsalizes Xenopus and zebrafish embryos (reviewed in ).
In this section, we apply the above criteria to review the evidence that the developmental effects of lithium are mediated by inhibition of GSK3. (i) We and others have shown that lithium indeed inhibits GSK3 in Xenopus oocytes and embryonic extracts. (ii) Alternative GSK3 inhibitors, including 6BIO (6-bromo-5′-indirubin-3′-oxime), the 25-mer peptide GID (GSK3-interaction domain) and a ruthenium-based organometallic (RuOH) mimic the developmental effects of lithium [17–19], as does the GSK3 inhibitor protein FRAT (frequently rearranged in advanced T-cell lymphomas)/GBP (GSK3-binding protein) . (iii) GSK3 loss-of-function mutations mimic the developmental effects of lithium in Dictyostelium , and dominant-negative GSK3 mimics lithium action in sea urchins, frogs and zebrafish (reviewed in ). (iv) Overexpression of GSK3 suppresses the dorsalizing effects of lithium in Xenopus (P. Klein, unpublished work). In metazoan embryos, the developmental effects of lithium inhibition of GSK3 are mediated through activation of canonical Wnt signalling, and thus blocking downstream Wnt signalling, for example by removing β-catenin, blocks the developmental effects of lithium (J. Heasman, personal communication) and conversely in vivo lithium treatment mimics the developmental phenotype of Wnt gain-of-function mutations in the mouse anterior heart field, leading to expansion of right heart and cardiac outflow precursors . Interestingly, lithium exposure in human embryos has been associated with increased risk for Ebstein's anomaly, a downward displacement of the tricuspid valve and other right heart anomalies proposed to be a consequence of lithium-mediated activation of Wnt signalling . Taken together, these data strongly support GSK3 as a critical target of lithium action in the development of diverse organisms.
Lithium also inhibits IMPase in vivo in Xenopus embryos . However, an alternative and much more potent IMPase inhibitor (L690,330) has no obvious effect on embryo development , indicating that inhibition of IMPase is not sufficient to account for the developmental effects of lithium. This is one of the few examples in which a specific IMPase inhibitor has been tested in an in vivo setting, and was possible because the compound, which does not readily cross plasma membranes, could be injected directly into embryonic Xenopus blastomeres. Genetic tests of IMPase function have not been carried out in Xenopus or zebrafish. Lithium effects in Xenopus can be blocked by co-injection of 0.3 M inositol, supporting the inositol-depletion hypothesis; however, this concentration of inositol also reverses the dorsalizing effect of dominant-negative GSK3 , suggesting either that inositol rescue has an indirect effect on dorsalizing pathways or, interestingly, that GSK3 somehow regulates inositol levels, as proposed by Greenberg and colleagues  and discussed below.
Effects of lithium on neuronal function
Loss of function of the ipp gene in Drosophila leads to defects in synaptic transmission in the larval neuromuscular junction, as well as accumulation of inositol phosphate species . Lithium inhibits the Drosophila IPPase completely at 2 mM and phenocopies the synaptic defects of the ipp mutants. Alternative IPPase inhibitors are not available, and rescue of lithium effects by overexpressing IPPase was not tested, but these results nevertheless strongly support that the effect of lithium in the neuromuscular junction of Drosophila larvae indeed occurs through inhibition of IPPase. A similar phenotype in other organisms has not been reported, however.
Mutations in the IMPase gene ttx-7 disrupts thermotaxis in Caenorhabditis elegans, and lithium also phenocopies this defect . This work was especially elegant, as either exogenous inositol or overexpression of IMPase rescues normal thermotaxis. These data make a strong case that disruption of this behaviour by lithium is mediated by inhibition of IMPase. It will be interesting to see whether lithium or loss of the IMPase gene also reduces synthesis of PtdIns, PtdInsP or PtdInsP2, as a decrease in PtdInsP2 by lithium has been observed in Dictyostelium and probably accounts for the lithium-induced chemotactic defects observed in this organism .
Lithium, valproic acid and carbamazepine, all of which are used to treat BD, stabilize neuron growth cones in cultured sensory neurons, suggesting that each drug modulates a common pathway that regulates growth cone dynamics [27a]. Furthermore, the effect of all three drugs was blocked by adding inositol. A reasonable hypothesis, proposed by the authors, is that growth cone stabilization by these BD drugs arises due to reduction in endogenous inositol. To test this hypothesis, it will be important to measure inositol, but this has not yet been done in this setting. However, lithium (directly) and valproic acid (indirectly) also inhibit GSK3 in neurons [28,29], alternative direct GSK3 inhibitors stabilize growth cones , and knockdown of Gsk3 also stabilizes growth cones , providing strong evidence that the lithium effect on neuronal growth cones occurs through inhibition of GSK3. An interesting hypothesis that could reconcile these findings, as articulated by Greenberg and colleagues, is that GSK3 may indirectly regulate inositol synthesis , so that inhibition would reduce inositol synthesis and addition of inositol would then rescue this defect whether arising from GSK3 inhibition or other means of reducing cellular inositol. Greenberg and colleagues provided strong data to support this model in yeast , and it will be intriguing to see whether similar regulation occurs in neurons.
Lithium-sensitive behaviours in mouse
Surprisingly few behaviours had been described for chronic lithium treatment. We therefore examined multiple established and reproducible behaviours in mice placed on a simple treatment protocol (lithium in the food) that achieves serum lithium levels of 1 mEq/l. In addition to the known attenuation of amphetamine-induced hyperactivity and enhancement of pilocarpine-induced seizures , we found that the forced swim test, the elevated zero maze, and holeboard/exploratory behaviours were affected by long-term dietary lithium . Importantly, overall activity, co-ordination and general measures of the state of the animal were unaffected. Furthermore, Beaulieu et al.  reported that the tail suspension test and dark to light emergence are affected by parenteral lithium administration.
Evidence that GSK3 inhibition mediates the behavioural effects of lithium
Lithium inhibits GSK3 in vivo in mouse and rat brain [2,33,34], and alternative GSK3 inhibitors mimic many of the behavioural effects of lithium (Table 1). Both AR-A014418, an ATP-competitive GSK3 inhibitor that crosses the blood–brain barrier, and the peptide inhibitor L803mts, which was injected into the ventricles, reduce immobility in the forced swim test [35,36]. The thiadiazolidinone TDZD-8 reduces immobility in a related behaviour, the tail suspension test, and reduces latency to emerge from dark to light areas . Lithium attenuates hyperactivity induced by amphetamine ; this amphetamine-induced behaviour is believed to be due to increased dopamine signalling, and is mimicked in DAT-KO (dopamine transporter knockout) mice . Furthermore, lithium and several GSK3 inhibitors, including SB216763, alsterpaullone, 6BIO and TDZD, reduce hyperactivity in DAT-KO mice. Open field activity is also attenuated in wild-type mice by intraperitoneal injection of lithium (as LiCl), TDZD or AR-A014418 [5,35]; as the open field is generally a measure of the overall state of the animal, these observations could suggest a non-specific effect of this mode of drug delivery on the state of the animal. However, an alternative explanation, which we favour, is that intraperitoneal injection achieves higher peak concentrations of lithium or other inhibitors, and that the reduced activity is a specific consequence of more potent inhibition of GSK3. Support for this explanation comes from our observation that, although neither oral lithium nor Gsk3b haploinsufficiency alone alters open field activity, oral lithium treatment of Gsk3b+/− mice does reduce activity in the open field (and also markedly reduces immobility in the forced swim test ). Furthermore, overexpression of a phosphorylation-resistant Gsk3 mutant increases locomotor activity and was proposed as a model of manic hyperactivity .
To provide genetic evidence to support GSK3 as the target of lithium, we and others have tested whether deletion of the Gsk3b gene in mice affects behaviour in a manner similar to lithium [5,32,37]. [Careful attention to the genetic background of the mice to be tested is essential to obtain reliable results , and therefore our behavioural studies with Gsk3b-knockout mice were performed in mice that had been backcrossed into a defined strain (C57/Bl6) for more than ten generations]. Gsk3b−/− mice die during gestation, but Gsk3b+/− mice are viable and develop apparently normally, despite 50% decrease in GSK3β protein levels throughout the cortex, hippocampus, hypothalamus and cerebellum . Gsk3b+/− mice behave similarly to lithium-treated mice in the forced swim test, tail suspension test, amphetamine-induced hyperactivity, exploratory behaviour and elevated zero maze [5,32,37]. Furthermore, expression in the brain of a β-catenin mutant that lacks the GSK3 phosphorylation sites (and is therefore stabilized) mimics the effects of lithium in the forced swim test and amphetamine-induced hyperactivity , although conditional loss of β-catenin has so far had only modest effects on lithium-sensitive behaviours . Finally, preliminary data show that the effects of lithium on the forced swim test, exploratory behaviour and elevated zero maze are reversed by overexpression of Gsk3b in the brain, strongly supporting the hypothesis that GSK3 is the specific target of lithium in these behaviours.
Evidence for inositol depletion in the behavioural effects of lithium
Chronic lithium also decreases inositol levels in mouse brain by 10–25% [42,43]. To test whether this partial reduction in inositol can account for the observed behavioural changes, an alternative approach to reducing inositol in the brain would be extremely helpful, but IMPase inhibitors that cross the blood–brain barrier are not currently available. However, homozygous knockout of the sodium–myo-inositol transporter 1 (SMIT1) gene in mouse reduces inositol levels in fetal brain by >90%, and yet has no effect on global phosphoinositide levels, indicating that the more modest inositol reduction observed with lithium is unlikely to reduce phosphoinositide synthesis . Consistent with this, lithium has never been shown to decrease PtdIns or PtdInsP2 in vivo [45,46]. Nevertheless, a decrease in PtdIns/PtdInsP2 could be restricted to specific areas within the brain that would not be detected by global phosphoinositide measurement. Therefore it is still worthwhile to test behaviours in SMIT1-knockout mice. Although SMIT1−/− mice die at gestation, the SMIT1+/− mice are viable. Inositol levels are decreased in the brains of adult SMIT1+/− mice by 33–37% (i.e. a greater reduction than with lithium) . Under these conditions of inositol depletion, there is no effect in the forced swim test, amphetamine-induced hyperactivity or sensitivity to pilocarpine-induced seizures [43,47], demonstrating that global inositol depletion to an extent greater than observed with lithium is not sufficient to cause lithium-sensitive behaviours. One caveat to these experiments, however, is that it is not clear that SMIT1 is expressed in neurons  or that inositol levels are specifically decreased in neurons of SMIT1 mutant mice.
More recent experiments show that the SMIT1−/− mice can be raised to adulthood if the mothers are supplemented with inositol. When inositol supplementation is discontinued, the adults show a ∼60% decrease in brain inositol. Under these conditions of more severe inositol depletion, the animals demonstrate reduced immobility (increased swimming activity) in the forced swim test and increased sensitivity to pilocarpine-induced seizures, paralleling the effects of lithium ; how this mutation affects animal state, for example in the open field, was not reported, and is an important concern, as increased baseline activity is a potential confounding factor in interpreting the forced swim test in IMPA1 mutant mice (see below). Furthermore, lithium treatment does not achieve this degree of inositol depletion, so the relevance of these observations to lithium action will need to be explored further.
Mouse knockouts have been reported for both IMPase genes, IMPA1 and IMPA2. Homozygous knockout of IMPA1 is lethal, but can be rescued by inositol supplementation of pregnant mothers . Although this mutation does not affect inositol levels in the adult brain, it does reduce IMPase activity by up to 65%. Therefore, although this mouse line is not a model of global inositol depletion, they may have localized decreases in inositol levels that would be difficult to measure, but could still mimic lithium-mediated inhibition of IMPase. These mice demonstrate increased swimming activity in the forced swim test and increased sensitivity to pilocarpine, similar to lithium (Table 1). However, deletion of IMPA1 also causes marked hyperactivity in the open field (and in the home cage); as lithium increases swimming activity in the forced swim test (traditionally reported as reduced immobility), the baseline hyperactivity in IMPA1−/− mice is a significant confounding factor in interpreting the forced swim test in this mutant line. Knockout of IMPA2 does not mimic lithium-sensitive behaviours, and does not reduce inositol or IMPase activity in brain , implying redundancy with IMPA1.
The study of lithium action in diverse model systems has led to a number of approaches to probe potential targets of lithium action. In the present review, we have focused on direct molecular targets and have reviewed the various approaches in terms of a rubric to validate these enzymes as relevant targets of lithium in selected biological contexts. The evidence supporting GSK3 as the relevant target of lithium in developing organisms including Dictyostelium, sea urchins, Xenopus and zebrafish is very strong, and in metazoans, it is highly likely that lithium exerts its effects on cell fate and patterning by inhibiting GSK3 and activating the Wnt signalling pathway. Conversely, inhibition of inositol phosphatases is a very likely explanation for the in vivo effects of lithium on synaptic transmission in invertebrates such as Drosophila (ipp) and C. elegans (ttx-7), and it will be interesting to see whether these observations apply to mammalian synaptic function. The direct target(s) responsible for the effects of lithium and other BD drugs on axonal growth cones in cultured neurons remain(s) unclear, with support for both inositol depletion and inhibition of GSK3; an intriguing hypothesis to reconcile these observations in this system is that GSK3 regulates inositol synthesis, an idea that is supported by data from yeast, but not yet tested in mammalian cells. Finally, global reduction in cerebral inositol to an extent greater than that observed with lithium does not recapitulate lithium effects in mouse behaviour, and although the SMIT1 and IMPA1 knockouts show some behavioural parallels to lithium treatment, we feel that the weight of the pharmacological and genetic evidence, including the effects of multiple GSK3 inhibitors and the Gsk3b+/− mutant, strongly supports GSK3 as the critical target of lithium action in multiple mouse behaviours.
Work on lithium action in the Klein laboratory is supported by grants from the National Institutes of Health/National Institute of Mental Health [grant number R01MH058324] and the American Federation for Aging Research.
Bipolar Disorder: Molecular and Cellular Biology: Biochemical Society Focused Meeting held at Royal Holloway, University of London, Egham, U.K., 23–24 April 2009. Organized and Edited by Robin Williams (Royal Holloway, Egham, U.K.) and Talvinder Sihra (University College London, U.K.).
Abbreviations: BD, bipolar disorder; 6BIO, 6-bromo-5′-indirubin-3′-oxime; DAT-KO, dopamine transporter knockout; GSK3, glycogen synthase kinase 3; IMPase, inositol monophosphatase; IPPase, inositol polyphosphate 1-phosphatase; PGM, phosphoglucomutase; PP, protein phosphatase; SMIT1, sodium–myo-inositol transporter 1; TDZD, thiadiazolidinone
- © The Authors Journal compilation © 2009 Biochemical Society