Alon Shamir

Valproate decreases inositol biosynthesis.

BACKGROUND Lithium and valproate (VPA) are used for treating bipolar disorder. The mechanism of mood stabilization has not been elucidated, but the role of inositol has gained substantial support. Lithium inhibition of inositol monophosphatase, an enzyme required for inositol recycling and de novo synthesis, suggested the hypothesis that lithium depletes brain inositol and attenuates phosphoinositide signaling. Valproate also depletes inositol in yeast, Dictyostelium, and rat neurons. This raised the possibility that the effect is the result of myo-inositol-1-phosphate (MIP) synthase inhibition. METHODS Inositol was measured by gas chromatography. Human prefrontal cortex MIP synthase activity was assayed in crude homogenate. INO1 was assessed by Northern blotting. Growth cones morphology was evaluated in cultured rat neurons. RESULTS We found a 20% in vivo reduction of inositol in mouse frontal cortex after acute VPA administration. As hypothesized, inositol reduction resulted from decreased MIP synthase activity: .21-.28 mmol/LVPA reduced the activity by 50%. Among psychotropic drugs, the effect is specific to VPA. Accordingly, only VPA upregulates the yeast INO1 gene coding for MIP synthase. The VPA derivative N-methyl-2,2,3,3,-tetramethyl-cyclopropane carboxamide reduces MIP synthase activity and has an affect similar to that of VPA on rat neurons, whereas another VPA derivative, valpromide, poorly affects the activity and has no affect on neurons. CONCLUSIONS The rate-limiting step of inositol biosynthesis, catalyzed by MIP synthase, is inhibited by VPA; inositol depletion is a first event shown to be common to lithium and VPA.

Human 1-D-myo-Inositol-3-phosphate Synthase Is Functional in Yeast

We have cloned, sequenced, and expressed a human cDNA encoding 1-d-myo-inositol-3-phosphate (MIP) synthase (hINO1). The encoded 62-kDa human enzyme converted d-glucose 6-phosphate to 1-d-myo-inositol 3-phosphate, the rate-limiting step for de novo inositol biosynthesis. Activity of the recombinant human MIP synthase purified from Escherichia coli was optimal at pH 8.0 at 37 °C and exhibited Km values of 0.57 mm and 8 μm for glucose 6-phosphate and NAD+, respectively. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{+}\) \end{document} and K+ were better activators than other cations tested (Na+, Li+, Mg2+, Mn2+), and Zn2+ strongly inhibited activity. Expression of the protein in the yeast ino1Δ mutant lacking MIP synthase (ino1Δ/hINO1) complemented the inositol auxotrophy of the mutant and led to inositol excretion. MIP synthase activity and intracellular inositol were decreased about 35 and 25%, respectively, when ino1Δ/hINO1 was grown in the presence of a therapeutically relevant concentration of the anti-bipolar drug valproate (0.6 mm). However, in vitro activity of purified MIP synthase was not inhibited by valproate at this concentration, suggesting that inhibition by the drug is indirect. Because inositol metabolism may play a key role in the etiology and treatment of bipolar illness, functional conservation of the key enzyme in inositol biosynthesis underscores the power of the yeast model in studies of this disorder.

IMPA1 is Essential for Embryonic Development and Lithium-Like Pilocarpine Sensitivity

Lithium has been the standard pharmacological treatment for bipolar disorder over the last 50 years; however, the molecular targets through which lithium exerts its therapeutic effects are still not defined. We characterized the phenotype of mice with a dysfunctional IMPA1 gene (IMPA1−/−) to study the in vivo physiological functions of IMPA1, in general, and more specifically its potential role as a molecular target in mediating lithium-dependent physiological effects. Homozygote IMPA1−/− mice died in utero between days 9.5 and 10.5 post coitum (p.c.) demonstrating the importance of IMPA1 in early embryonic development. Intriguingly, the embryonic lethality could be reversed by myo-inositol supplementation via the pregnant mothers. In brains of adult IMPA1−/− mice, IMPase activity levels were found to be reduced (up to 65% in hippocampus); however, inositol levels were not found to be altered. Behavioral analysis of the IMPA1−/− mice indicated an increased motor activity in both the open-field test and the forced-swim test as well as a strongly increased sensitivity to pilocarpine-induced seizures, the latter supporting the idea that IMPA1 represents a physiologically relevant target for lithium. In conclusion the IMPA1−/− mouse represents a novel model to study inositol homeostasis, and indicates that genetic inactivation of IMPA1 can mimic some actions of lithium.

Mitochondrial DNA HV lineage increases the susceptibility to schizophrenia among Israeli Arabs

Haplotypes and haplogroups are linked sets of common DNA variants, acting as susceptibility or protective factors to complex disorders. Growing evidence suggests that dysfunction of mitochondrial bioenergetics contributes to the schizophrenia phenotype. We studied mitochondrial DNA haplogroups in schizophrenia patients. Since mitochondria are inherited from the mothers, we used healthy fathers as an ideal case-control group. Analysis of the distribution of mitochondrial haplogroups in schizophrenia patients compared to their healthy fathers (202 pairs) resulted in an over-representation of the mtDNA lineage cluster, HV, in the patients (p=0.01), with increased relative risk (odds ratio) of 1.8. Since mitochondrial DNA is small relative to nuclear DNA, a total mitochondrial genome analysis was possible in a hypothesis-free manner. However, mitochondrial DNA haplogroups are highly variable in human population and it will be necessary to replicate our results in other human ethnic groups.

The effect of lithium on expression of genes for inositol biosynthetic enzymes in mouse hippocampus; a comparison with the yeast model

In the de novo synthesis of inositol, the conversion of D-glucose-6-phosphate to L-myo-inositol-1-phosphate (MIP) is catalyzed by MIP synthase. Little is known about mammalian MIP synthase and nothing is known about its regulation. The second step in inositol biosynthesis is the conversion of MIP to inositol by inositol-monophosphatase (IMPase), a common step to inositol production via the de novo pathway and its recycling from inositol phosphates. Because lithium inhibits IMPase both in yeast and in mammals, and the drug upregulates yeast MIP synthase (INO1) and downregulates IMPase (INM1), the present study was undertaken to determine whether chronic in vivo therapeutic lithium concentrations affect MIP synthase and IMPase expression in mouse frontal cortex and hippocampus. Mice were treated with food containing LiCl (2.5 g/kg) for 10 days. RNA was purified from the brain areas and mRNA amplified using RT-PCR. Expression of MIP synthase and IMPA1 (one of the genes coding for IMPase) but not IMPA2 was upregulated in mouse hippocampus. None of the genes were affected in the frontal cortex. In yeast, when inositol is limiting, the heterodimeric transcriptional activator Ino2p/Ino4p derepresses expression of INO1 by binding to the upstream activation sequence UAS(INO). Using the TFSEARCH program, we found that the promoter of the virtual human MIP synthase gene contains upstream stimulating factor (USF) elements with a similar core binding sequence. The fact that lithium treatment upregulates both MIP synthase and IMPA1 mRNA levels in mouse hippocampus may reflect a compensatory response of both genes to inositol depletion.

Lack of Lithium-Like Behavioral and Molecular Effects in IMPA2 Knockout Mice

Lithium is a potent mood-stabilizing medication in bipolar disorder. Despite 50 years of clinical use, the mechanism of action is unknown. Multiple effects have been attributed to lithium including the uncompetitive inhibition of inositol monophosphatase (IMPase). IMPA2, one of the genes that encode IMPase, is located in a region with linkage to bipolar disorder. Owing to the role of IMPase in cell signaling and the possibility that this enzyme is a target for mood-stabilizing drugs, we generated IMPA2−/− mice. Possible involvement of IMPase in complex behaviors related to affective disorders was assessed by monitoring the behavior of the IMPA2−/− mice in the forced swim test, the tail suspension test (TST), the elevated zero-maze and open field test. It has been described that chronically lithium-treated mice exhibit reduced immobility time in the forced swim test and decreased exploratory behavior. We found increased rearing of IMPA2−/− mice in the open field, suggesting an increased exploratory behavior. Although immobility time of IMPA2−/− female but not male mice in the forced swim test was reduced, no difference was found between male and female IMPA2−/− and IMPA2+/+ mice in the TST and overall there was no clear effect of the deletion of IMPA2 on depression-like behavior. Frontal cortex IMPase activity and inositol levels in the IMPA2−/− mice did not differ from IMPA2+/+ mice, but kidney inositol levels were reduced. In conclusion, phenotypic characterization of the IMPA2−/− mouse indicates that deleting IMPA2 does not mimic the effects of lithium treatment.

The Chromatin-binding Protein HMGN1 Regulates the Expression of Methyl CpG-binding Protein 2 (MECP2) and Affects the Behavior of Mice

Background: HMGN1 is a chromatin-binding protein that modulates the cellular transcription profile. Results: Mice that either overexpress or lack HMGN1 have altered behavioral phenotypes. HMGN1 is a negative regulator of MeCP2, a protein involved in neurodevelopmental disorders. Conclusion: Misregulation of HMGN1 protein levels lead to behavioral changes in mice. Significance: HMGN1 is an epigenetic factor that contributes to the development of neurodevelopmental disorders. High mobility group N1 protein (HMGN1), a nucleosomal-binding protein that affects the structure and function of chromatin, is encoded by a gene located on chromosome 21 and is overexpressed in Down syndrome, one of the most prevalent genomic disorders. Misexpression of HMGN1 affects the cellular transcription profile; however, the biological function of this protein is still not fully understood. We report that HMGN1 modulates the expression of methyl CpG-binding protein 2 (MeCP2), a DNA-binding protein known to affect neurological functions including autism spectrum disorders, and whose alterations in HMGN1 levels affect the behavior of mice. Quantitative PCR and Western analyses of cell lines and brain tissues from mice that either overexpress or lack HMGN1 indicate that HMGN1 is a negative regulator of MeCP2 expression. Alterations in HMGN1 levels lead to changes in chromatin structure and histone modifications in the MeCP2 promoter. Behavior analyses by open field test, elevated plus maze, Reciprocal Social Interaction, and automated sociability test link changes in HMGN1 levels to abnormalities in activity and anxiety and to social deficits in mice. Targeted analysis of the Autism Genetic Resource Exchange genotype collection reveals a non-random distribution of genotypes within 500 kbp of HMGN1 in a region affecting its expression in families predisposed to autism spectrum disorders. Our results reveal that HMGN1 affects the behavior of mice and suggest that epigenetic changes resulting from altered HMGN1 levels could play a role in the etiology of neurodevelopmental disorders.

Inositol Monophosphatase Activity in Brain and Lymphocyte-Derived Ceii Lines of Bipolar Patients

Background: Inositol monophosphatase (IMPase) activity was reported to be low in lymphocyte-derived cell lines of bipolar patients. Methods: IMPase activity was measured spectrophotometrically as inorganic phosphate liberated from inositol-1-phosphate. Results: The previously reported reduction was replicated in a new, small group of bipolar patients. The reduction is not present in cell lines of unipolar or schizophrenic patients. IMPase activity in postmortem frontal and occipital cortical samples of unipolar, bipolar and schizophrenic patients was not different from controls. Conclusions: A reduction in lymphocyte-derived IMPase activity without a parallel reduction in cortical IMPase activity could be due to the fact that most leukocyte IMPase activity is the product of the IMPA-2 gene.

Characterization of two genes, Impa1 and Impa2 encoding mouse myo-inositol monophosphatases.

The enzyme myo-inositol monophosphatase (Impa) catalyzes the synthesis of free myo-inositol from various myo-inositol monophosphates in the phosphatidylinositol signaling system. Impa is a lithium-blockable enzyme that has been hypothesized to be the biological target for lithium-salts used as mood-stabilizing drugs in the treatment of manic-depressive (bipolar) illness. As an initial step to explore the functional consequences of reduced or absent Impa activity in an animal model we here report the isolation of two Impa-encoding mouse genes, Impa1 and Impa2. Impa1 spans approximately 17.5 kb and contains nine exons of 46--1354 bp encoding a protein of 277 amino acids. Impa2 spans at least 19.5 kb and contains eight exons of 46--444 bp size encoding a protein of 290 amino acids. The genomic structure including the positions of the exon-intron splice sites seems to be conserved among myo-inositol monophosphatase genes in mammalian species. One or more Impa-like genes do also exist in evolutionary more distant species like invertebrates, plants and bacteria. The proteins encoded by the non-vertebrate genes seem to be equally related to Impa1 and Impa2. We therefore suggest that the Impa1 and Impa2 genes duplicated from a common ancestral gene after the evolutionary divergence of vertebrates.

Only tryptophan hydroxylase (TPH)-2 is relevant to the CNS

In the July issue of this journal Sekizawa et al. [2004] reported a relationship between the tryptophan hydroxylae (TPH) gene polymorphism and susceptibility to childhood onset schizophrenia in a Japanese cohort. The report, related to the TPH1 gene, adds to others showing an association of polymorphic forms of TPH1 with schizophrenia, unipolar depression, bipolar disorder, alcoholism, drug abuse, and suicidal behavior [Arango et al., 2003; Rujescu et al., 2003]. Astonishingly, the authors ignore the recently discovered additional mammalian TPH gene, TPH2, [Walther et al., 2003] although it is now well established that TPH2 is predominantly expressed in the brain, both in serotonergic regions [Zill et al., 2004a] and in other areas, including regions considered to be involved in the etiology of psychiatric disorders [Walther and Bader, 2003; Zill et al., 2004b], while TPH1 is expressed in peripheral tissues [Walther and Bader, 2003] which is not accepted as likely to be involved in psychiatric disorders. TPH2, located on the long arm of human chromosome 12 that has been reported as a probable susceptibility region for affective disordersrelated genes [Abkevich et al., 2003], has recently been reported to be in association with affective disorders [Harvey et al., 2004; Zill et al., 2004a], suggesting the involvement of TPH2 in these illnesses. We believe that these novel findings justify a new concept of TPH2 rather than TPH1 as a candidate gene for 5-HT-related affective disorders as well as re-evaluation of the interpretation of the association studies dealing with TPH1 and psychiatric illnesses.