Mood stabilizers target cellular plasticity and resilience cascades

Bipolar disorder is a devastating disease with a lifetime incidence of about 1% in the general population. Suicide is the cause of death in 10 to 15% of patients and in addition to suicide, mood disorders are associated with many other harmful health effects. Mood stabilizers are medications used to treat bipolar disorder. In addition to their therapeutic effects for the treatment of acute manic episodes, mood stabilizers are useful as prophylaxis against future episodes and as adjunctive antidepressant medications. The most established and investigated mood-stabilizing drugs are lithium and valproate but other anticonvulsants (such as carbamazepine and lamotrigine) and antipsychotics are also considered as mood stabilizers. Despite the efficacy of these diverse medications, their mechanisms of action remain, to a great extent, unknown. Lithium’s inhibition of some enzymes, such as inositol monophosphatase and gycogen synthase kinase-3, probably results in its mood-stabilizing effects. Valproate may share its anticonvulsant target with its mood-stabilizing target or may act through other mechanisms. It has been shown that lithium, valproate, and/or carbamazepine regulate numerous factors involved in cell survival pathways, including cyclic adenine monophospate response element-binding protein, brain-derived neurotrophic factor, bcl-2, and mitogen-activated protein kinases. These drugs have been suggested to have neurotrophic and neuroprotective properties that ameliorate impairments of cellular plasticity and resilience underlying the pathophysiology of mood disorders. This article also discusses approaches to develop novel treatments specifically for bipolar disorder.     

Lithium Modulates Cancer Cell Growth, Apoptosis, Gene Expression and Cytokine Production in HL-60 Promyelocytic Leukaemia Cells and Their Drug-Resistant Sub-clones

Lithium has been an FDA-approved and preferred drug for the treatment of mood disorders for many years, and cumulative evidence has pointed towards its potential use as an anti-cancer agent. Previous studies in our laboratory have demonstrated that lithium induces apoptotic cell death in HL-60 promyelocytes at concentrations of 10?mM and above. A lithium-tolerant HL-60 sub-clone, resistant to up to 15?mM lithium, was also generated and its growth profile reported. Treatment of cells with lithium resulted in a dose-dependent induction of p53, retinoblastoma (Rb) and bax expression which was accompanied by concomitant inhibition of bcl-2 expression as demonstrated using immunohistochemical microscopy. These results seem to suggest that lithium induced cell death in these cells by inhibiting expression of anti-apoptotic protein, bcl-2, while inducing higher expression of its pro-apoptotic counterparts which include bax. Expression of bax and bcl-2 is also linked to expression of inflammation-regulating cytokines. Using ELISA assays, lithium was demonstrated to induce production of pro-inflammatory cytokines, IL-6 and TNF-??, while inhibiting release of anti-inflammation-related IL-2 and IL-10 in a dose-dependent fashion. Our findings identify a critical function for lithium in modulating pro- versus anti-apoptotic gene expression and pro- versus anti-inflammatory cytokines in vitro and provide a rationale for suggesting a promising role of lithium in regulation of inflammation and cancer growth.     

The Role of Glial Adenosine Receptors in Neural Resilience and the Neurobiology of Mood Disorders

Adenosine receptors were classified into A₁- and A₂-receptors in the laboratory of Bernd Hamprecht more than 25 years ago. Adenosine receptors are instrumental to the neurotrophic effects of glia cells. Both microglia and astrocytes release after stimulation via adenosine receptors factors that are important for neuronal survival and growth. Neuronal resilience is now considered as of pivotal importance in the neurobiology of mood disorders and their treatment. Both sleep deprivation and electroconvulsive therapy, two effective therapeutic measures in mood disorders, are associated with an increase of adenosine and upregulation of adenosine A₁-receptors in the brain. Parameters closely related to adenosine receptor activation such as cerebral metabolic rate and delta power in the sleep EEG provide indirect evidence that adenosinergic signaling may be associated with the therapeutic response to these measures. Thus, neurotrophic effects evoked by adenosine receptors might be important in the mechanism of action of ECT and perhaps also sleep deprivation.     

Lithium increases expression of p21WAF/Cip1 and survivin in human glioblastoma cells

Lithium is the most widely prescribed mood stabilizer, but the precise molecular mechanisms underlying its therapeutic function are not yet fully elucidated. Recent preclinical and clinical evidence indicates its neuroprotective and neurotrophic effects. As a tight coupling of function and metabolism in the central nervous system between glial cells and neurons has recently been detected, lithium's effect on glial cells may participate also in the total beneficial effects of this drug. The aim of the present study was to analyze molecular mechanisms induced in human glioblastoma A1235 cells by the treatment with lithium, especially its influence on the expression of apoptosis-related genes. Lower levels of lithium (0.5 mmol/L and 2 mmol/L) did not cause any cytotoxicity or changes in the cell cycle phase distribution following 72 h incubation. However, a higher dose (20 mmol/L) was cytostatic for glioblastoma cells, and caused accumulation of cells in G₂/M phase of the cell cycle. The treatment with lithium did not alter the levels of Bcl-2 or procaspase-3 and did not cleave PARP, but increased the levels of p21^WAF/Cip1 and survivin. Thus, increased expression of p21^WAF/Cip1 (a protein with antiapoptotic function), and survivin (a protein that supports the growth of cells by suppression of apoptosis and promotion of cell proliferation) may be the early events in the long-term cell response to lithium that are involved in the beneficial effects of this drug.     

Lithium, a common drug for bipolar disorder treatment, regulates amyloid-beta precursor protein processing

Lithium is one of the most widely used mood-stabilizing agents for the treatment of bipolar disorder. Although the underlying mechanism(s) of this mood stabilizer remains controversial, recent evidence linking lithium to neurotrophic/neuroprotective effects (Choi and Sung (2000) 1475, 225-230; Davies et al. (2000) 351, 95-105) suggests novel benefits of this drug in addition to mood stabilization. Here, we report that both lithium as well as valproic acid (VPA) inhibit beta-amyloid peptide (Abeta) production in HEK293 cells stably transfected with Swedish amyloid precursor protein (APP)(751) and in the brains of the PDAPP (APP(V717F)) Alzheimer's disease transgenic mouse model at clinically relevant plasma concentrations. Both lithium and VPA are known to be glycogen synthase kinase-3 (GSK3) inhibitors. Our studies reveal that GSK3beta is a potential downstream kinase, which modulates APP processing because inhibition of GSK3 activity by either a dominant negative GSK3beta kinase-deficient construct or GSK3beta antisense oligonucleotide mimics lithium and VPA effects. Moreover, lithium treatment abolished GSK3beta-mediated Abeta increase in the brains of GSK3beta transgenics and reduced plaque burden in the brains of the PDAPP (APP(V717F)) transgenic mice.     

Mood stabilizing drug lithium increases expression of endoplasmic reticulum stress proteins in primary cultured rat cerebral cortical cells

The mood stabilizing drug lithium is a highly effective treatment for bipolar disorder. Previous studies in our laboratory found that chronic treatment with the mood stabilizing drug valproate in rat brain increased the expression of endoplasmic reticulum (ER) stress proteins GRP78, GRP94 and calreticulin. We report here that in primary cultured rat cerebral cortical cells, expression of GRP78, GRP94 and calreticulin are increased not only by valproate, but also by lithium after chronic treatment for 1 week at therapeutically relevant concentrations. However, two other mood stabilizing drugs carbamazepine and lamotrigine had no effect on expression of GRP78, GRP94 or calreticulin. Chronic treatment with lithium for 1 week increased both mRNA and protein levels of ER stress proteins. In contrast to a classic GRP78 inducer thapsigargin, an inhibitor of the ER Ca2+ -ATPase, chronic treatment with lithium or valproate for 1 week modestly increased GRP78 expression in neuronal cells, had no effect on basal intracellular free Ca2+ concentration and does not induce cell death. These results indicate that lithium and valproate may increase expression of GRP78, GRP94 and calreticulin in primary cultured rat cerebral cortical cells without causing cell damage. These results also suggest that the mechanism of GRP78 increase induced by lithium and valproate may be different from that of thapsigargin.     

Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells

Protein phosphorylation plays an indispensable role in cellular regulation of mitosis, metabolism, differentiation, and death. We previously reported that the protein phosphatase inhibitor okadaic acid (OKA) induces apoptosis in renal epithelial cells in culture. In the present study, we examined the role of phosphotidylinositol 3 (PI3) kinase signaling in okadaic acid-induced apoptosis by pre-treating normal rat kidney renal epithelial cells expressing human bcl-2 with the PI3 kinase inhibitors, LY294002 and wortmannin, followed by apoptosis-inducing concentrations of okadaic acid. Given the reported cell survival activity of PI3 kinase signaling mostly attributed to Akt kinase activation, we hypothesized that inhibition of PI3 kinase would enhance okadaic-induced apoptosis. Surprisingly, our data show that pretreatment with LY294002, but not wortmannin, attenuated okadaic acid-induced apoptosis. In contrast, to LY294002, wortmannin enhanced apoptosis. Interestingly, we also found that LY294002 treatment increased bcl-2 protein levels in normal rat kidney epithelial cells expressing bcl-2 (NRK-bcl-2). In untreated cells, bcl-2 appeared to be mainly perinuclear, coincident with the nuclear membrane, or in the cytosol. In OKA treated cells that were pre-treated with Ly294002, bcl-2 was highly co-localized with mitochondria, but in cells treated with okadaic acid alone, bcl-2 was associated with fragmented chromatin. In this model, it appears that LY294002 may exert anti-apoptotic effects by a previously unreported treatment related increase in bcl-2. Although it is widely accepted that bcl-2 protein can inhibit apoptosis, we propose that the subcellular location of bcl-2 is an important determinant in whether bcl-2 effectively inhibits apoptosis.     

Bcl-2 overexpression decreases BCNU sensitivity of a human glioblastoma line through enhancement of catalase activity

The aim of this study was to evaluate the role of bcl-2 in 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sensitivity of the ADFS human glioblastoma cell line in vitro and in vivo. To this end, the ADFS line expressing a low level of the bcl-2 protein was transfected with a bcl-2 expression vector. We found that bcl-2 overexpressing clones were less sensitive to in vitro BCNU treatment than the control clone. Cell cycle analysis demonstrated that while BCNU induced a consistent block in S/G2-M phases of the cell cycle in the control clone, it did not affect the cell cycle phase distribution of the two bcl-2 transfectants. The different sensitivity to BCNU was unrelated to the ability of bcl-2 to inhibit apoptosis, while bcl-2 appeared to protect bcl-2 transfectants from BCNU toxicity through an increase of catalase activity. The ability of the catalase inhibitor, sodium azide, to increase the BCNU sensitivity of the bcl-2 transfectants to levels of the BCNU-treated control clone substantiated the role of the catalase activity. The effect of bcl-2 in reducing sensitivity to BCNU was also confirmed by in vivo experiments. Xenografts of bcl-2 overexpressing tumors were less sensitive to BCNU treatment than xenografts originating from control cells.     

Lithium enhances the neuronal differentiation of neural progenitor cells in vitro and after transplantation into the avulsed ventral horn of adult rats through the secretion of brain-derived neurotrophic factor

This study was undertaken to elucidate the molecular mechanisms by which lithium regulates the development of spinal cord-derived neural progenitor cells (NPCs) in vitro and after transplanted in vivo . Our results show that lithium at the therapeutic concentration significantly increases the proliferation and neuronal differentiation of NPCs in vitro. Specific ELISAs, western blotting, and quantitative real-time RT-PCR assays demonstrate that lithium treatment significantly elevates the expression and production of brain-derived neurotrophic factor (BDNF) by NPCs in culture. Application of a BDNF neutralizing antibody in culture leads to a marked reduction in the neurogenesis of lithium-treated NPCs to the control level. However, it shows no effects on the proliferation of lithium-treated NPCs. These findings suggest that the BDNF pathway is possibly involved in the supportive role of lithium in inducing NPC neurogenesis but not proliferation. This study also provides evidence that lithium is able to elevate the neuronal generation and BDNF production of NPCs after transplantation into the adult rat ventral horn with motoneuron degeneration because of spinal root avulsion, which highlights the therapeutic potential of lithium in cell replacement strategies for spinal cord injury because of its ability to promote neuronal differentiation and BDNF production of grafted NPCs in the injured spinal cord.     

Lithium selectively increases neuronal differentiation of hippocampal neural progenitor cells both in vitro and in vivo

Lithium has been demonstrated to increase neurogenesis in the dentate gyrus of rodent hippocampus. The present study was undertaken to investigate the effects of lithium on the proliferation and differentiation of rat neural progenitor cells in hippocampus both in vitro and in vivo. Lithium chloride (1-3 mM) produced a significant increase in the number of bromodeoxyuridine (BrdU)-positive cells in high-density cultures, but did not increase clonal size in low-density cultures. Lithium chloride at 1 mM (within the therapeutic range) also increased the number of cells double-labeled with BrdU antibody and TuJ1 (a class III beta-tubulin antibody) in high-density cultures and the number of TuJ1-positive cells in a clone of low-density cultures, whereas it decreased the number of glial fibrillary acidic protein-positive cells in both cultures. These results suggest that lithium selectively increased differentiation of neuronal progenitors. These actions of lithium appeared to enhance a neuronal subtype, calbindin(D28k)-positive cells, and involved a phosphorylated extracellular signal-regulated kinase and phosphorylated cyclic AMP response element-binding protein-dependent pathway both in vitro and in vivo. These findings suggest that lithium in therapeutic amounts may elicit its beneficial effects via facilitation of neural progenitor differentiation toward a calbindin(D28k)-positive neuronal cell type.     

Effects of coumarin and 7OH-coumarin on bcl-2 and Bax expression in two human lung cancer cell lines in vitro

Coumarin and its derivative 7-hydroxycoumarin (7-OHC) have antitumor and antimetastatic properties. The purpose of this study was to investigate the possible effects of these compounds on expression of the bcl-2 and Bax oncoproteins in two human lung cancer cell lines, A427 and Calu-1. The cells were cultured in vitro for 24 h in RPMI 1640 with 1.5% (v/v) ethanol, 1.0 mM ethanolic coumarin or 1.0 mM ethanolic 7-OHC. Viability was determined in each cell line by an MTT assay. Total protein was extracted from cell lysates and the bcl-2 and Bax oncoproteins were identified. Western blotting showed a decrease in bcl-2 and an increase in Bax in A427 cells cultured with coumarin or 7-OHC. Neither drug changed bcl-2 expression in Calu-1 cells compared to solvent controls, and Bax expression was only slightly increased by coumarin. We conclude that 7-OHC is a more potent inhibitor of cell proliferation than coumarin and has more marked effects on oncoprotein expression. Also, the A427 cell line was more sensitive to the drugs than Calu-1.     

Bcl-2 Protects Neural Cells from Cyanide/Aglycemia-Induced Lipid Oxidation, Mitochondrial Injury, and Loss of Viability

Abstract: The protooncogene bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that the mechanism of action of Bcl-2 involves antioxidant activity. The involvement of free radicals in ischemia/reperfusion injury to neural cells has led us to investigate the effect of Bcl-2 in a model of delayed neural cell death. We have examined the survival of control and bcl-2 transfectants of a hypothalamic tumor cell line, GT1-7, exposed to potassium cyanide in the absence of glucose (chemical hypoxia/aglycemia). After 30 min of treatment, no loss of viability was evident in control or bcl-2 transfectants; however, Bcl-2-expressing cells were protected from delayed cell death measured following 24–72 h of reoxygenation. Under these conditions, the rate and extent of ATP depletion in response to treatment with cyanide in the absence of glucose and the rate of recovery of ATP during reenergization were similar in control and Bcl-2-expressing cells. Bcl-2-expressing cells were protected from oxidative damage resulting from this treatment, as indicated by significantly lower levels of oxidized lipids. Mitochondrial respiration in control but not Bcl-2-expressing cells was compromised immediately following hypoxic treatment. These results indicate that Bcl-2 can protect neural cells from delayed death resulting from chemical hypoxia and reenergization, and may do so by an antioxidant mechanism. The results thereby provide evidence that Bcl-2 or a Bcl-2 mimetic has potential therapeutic application in the treatment of neuropathologies involving oxidative stress, including focal and global cerebral ischemia.     

Variable functions of bcl-2 in mediating bioreactor stress- induced apoptosis in hybridoma cells

It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2 transfected cell line exhibited a nearly five fold increase in viable cell number. This finding indicates that under apoptosis-suppressed conditions, shear stress can stimulate cell growth. Batch cultivation of both control and bcl-2 transfected cells in 350 and 400 mOsm media resulted in suppression of cell growth, athough the effect was most marked in the control cell line. Adaptation of control cells to 400 mOsm proved to be impossible to achieve. However, the bcl-2 transfected cells exhibited resistance to the osmotic stress resulting in long term adaptation to a high salt environment. Specific productivity of bcl-2 transfected cells grown in high osmolarity medium was 100% higher than that produced by non- adapted bcl-2 transfected cells grown in normal osmolarity medium. These results demonstrate that bcl-2 has a beneficial effect on hybridoma cultivation under a wide range of culture stresses. This revised version was published online in August 2006 with corrections to the Cover Date.     

The interaction between dopamine D2-like and beta-adrenergic receptors in the prefrontal cortex is altered by mood-stabilizing agents

Several studies have suggested the involvement of biogenic monoaminergic neurotransmission in bipolar disorder and in the therapy for this disease. In this study, the effects of the mood-stabilizing drugs lithium, carbamazepine or valproate on the dopaminergic and adrenergic systems, particularly on D2-like and beta-adrenergic receptors, were studied both in cultured rat cortical neurones and in rat prefrontal cortex. In vitro and in vivo data showed that stimulation of beta-adrenergic receptors with isoproterenol increased cyclic adenosine monophosphate (cAMP) levels and this effect was significantly inhibited by lithium, carbamazepine or valproate. The activation of dopamine D2-like receptors with quinpirole decreased the isoproterenol-induced rise in cAMP in control conditions. This inhibition was observed in vivo after chronic treatment of the rats with carbamazepine or valproate, but not after treatment with lithium or in cultured rat cortical neurones after 48 h exposure to the three mood stabilizers. Dopamine D2 and beta1-adrenergic receptors were found to be co-localized in prefrontal cortical cells, as determined by immunohistochemistry, but western blot experiments revealed that receptor levels were differentially affected by treatment with the three mood stabilizers. These data show that mood stabilizers affect D2 receptor-mediated regulation of beta-adrenergic signalling and that each drug acts by a unique mechanism.     

lpr T cells have lower ability to maintain bcl-2 expression ex vivo

Autoimmune-prone lpr mice develop lymphoproliferative disorders, whereas their lymphocytes show accelerated apoptosis in culture. To elucidate whether the bcl-2 protein, a repressor of apoptosis, is critical to the discrepancy between in vivo and in vitro survival, we examined bcl-2 expression in T cells from +/+ and lpr mice during culture. The expression levels of bcl-2 in cultured T cells from lpr mice were significantly down-modulated compared to those from +/+ mice and freshly obtained T cells. Besides, the reduction of bcl-2 protein levels was inhibited in T cells cultured in the presence of T cell receptor (TCR) signalling. These results suggest that lpr T cells might be susceptible to apoptosis in vitro due to down-modulation of bcl-2 by withdrawal of TCR signalling.     

Shift of the Cellular Oxidation-Reduction Potential in Neural Cells Expressing Bcl-2

Abstract: Expression of the protooncogene bcl-2 inhibits both apoptotic and in some cases necrotic cell death in many cell types, including neural cells, and in response to a wide variety of inducers. The mechanism by which the Bcl-2 protein acts to prevent cell death remains elusive. One mechanism by which Bcl-2 has been proposed to act is by decreasing the net cellular generation of reactive oxygen species. To evaluate this proposal, we measured activities of antioxidant enzymes as well as levels of glutathione and pyridine nucleotides in control and bcl-2 transfectants in two different neural cell lines—rat pheochromocytoma PC12 and the hypothalamic GnRH cell line GT1-7. Both neural cell lines overexpressing bcl-2 had elevated total glutathione levels when compared with control transfectants. The ratios of oxidized glutathione to total glutathione in PC12 and GT1-7 cells overexpressing bcl-2 were significantly reduced. In addition, the NAD⁺/NADH ratio of bcl-2 -expressing PC12 and GT1-7 cells was two- to threefold less than that of control cell lines. GT1-7 cells overexpressing bcl-2 had the same level of glutathione peroxidase, catalase, superoxide dismutase, and glutathione reductase activities as control cells. PC12 cells overexpressing bcl-2 had a twofold increase in superoxide dismutase and catalase activity when compared with matched control transfected cells. The levels of glutathione peroxidase and glutathione reductase in PC12 cells overexpressing bcl-2 were similar to those of control cells. These results indicate that the overexpression of bcl-2 shifts the cellular redox potential to a more reduced state, without consistently affecting the major cellular antioxidant enzymes.     

By Blocking Apoptosis,Bcl-2 in p38-Dependent Manner Promotes Cell Cycle Arrest and Accelerated Senescence After DNA Damage and Serum Withdrawal

E1A+ras-transformed rodent fibroblasts are unable to be arrested in the cell cycle and die by apoptosis in response to cytostatics, ionizing radiation (IR), or serum withdrawal. Overexpression of the human antiapoptotic gene bcl-2 suppresses apoptosis and induces reversible cell cycle arrest after IR or serum withdrawal and cell senescence after adriamycin treatment. Bcl-2-sustained adriamycin-induced cell senescence requires p38 MAPK, since the knockout of p38 MAPK abrogated anti-apoptotic and senescence-inducing effects of Bcl-2 in adriamycin-treated cells. Moreover, resistance to apoptosis and cell cycle arrest were not observed in p38 -/- E1A+ras+bcl-2-transformants following IR or serum deprivation. However, the pro-apoptotic effect of nocodazole in E1A+ras-transformed cells can not be prevented by Bcl-2 overexpression independently of the presence of p38 MAPK. These results allow us to conclude that p38 is necessary for Bcl-2-induced inhibition of apoptosis, induction of cell cycle arrest and accelerated senescence after DNA damage and serum starvation, but not after nocodazole treatment.