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implicated in mood stabilization for the treatment of bipolar disorder.

Lithium and phosphoinositide/protein kinase Csignaling pathway

Lithium has been shown to be an inhibitor of anumber of structurally similar magnesium-dependentphosphomonoesterases at K i values within thetherapeutically relevant range of concentrations(0.81.2 mM). 16,17 It was recognized over two decadesago that lithium is a potent inhibitor of the intracel-lular enzyme, inositol monophosphatase (IMPase)(K i 0.8 mM), which converts inositol monopho-sphate to inositol. 1820 Biochemical and Geneticstudies subsequently identified the upstream inositolpolyphosphatase as an additional target for

lithium. 21,22 Thus, receptor G-protein-coupled PIhydrolysis has been extensively investigated as a sitefor the action of lithium as a mood stabilizer (seereview Manji and Lenox; 23 see Figure 1). Furthermore,since there is evidence that the mode of enzymeinhibition of IMPase is un competitive, the preferen-tial site of action for lithium was proposed to be themost overactive receptor-mediated neuronal path-ways undergoing the highest rate of phosphatidyl(4,5) bisphosphate (PIP 2 ) hydrolysis. 24,25 While it wasposited that lithium produces its therapeutic effectsvia a consequent reduction in relative concentrationsof myo- inositol and PIP 2 concentrations, data-drivensupport for this hypothesis has been highly depen-dent upon the cell and animal models underinvestigation. 2631 Much of this inconsistency may be related to the relatively small size of the signal-

Figure 1 Lithium-responsive signal transduction pathways focused on PI/PKC and GSK-3 signaling. Lithium directlyinhibits the enzyme inositol monophosphate phosphatase (IMPase; K i E 0.8mM). Binding of ligands to G-protein-coupledreceptors (GPCRs) activates PLC and causes hydrolysis of PIP 2 to inositol-1,4,5 trisphosphate (IP 3) and DAG. IP 3 stimulatesCa2 release from cellular store, and DAG activates DAG-dependent protein kinase C (PKC). In the presence of receptorligands, long-term inhibition of IMPase results in a depletion of myo -inositol ( myo -I) and an accumulation of DAG followed by a downregulation of PKC isozymes a and e. On the other hand, lithium is a potent inhibitor of glycogen synthase kinase-3 b(GSK-3b ; K i E 2 mM) leading to the stabilization of b-catenin, which enters the nucleus to activate LEF/TCF-dependent genes.Lithium-responsive gene network is hypothetically proposed to be activated through PKC and GSK-3 signaling pathways andtheir crosstalk at therapeutically relevant concentrations. Abbreviations: A, receptor agoinst; Akt/PKB, a serine/threoninekinase; APC, adenomatous polyposis protein; axin, a homolog of Drosophila product of the fused locus; CMP-PA, cytidinemonophosphate-phosphatidate; DAG, diacylglycerol; Dsh, dishevelled; G, G protein; GBP, GSK-3 binding protein; GSK-3,glycogen synthase kinase-3; IP3, inositol-1,4,5-trisphosphate; LEF/TCF, LEF(lymphoid enhancer element)/TCF(T cell factor-1) family transcription factors; LRE, lithium-responsive promoter element; MUNC, a synaptic protein that contains DAG binding domain; PI, phosphatidylinositol; PIP 2 , phosphatidylinositol 4,5 bisphosphate; PP2A, protein phosphatase 2A; PLC,phospholipase C; Wnt, a secreted glycoprotein ligand for Wnt/ b-catenin signaling pathway.

Molecular basis of lithium actionRH Lenox and Le Wang

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dependent pools of myo- inositol and PIP 2 , as well asthe fact that the lithium-induced reduction of myo-inositol appears to be dependent upon multiplefactors including cell-type and tonic activity of thereceptor-coupled PI signaling pathway. 3133 It is of interest in this regard that a significant lithium-induced reduction in myo -inositol levels in the rightfrontal lobe has been observed in bipolar patientsprior to clinical response using proton magneticresonance spectroscopy. 34 However, these and otherdata suggest that while inhibition of IMPase mayrepresent an initial effect of lithium, reducing myo-inositol levels may be more critical to the specificityof the cellular site of action for lithium than its long-term therapeutic efficacy (see review, Lenox andManji). 2

Such data in turn supported the hypothesis thatshort-term lithium exposure in selective neuronalpopulations in brain undergoing heightened activa-tion would result in diacylglycerol (DAG)-mediated

activation of protein kinase C (PKC), subsequentdownregulation of PKC isozymes, and long-termdownstream changes in brain. 5,35 PKC isozymes have been implicated in the regulation of neurotransmitterrelease, neuronal excitability, and long-term changesin PKC-regulated protein function, neuroplasticity,and gene expression. PKC activity is tightly regulated by virtue of not only the distinct distribution of itsfamily of isozymes, but its required translocation tothe membrane associated with phosphorylation and binding to a receptor for activated C-kinase (RACK)proteins, and ultimate autocatalysis upon prolongedactivation. Several laboratories including our ownhave demonstrated that short-term lithium treatmentactivates PKC, and long-term lithium treatment down-regulates PKC isozymes in a PI-signaling-dependentmanner in brain. 5,35 Studies of chronic lithium (1 mM)exposure in both in vivo and in vitro models from ourlaboratory and others have demonstrated a reductionin PKC isozymes a and e in rat subiculum and in CA1regions of the hippocampus, which has been repli-cated in immortalized hippocampal cell modelsystem. 19,36,37 Furthermore, administration of myo-inositol to rats has been reported to reverse thedownregulation of PKC e in brain following chroniclithium. These studies have led to investigationsexamining PKC regulation in man (see reviews, Manji

and Lenox,19,35

Jope and Song,38

Wang et al ,39

Soareset al 40 ). Recent studies using human post-mortem brain homogenates revealed an increased associationof the receptor for activated C kinase-1 (RACK1) withPKC isozymes in frontal cortex of subjects with BPD,suggesting that interactions between these proteinsmay be also altered in bipolar disease. 37 In addition,there is intriguing evidence that tamoxifen, a drugknown to inhibit PKC in vitro , was effective in aninitial clinical study of acutely manic patients withBPD.41 Thus, it appears that chronic lithium-depen-dent modulation of receptor-coupled PIPKC signal-ing pathways may contribute to the long-term actionof lithium in the brain.

Lithium and Wnt signaling pathway

Glycogen synthase kinase 3 b (GSK-3b) is a serine/threonine kinase that is constitutively active in cellsand negatively regulates its substrates, one of which isb-catenin, a downstream effector of the Wnt signalingpathway controlling dorsalventral axis specificationand cell fate determination in various organismsincluding Dictyostelium , sea urchins, zebrafish, andXenopus. 17 GSK-3 activity is regulated through multi-ple proteins in a complex (Figure 1), where GSK-3 isactivated in the presence of axin, adenomatouspolyposis coli (APC), dishelveled (Dsh) and pro-motes phosphorylation of b-catenin for ubiquitinproteosome-mediated degradation. 43 On the contrary,GSK-3 binding protein (GBP), which is requiredfor axis formation in Xenopus , could join the comp-lex to inhibit GSK-3 activity, in part, by preventingaxin from binding GSK-3. 44 When b-cateninaccumulates in cytoplasm, it translocates into nu-

cleus and activates T-cell factor (TCF) and lymphoidenhancer element (LEF)-dependent gene transcrip-tion, through which the Wnt pathway controlsnumerous developmental processes. The full reper-toire of the genes containing LEF/TCF responsiveelements in their promoters has not been defined.Patterning genes Ultrabithorax in Drosophila, sia-mois , and nodal-related gene-3 in Xenopus ,4547 hu-man genes c-myc and cyclin D1 ,4850 and connexin43and E-cadherin 51,52 represent major LEF/TCF-respon-sive genes discovered to date. Other possibletargets of GSK-3 may include AP-1, CREB, NF- kB,heat shock protein 1, and CCAAT/enhancer binding proteins. 53 LEF1 was recently found to becritical to the generation of dentate gyrus granulecells and the development of the hippocampus inmice. 4547,54 In addition, GSK-3 could phosphorylateNF-AT, facilitating its export from the nucleus,and thus antagonizing NF-AT-dependent gene tran-scription. 55

Lithium mimics Wnt signals by inhibition of GSK-3 both in vitro and in vivo . This suggests that the often-observed developmental effects of lithium, whichcould not be explained by inhibition of IMPase, may be in fact a result of inhibition of GSK-3 b .56,57 Valproicacid also inhibits GSK-3, 58 but its control over theb-catenin stability differs from lithium. While lithium

stabilizes b-catenin via inhibition of GSK-3, VPA-induced b -catenin accumulation is through increasedexpression of b-catenin rather than stabilization of the protein. 59 GSK-3 has also been implicated insynaptic function. For example, GSK-3 b inhibitioninduces the synapsin I clustering in the formation of synapses and neurotransmitter release. 60 In addition,GSK-mediated phosphorylation of MAP-B and tauproteins regulate microtubule assembly and stabiliza-tion at synapses. 57,6163 Clinical studies have demon-strated that specific GSK-3 inhibitors mimic thetherapeutic action of mood stabilizers and mighttherefore be plausible drugs in treating bipolarpatients. 64


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development. 8890 Within brain and neurons in cul-ture, MARCKS protein is expressed in neurites andsynaptosomes, and is colocalized with synapticvesicles. 9193 PKC-MARCKS signaling has been im-plicated in vesicular trafficking of neurotransmitter inliving neurons, 94 and in the regulation of variousprocesses including synaptic transmission at nerveterminals. 80,81 Electrophysiological and behavioralstudies in heterozygote mutant mice have shown that50% reduction in MARCKS significantly affectslong-term potentiation (LTP) and hippocampallydependent behavior. 9597 These data indicate thatMARCKS plays an important role in the mediationof neuroplastic processes in the developing andmature CNS.

Chronic treatment with lithium at therapeuticallyrelevant concentrations significantly downregulatesMARCKS protein and mRNA (50%) in both rat brainand immortalized hippocampal cells. 27,98,99 Directactivation of PKC by phorbol esters in immortalized

hippocampal cells also downregulates the MARCKSprotein, 100 suggesting a role for PKC in the regulationof MARCKS gene ( Macs ) expression in brain. Thelithium-induced downregulation of MARCKS proteinis dependent upon the myo- inositol concentrationand the level of activation of receptor-coupled PIsignaling. 31 Addition of inositol completely reversesthe lithium-induced downregulation of MARCKS,indicating that the downregulation of MARCKS wasmediated via the phosphoinositol pathway. 31 Further-more, lithium-induced down regulation of MARCKSis only apparent after chronic, but not acute , admin-istration and persists beyond abrupt withdrawal of the drug for an extended period of time; parallelingthe clinical time course for the therapeutic effects of lithium during initial treatment as well as itsdiscontinuation. Of interest, the structurally unre-lated mood-stabilizer, valproic acid (VPA), shares theproperty of lithium in reducing the MARCKS expres-sion in hippocampus, but carbamazepine (CBZ) andother psychotropic agents (antianxiety, analgesic,antipsychotic, antidepressant, calcium-channel blocker) as well as other monovalent cations such asrubidium do not alter MARCKS regulation. 101,102 Onthe other hand, VPA-induced downregulation of MARCKS is inositol-independent, and has an addi-tive effect on MARCKS regulation, consistent with

clinical data supporting greater efficacy of thecombined treatment in refractory bipolar patients. 101These findings point to a common mechanism, inwhich MARCKS may be a shared target for bothlithium and VPA. Thus, we have proposed that thereduction of MARCKS protein following long-termlithium administration alters pre/postsynaptic membrane structure and stabilizes aberrant neuronalsignaling in key brain regions of patients withBPD.3,5 The regulation of MARCKS expression repre-sents a highly valued target for the long-term action of mood stabilizers, not only by virtue of its underlyingneurobiological properties but also in light of itspharmacological sensitivity and selectivity for struc-

turally unrelated drugs with efficacy in the prophy-lactic treatment of BPD. While MARCKS remains anattractive target for illustrative purposes in defining alithium-responsive gene network, it is evident thatother targets, such as prolyl oligopeptidase (POase),which appears to mediate the common effect of lithium, CBZ and VPA on growth cone stability andhas been implicated in affective disorders, may atsome point represent a similar opportunity. 103

Perspective: linking the lithium-responsive genesas a network

The mechanisms underlying the prophylactic treat-ment of BPD by lithium are likely to be hardwired inthe genomic DNA. Despite all the reports of howindividual gene expression can be modulated inresponse to lithiums exposure, there is as yet nostrategy available for the identification of the lithium-responsive genomic regulatory network in brain.

Many studies have focused on determining the effectof lithium on one or a few genes at a time, anapproach that is not adequate for the analysis of largeregulatory control system organized as networks.Gene-specific expression is controlled by specificcis -regulatory target sequence embedded in promotersand the cognate binding transcription factors encoded by a set of regulatory genes. The functional linkages of such a genomic regulatory network include elementsthat exhibit multiple interactions between the outputsof regulatory genes and corresponding cis-regulatorygenomic sequences. Thus, identifying networks of transcription factors and the genes they regulate inresponse to lithium exposure is important to under-stand the biological responsiveness of an organism tolithium and the prophylactic properties of its actionin brain.

Studies in our laboratory have demonstrated thatthe lithium-induced reduction of MARCKS proteinwas accompanied by a downregulation of MARCKSmRNA with no evidence for a change in the half-lifeof the mRNA. 99 Furthermore, synthesis of nascentRNA for MARCKS and the Macs promoter activitywere also found to be significantly reduced inchronic, but not acute, lithium-treated immortalizedhippocampal cells. Both reductions were signifi-cantly enhanced in the presence of activation of

receptor-coupled PI signaling.99

We have identifiedfor the first time a lithium-responsive promotersequence located in the upstream region ( 993/

713) of the Macs promoter. 99 The mutant promoterlacking the 993/ 713 fragment not only did notrespond to chronic lithium expose but also had asignificantly reduced promoter activity, suggestingthat chronic lithium represses the transcriptionalactivator(s) bound to this region. Until now, however,no lithium-responsive transcription factor directly bound to this region has been identified. Whilespecific transcription factors that bind to lithium-responsive promoter element (LRE) remain to beidentified (Figure 2), current data suggest that the


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mechanism by which chronic lithium represses theMacs gene transcription are likely through: (a)lithium-induced downregulation of DNA bindingactivity and/or expression of transcription activator(s)interacting with LRE; (b) lithium-induced down-regulation of activator function of transcriptionactivator(s) acting at LRE; (c) lithium-induced disrup-tion of the coordinate interaction between the distalactivating sequence-bound activator(s) and the prox-imal core promoter sequence-bound basal transcrip-tional machinery. The lithium-responsive 993/ 713fragment contains significant enhancer/activator DNAelements that are necessary to sustain the optimalMacs gene transcription in cells. 104 While an atypicalSp1 site, characterized by the presence of a prominentGA-rich sequence, was identified within the 993/

713, a classical Sp1 site was found in the middle of the GC-rich box close to a potential Z-DNA-formingsegment near the transcription initiation site. 104Presence of Z-DNA forming segment signifies both

compositional and conformational changes influen-cing the genomic landscape, the accessibility of cis-acting elements, and the activation of gene transcrip-tion in the core promoter region. 105 As summarized inFigure 2, Macs promoter lacks typical TATA box,and in the absence of a TATA box multiple Sp1 sitesin the promoter may control the transcription of Macsgene.

A recent microarray experiment further showedthat the expression of as many as 37 genes from 4132rat genes is altered during the chronic lithiumtreatment. 106 If these data were extrapolated to theentire genome, there would be as many as 750 genesthat could potentially be regulated by lithium.However, the majority of such studies using differ-ential display and microarray analysis do not distin-guish between direct activation of target genes by atranscription factor and indirect effects resulting fromone transcription factor inducing the expression of asecond. Furthermore, the functional value of the vastmajority of these potential targets remains unknown.Using our knowledge regarding a high-value pharma-cologically relevant target such as MARCKS for theaction of chronic lithium in brain to identify lithium-responsive elements that could serve to link lithium-responsive genes as a regulatory network is bothintriguing and challenging. To establish such a genenetwork, some studies have coupled the overexpres-

sion of a given transcription factor with microarrayexperiments. However, the genes affected by theoverexpressed transcription factors may not representthe true targets of those factors under physiologicalconditions. Recently, using finite perturbations of gene expression in conjunction with microarrayexperiments that may mimic physiological conditionshas been advocated; thus differing from the drastic

Figure 2 Lithium-responsive promoter of the Macs gene. The Promoter region upstream of Macs is pharmacologicallyresponsive to chronic, but not acute, lithium exposure. A hypothetical activator complex binds to the upstream region of Macs promoter and crosstalk with proteins in proximal promoter region including TBP, TAFs, Sp1, hypothetical tetheringproteins, etc. The gray rectangle box represents cis-regulatory enhancer elements (LRE) that may interact with specificlithium-responsive transcription factors. Long-term treatment with lithium is postulated to repress the Macs transcriptionthrough: (a) lithium-induced reduction in the DNA binding of the transcription activator(s) acting at the LRE; (b) lithium-induced alteration in the expression and/or the transactivation function of lithium-responsive transcription activator(s)acting at the LRE; (c) lithium-induced disruption of the interaction between enhancer complex formed at LRE and theproximal transcription initiator. In the figure, # refers to potential intermediary factors mediating the indirect action of chronic lithium on the Macs promoter.


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changes incurred during transgenic overexpression orknockouts. 107 In such studies, one can modulatesingle gene expression using techniques such asRNA interference (RNAi) one at a time and in asystematic fashion among all the genes of interest.Once the perturbed systems are in a new steady state,the levels of gene expression is measured against thatof reference by microarray or Taqman RCR. Theresulting data are arranged as a regulatory strengthmatrix, from which a regulatory network exhibitinggenegene interactions could be inferred using ap-proaches adapted from metabolic control analysis. 108The method, however, lacks genome-wide efficiencyand does not provide direct evidence of in vivointeraction of given transcription factors with theirrespective cis-regulatory elements across the genome.To address these questions, a new technology knownas chIpchip was developed, in which chromatinimmunoprecipitation (chIp) is coupled with micro-array experiment (chip) to accelerate the genome-

wide detection of DNAprotein interactions in realtime and in real space. Using such a combinedapproach as chIpchip, genome-wide mapping of

DNA binding sites for transcription factors (STE12,GAL4, RAP1, SCB, MCB, MCM1, SFF, and SW15) has been achieved in yeast. 109,110 This experimentalapproach has also been successfully extended tohuman genome, where DNA binding sites for E2Fand GATA-1 transcription factors are mapped genomewide. 111113

A lithium-responsive gene network will offer anopportunity to define a pathway associated with thelong-term prophylactic properties of lithium, distinctfrom its side-effect profile, which will drive thediscovery of novel agents for stabilization of moodin patients with BPD (Figure 3). Recently, a regulatorygene network that directs specific developmentalevents has been identified in developing sea urchinembryo. 114,115 This provides a heuristic model forconstructing a lithium-responsive gene network asa means to identify signature genes that directthe therapeutic or nontherapeutic action of lithium.While chromosome immunoprecipitation could start

with antibodies against several known lithium-responsive transcription factors such as AP-1, CREB,NF-kB, LEF/TCF, these are general transcription

Figure 3 Model for a gene network for lithium-responsive genes: defining the therapeutic effect of chronic lithium. A genenetwork can be reconstructed by perturbation microarray and chlpchip methods (see test for details). A lithium-responsivegene network will consist of a distinctive subset of genes that share a lithium-responsive element (LRE) as defined within theMacs gene which is regulated by specific transcription factors referred to as TFs that are yet to be identified. Such a lithium-responsive gene network (TF1) may subserve critical processes in the brain underlying cytoskeletal remodeling andregulation of synaptic signaling, while a different subset of genes defined by lithium-responsive elements regulated bytranscription factors (TF2, TF3) may underlie processes such as cell proliferation. Since these different network of genesunderlie different physiological sequela of chronic lithium, modulation of gene expression involved in the cytoskeletalrestructuring and neuroplasticity by lithium may prove to be fundamental to the mood-stabilizing properties of lithium in the brain, whereas regulation of genes involved in cell proliferation and immune response may be linked to lithiumsleukocytotic and antiviral effects.


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factors that elicit far more gene expression thannecessary for the therapeutic action of lithium. Thus,identifying lithium-responsive transcription factorsor promoter elements from highly valued targets suchas the Macs gene becomes extremely desirable. Wedemonstrated that Macs promoter is pharmacologi-cally responsive to chronic, but not acute, lithiumtreatment at therapeutically relevant concentrations,and possesses pharmacological properties consistentwith its expressed protein and clinical properties of lithium. 99 We further showed that the sites for lithiumresponsiveness lie within an enhancer element of thepromoter that has the potential to interact witha unique lithium-responsive activator complex(Figure 3). 104 This raises the possibility that uniquetranscription factors sensitive to lithium treatmentmay be identifiable through this promoter. Whilegenes like Macs involved in the cytoskeletal restruc-turing, synaptic transmission, and neuroplasticitymay relate preferentially to the long-term mood-

stabilizing properties of chronic lithium in the brain,genes underlying cell proliferation and immuneresponse may serve to identify pathways associatedwith lithiums leukocytotic and antiviral effects(Figure 3). Thus, defining a lithium-responsive genenetwork will provide the data for pathway mapping of novel targets with better-defined mood-stabilizingproperties for the long-term treatment of BPD.

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