Bipolar affective disorder (manic-depressive illness)is a chronic, severe, debilitating illness affecting 1-2% of the population. The Food and Drug Administration-approved drugs lithium and valproate are not completely effective in the treatment of this disorder, and the mechanisms underlying their therapeutic effects have not been established. We are employing genetic and molecular approaches to identify common targets of lithium and valproate in the yeast Saccharomyces cerevisiae. We show that both drugs affect molecular targets in the inositol metabolic pathway. Lithium and valproate cause a decrease in intracellular myo-inositol mass and an increase in expression of both a structural (INO1) and a regulatory (INO2) gene required for inositol biosynthesis. The opi1 mutant, which exhibits constitutive expression of INO1, is more resistant to inhibition of growth by lithium but not by valproate, suggesting that valproate may inhibit the Ino1p-catalyzed synthesis of inositol 1-phosphate. Consistent with this possibility, growth in valproate leads to decreased synthesis of inositol monophosphate. Thus, both lithium and valproate perturb regulation of the inositol biosynthetic pathway, albeit via different mechanisms. This is the first demonstration of increased expression of genes in the inositol biosynthetic pathway by both lithium and valproate. Because inositol is a key regulator of many cellular processes, the effects of lithium and valproate on inositol synthesis have far-reaching implications for predicting genetic determinants of responsiveness and resistance to these agents.Bipolar disorder, or manic-depressive illness, is a common condition with a lifetime prevalence of 1-2% (1). It is characterized by recurring bouts of mania and depression, which have deleterious effects on career and interpersonal relationships. Approximately 15% of those afflicted commit suicide, and mortality rates because of physical disorders are also increased (2, 3). For decades, lithium has been the most effective agent for the treatment of bipolar illness (4). Despite the marked benefit that many patients obtain from lithium therapy, 20 -40% of patients fail to show a satisfactory antimanic response to lithium, and many patients suffer significant morbidity (5). More recently, the branched fatty acid valproate has been used for treatment of bipolar disorder (6). Like lithium, it is not completely effective, and the molecular mechanisms underlying its therapeutic effects have not been elucidated. Lithium and valproate exert a variety of biochemical effects, only some of which are likely to be related to their therapeutic mechanisms of action. Identifying common targets of lithium and valproate is an approach that may more directly address the therapeutic mechanisms underlying their efficacy (7-11).The inositol depletion hypothesis proposes that lithium acts by depletion of inositol from the brain. This is based on the observed uncompetitive inhibition of inositol monophosphatases by lithium, resulting in decreased inositol, an i...

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In a genetic screen for Saccharomyces cerevisiae mutants hypersensitive to the inositol-depleting drugs lithium and valproate, a loss of function allele of TPI1 was identified. The TPI1 gene encodes triose phosphate isomerase, which catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate. A single mutation (N65K) in tpi1 completely abolished Tpi1p enzyme activity and led to a 30-fold increase in the intracellular DHAP concentration. The tpi1 mutant was unable to grow in the absence of inositol and exhibited the "inositol-less death" phenotype. Similarly, the pgk1 mutant, which accumulates DHAP as a result of defective conversion of 3-phosphoglyceroyl phosphate to 3-phosphoglycerate, exhibited inositol auxotrophy. DHAP as well as glyceraldehyde 3-phosphate and oxaloacetate inhibited activity of both yeast and human myo-inositol-3 phosphate synthase, the rate-limiting enzyme in de novo inositol biosynthesis. Implications for the pathology associated with TPI deficiency and responsiveness to inositol-depleting anti-bipolar drugs are discussed. This study is the first to establish a connection between perturbation of glycolysis and inhibition of de novo inositol biosynthesis.Bipolar disorder, also called manic-depressive illness, is a severe psychiatric illness with a prevalence of about 1.5% (1). Lithium and valproate (VPA) 3 are two FDA-approved drugs for the treatment of bipolar disorder. Neither drug is completely effective, but the development of new therapies is hindered by the fact that the mechanisms underlying the therapeutic effects of lithium and VPA are not known. The inositol depletion hypothesis has been proposed to explain the therapeutic effects of lithium in the treatment of bipolar disorder (2). This hypothesis is based on the evidence that inositol monophosphatase is inhibited by therapeutic concentrations of lithium, which can, thus, disrupt the phosphoinositide cycle. More recently, VPA has also been linked to inositol depletion. VPA was found to decrease the concentration of myo-inositol in rat brain after chronic administration (3). Both lithium and VPA cause a decrease in intracellular inositol in yeast (4). Moreover, a recent study showed that lithium, VPA, and carbamazepine, another mood-stabilizing drug, decreased growth cone collapse and increased growth cone area in sensory neurons in culture. These effects were abolished by the addition of inositol (5). These studies suggested that inositol metabolism may be associated with the mechanism of action of lithium and VPA. Surprisingly, very little is known about the molecular control of inositol de novo biosynthesis in human cells.The de novo biosynthesis of inositol has been extensively characterized in the yeast Saccharomyces cerevisiae. Isolation of spontaneous mutants unable to grow in the absence of inositol in S. cerevisiae was first carried out three decades ago (6). Inositol auxotroph mutants undergo inositol-less death, the abrupt decrease in viable cells when deprived of inositol (7). In ...

. We report here that both drugs decrease the relative rate of membrane phosphatidylinositol synthesis and, to a lesser but still significant degree, the steady state relative phosphatidylinositol composition. In addition, both drugs increase the rate of phosphatidylcholine (PC) synthesis. Finally, valproate, but not lithium, increases expression of phosphatidylcholine pathway genes CHO1 and OPI3 . The overall effect on membrane phospholipid composition is a reduction in the phosphatidylinositol/ phosphatidylcholine ratio by both drugs. Because maintenance of the appropriate phosphatidylinositol/ phosphatidylcholine ratio is required for secretory vesicle formation, a decrease in this ratio may have far-reaching implications for understanding the therapeutic mechanisms of action of these drugs.

Myo-inositol exerts behavioral effects in animal models of psychiatric disorders and is effective in clinical trials in psychiatric patients. Interestingly, epi-inositol exerts behavioral effects similar to myo-inositol, even though epi-inositol is not a substrate for synthesis of phosphatidylinositol. We postulated that the behavioral effects of epi-inositol may be due to its effects on gene expression. Yeast INO1 expression was measured in northern blots. INM1 was determined by ␤-galactosidase activity in a strain containing the fusion gene INM1-lacZ integrated into the genome. Epi-inositol affects regulation of expression of the INO1 gene (encoding inositol-1-P synthase), even though it cannot support growth of an inositol auxotroph (suggesting that, as in mammalian cells, it is not incorporated into phosphatidylinositol). Like myo-inositol, although to a lesser extent, epi-inositol causes a significant reduction in INO1 expression, and reverses the lithium-or valproate-induced increase in INO1 expression. However, it does not affect regulation of INM1 (encoding inositol monophosphatase), the expression of which is up-regulated by myo-inositol. The observed regulatory effects of epi-inositol on expression of the most highly regulated gene in the inositol biosynthetic pathway may help to explain how this inositol isomer can exert behavioral effects without being incorporated into phosphatidylinositol.