Molecular effects of lithium are partially mimicked by inositol-monophosphatase (IMPA)1 knockout mice in a brain region-dependent manner.

We have previously shown that homozygote knockout (KO) of inositol-monophosphatase1 (IMPA1) results in lithium (Li)-like behavior. We now aimed to find out whether Li-treated mice and IMPA1 KO mice exhibit neurochemical similarity at the gene- and protein-expression level. Hippocampal and frontal cortex B-cell lymphoma (Bcl-2), Bcl-2-associated X protein (BAX), P53, Perodoxin2 (PRDX2), myristoylated alanine-rich C kinase substrate (MARCKS) and neuropeptide Y (NPY) mRNA levels, and hippocampal, frontal cortex and hypothalamic cytokine levels, all previously reported to be affected by lithium treatment, were measured in three groups of mice: wildtype (WT) on regular-food (RF), WT on Li-supplemented food (Li-treated) and IMPA1-KOs. Hippocampal and frontal cortex Bcl-2 and MARCKS were the only genes commonly affected (downregulated) by Li and IMPA1 KO; Bcl-2 - by 28% and 19%, respectively; MARCKS - by about 20% in both regions. The effect of Li and of IMPA1 KO on cytokine levels differed among the three brain areas studied. Only in the hippocampus both interventions exerted similar effects. Frontal cortex cytokine levels were unaffected neither by Li nor by IMPA1 KO. Similar changes in Bcl-2 and MARCKS but not in PRDX2 and NPY following both Li-treatment and IMPA1 KO suggest a mechanism different than inositol-monophosphatase1 inhibition for Li׳s effect on the latter genes. The cytokine levels results suggest that the mechanism mediating Li׳s effect on the inflammatory system differs among brain regions. Only in the hippocampus the results favor the involvement of the phosphatidylinositol (PI) cycle.

Behavioural phenotyping of sodium-myo-inositol cotransporter heterozygous knockout mice with reduced brain inositol.

Inositol plays a key role in dopamine, serotonin, noradrenaline and acetylcholine neurotransmission, and inositol treatment is reported to have beneficial effects in depression and anxiety. Therefore, a reduction in brain intracellular inositol levels could be a cause of some psychiatric disorders, such as depression or anxiety. To determine the behavioural consequences of inositol depletion, we studied the behaviour of sodium-dependent myo-inositol cotransporter-1 heterozygous knockout mice. In heterozygous mice, free inositol levels were reduced by 15% in the frontal cortex and by 25% in the hippocampus, but they did not differ from their wild-type littermates in cholinergic-mediated lithium-pilocarpine seizures, in the apomorphine-induced stereotypic climbing model of dopaminergic system function, in the Porsolt forced-swimming test model of depression, in amphetamine-induced hyperactivity, or in the elevated plus-maze model of anxiety. Reduction of brain inositol by more than 25% may be required to elicit neurobehavioural effects.

Reduced inositol content in lymphocyte-derived cell lines from bipolar patients.

OBJECTIVES: The study aimed to determine whether low inositol content and uptake previously reported in brain and peripheral tissue of bipolar patients are also reflected in lymphocyte-derived cell lines from these patients. METHODS: Inositol content and uptake were studied in lymphocyte-derived cell lines grown in vitro for at least five generations to eliminate influences of drug treatment. Inositol content was studied gas chromatographically and inositol uptake by following 3H-inositol incorporation at various concentrations. RESULTS: Inositol levels of cell lines derived from bipolar patients were significantly lower than those of cell lines from controls. CONCLUSIONS: Low inositol content in lymphocyte-derived cell lines from bipolar patients corroborates previous findings in frontal cortex and in lymphoblastoid cell lines and are consistent with the notion that the phosphatidylinositol signaling system is involved in the pathophysiology of this disorder.

Low GSK-3 activity in frontal cortex of schizophrenic patients.

Glycogen synthase kinase-3 (GSK-3) (EC 2.7.1.37) is a protein kinase highly abundant in brain and involved in signal transduction cascades of multiple cellular processes, particularly neurodevelopment. Two forms of the enzyme, GSK-3alpha and -3beta have been previously identified. We have previously reported reduced GSK-3beta protein levels in postmortem frontal cortex of schizophrenic patients. In an attempt to explore whether reduction of GSK-3beta levels is brain region specific we examined it in occipital cortex. In order to find out if the reduction in frontal cortex is reflected in altered activity we measured GSK-3 enzymatic activity in this brain region. Western-blot analysis of GSK-3beta was carried out in postmortem occipital cortex of 15 schizophrenic, 15 bipolar, and 15 unipolar patients, and 15 normal controls. GSK-3 activity was measured by quantitating the phosphorylation of the specific substrate phospho-CREB in the frontal cortex specimens. GSK-3beta levels in occipital cortex did not differ between the four diagnostic groups. GSK-3 activity in the frontal cortex of schizophrenic patients was 45% lower than that of normal controls (0.196+/-0.082 and 0.357+/-0.084 pmol/mg proteinxmin, respectively; Kruskal-Wallis analysis: chi-square=8.27, df=3, p=0.04). The other two diagnostic groups showed no difference from the control group. Our results are consistent with the notion that schizophrenia involves neurodevelopmental pathology.

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.

Inositol monophosphatase activity in brain and lymphocyte-derived cell 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.

Scyllo-inositol in post-mortem brain of bipolar, unipolar and schizophrenic patients.

Inositol levels measured in postmortem brain of unipolar, bipolar and schizophrenic patients, suicide victims and normal controls showed no difference in scyllo-inositol levels in frontal or occipital cortex between any of the groups. We could not replicate previous reports of low myo-inositol levels in the frontal cortex of unipolar, bipolar and schizophrenic patients and suicide victims. There was no correlation between myo-inositol levels and estimated chlorpromazine equivalents in neuroleptic-treated subjects, and no effect of chronic haloperidol treatment on rat brain myo-inositol levels.

Low GSK-3beta immunoreactivity in postmortem frontal cortex of schizophrenic patients.

OBJECTIVE: Glycogen synthase kinase-3 (GSK-3) is a protein kinase that is highly abundant in the brain. It is involved in signal transduction cascades of multiple cellular processes, particularly neurodevelopment. In an attempt to explore possible involvement of GSK-3beta in psychiatric disorders, the authors examined its levels in postmortem brain tissue. METHOD: Western blot analysis was performed to measure GSK-3beta in the frontal cortex of 14 schizophrenic patients, 15 patients with bipolar disorder, 15 patients with unipolar depression, and 14 normal comparison subjects. RESULTS: GSK-3beta levels were 41% lower in the schizophrenic patients than in the comparison subjects. Other diagnostic groups did not differ from the comparison subjects. CONCLUSIONS: These results are consistent with the notion that schizophrenia involves neurodevelopmental pathology. It remains to be investigated whether the active fraction of GSK-3beta, or its activity, is also low in frontal cortex of schizophrenic patients and if this is also reflected in other brain regions.

Behavioral reversal of lithium effects by four inositol isomers correlates perfectly with biochemical effects on the PI cycle: depletion by chronic lithium of brain inositol is specific to hypothalamus, and inositol levels may be abnormal in postmortem brain from bipolar patients.

The inositol depletion hypothesis of lithium (Li) action has been criticized, because depletion of inositol after chronic Li treatment has not been reproducible, effects of inositol to reverse Li-induced behaviors occurred also with epi-inositol, a unnatural isomer, and because inositol is ubiquitous in brain and hard to relate to the pathogenesis of affective disorder. Therefore, we review our studies showing that lithium depletion of brain inositol occurs chronically in the hypothalamus, a region not previously examined; that behavioral effects of four different inositol isomers including epi-inositol correlate perfectly with their biochemical effects; and that inositol in postmortem human brain is reduced by 25% in frontal cortex of bipolars and suicides as compared with controls. Because inositol in postmortem brain is reduced and not increased in bipolar patients, the relationship between inositol, lithium, and affective disorder is complex.

Lithium-induced inositol depletion in rat brain after chronic treatment is restricted to the hypothalamus.

The influence of acute and chronic lithium administration on inositol content was examined in five different regions of the rat brain: caudate, cerebellum, cortex, hippocampus and hypothalamus. After acute administration of lithium at doses of 3, 6 or 10 mEq kg-1, no significant reductions of inositol were found in any brain region. Also no significant changes were observed in cortex, caudate, hippocampus and cerebellum after chronic treatment with lithium-containing diet, which led to brain concentrations of lithium in the therapeutic range. However, a moderate but significant reduction of inositol was under these conditions observed in the hypothalamus. At basal conditions, ie in control rats not treated with lithium, the inositol content in various brain areas was different, the hypothalamus containing the highest inositol concentration (4.4 mmol kg-1 wet weight) and the cortex the lowest (2.3 mmol kg-1 wet weight). It is concluded that chronic lithium treatment at therapeutically relevant brain concentrations does not evoke major changes in the inositol content of the brain but induces a moderate decrease which is restricted to the hypothalamus. The results are discussed with respect to the potential function of the hypothalamus in affective disorders.

Reduced frontal cortex inositol levels in postmortem brain of suicide victims and patients with bipolar disorder.

OBJECTIVE: This study aimed to evaluate aspects of second messenger function in the brain of suicide victims and patients with bipolar disorder. METHOD: Inositol and its synthetic enzyme, inositol monophosphatase, were measured in postmortem brain samples of 10 suicide victims, eight patients with bipolar affective disorder, and 10 normal comparison subjects. RESULTS: The frontal cortex inositol levels of the suicide victims and the patients with bipolar disorder were significantly less than those of the normal comparison group. No differences in cerebellum or occipital cortex inositol levels were found among the three groups. The groups also showed no differences in inositol monophosphatase activity in any brain area. CONCLUSIONS: These results could suggest a deficiency of second messenger precursor in patients with bipolar disorder and suicide victims.

Oral maternal inositol supplementation does not increase rat conceptus inositol levels.

Lithium (Li) is an effective drug for prophylaxis and treatment of major affective disorders. It is teratogenic both to animals and human beings. Depletion of inositol is associated both with lithium side effects and teratogenesis. There is no direct evidence showing that in humans Li-teratogenesis is associated with the phosphoinositol (PI) cycle. It is conceivable that the teratogenic effect of Li in humans is also associated with inositol depletion and therefore is amenable to inositol supplementation. To test the hypothesis that oral inositol may cross the placental barrier and may be useful as a protective supplement to lithium therapy during pregnancy, we studied the effect of 2.5% inositol in drinking water on embryonic inositol levels in rats. There was no effect on fetal inositol concentration. However, weight of embryos of mothers receiving inositol was significantly higher. These data do not support the concept that inositol supplementation may be useful in preventing human Li-induced teratogenesis.

CSF inositol in schizophrenia and high-dose inositol treatment of schizophrenia.

Inositol is a key metabolite in the phosphatidylinositol cycle, which is a second messenger system for serotonin-2 receptors that have been implicated in the pathophysiology of schizophrenia. Cerebrospinal fluid inositol levels were measured in 20 schizophrenic patients and 19 age- and sex-matched controls and no difference was found. However, the patients were all neuroleptic-treated. A controlled double-blind crossover trial of 12 g daily of inositol for a month in 12 anergic schizophrenic patients, twice the dose given before in schizophrenia, did not show any beneficial effects. However, the number of patients studied was small and the length of time of inositol administration may not have been sufficient.

High-dose peripheral inositol raises brain inositol levels and reverses behavioral effects of inositol depletion by lithium.

Lithium (Li) reduces brain inositol levels. Berridge has suggested that this effect is related to Li's mechanism of action. It had previously been shown that pilocarpine causes a limbic seizure syndrome in lithium treated rats, and that these lithium-pilocarpine seizures are reversible by intracerebroventricular inositol administration to rats. We now show that although inositol passes the blood-brain barrier poorly, large doses of intraperitoneal (IP) inositol can also reverse Li-pilocarpine seizures. Using gas chromatography, IP inositol can raise brain inositol levels. Demonstration that inositol enters brain after peripheral administration provides a basis for possible pharmacological intervention in psychiatric disorders at the level of second messengers linked to the phosphatidylinositol cycle.

Erythrocytes with covalently bound fibrinogen as a cellular replacement for the treatment of thrombocytopenia.

Thrombocytopenia in general, and autoimmune thrombocytopenia in particular, is a disease of high prevalence with a non-satisfactory regime of treatment. The present study aimed to explore the feasibility of an alternative treatment, based on the rationale that autologous erythrocytes modified to bear covalently bound fibrinogen would participate passively in the aggregation of the remaining platelets, thus augmenting the haemostatic needs, while resisting the autoimmune reaction directed towards the platelets. Several procedures for the cross-linking of fibrinogen to red blood cells (RBCs) were tested. Formaldehyde (33 microM) for 10 min at 23 degrees C attached 58 fibrinogen molecules per erythrocyte. These erythrocytes were indistinguishable from untreated erythrocytes in the following properties: osmotic fragility, bound haemoglobin, sedimentation rate, acetylcholinesterase activity, phagocytosis by macrophages, rosette formation with K562 cells. It is shown that RBCs cross-linked with fibrinogen are capable of participating in the in vitro aggregation of platelets and are indeed effective in the in vivo process of arrest of bleeding in an animal model of autoimmune thrombocytopenia.

Role of inositol-1-phosphatase inhibition in the mechanism of action of lithium.

Li inhibition of noradrenergic adenylate cyclase may be due to inhibition by Li of agonist-induced increases in GTP binding to G-protein. Such inhibition by Li of G-protein function could have effects on phosphatidyl-inositol-mediated second messenger systems as well as on cyclic AMP-mediated systems. However, Sherman, Berridge and others have proposed that Li affects phosphatidylinositol metabolism by inhibiting inositol-1-phosphatase. We recently have been able to measure inositol-1-phosphatase in human red blood cells. Preliminary data on patients treated with Li compared with controls suggests that the enzyme is indeed inhibited in vivo in patients undergoing Li treatment. However, a series of experiments in rats on addition of inositol to Li treatment did not find that inositol could reverse Li effects. Chronic oral high dose inositol does not reverse Li-induced polyuria (measured by polydipsia), Li-induced weight loss or Li-induced depression of exploratory behavior. These results suggest that Li inhibition of inositol-1-phosphatase indeed occurs in vivo. However, the physiological significance of inositol-1-phosphatase inhibition is not yet established.