Opposed effects of lithium on the MEK-ERK pathway in neural cells: inhibition in astrocytes and stimulation in neurons by GSK3 independent mechanisms

Lithium is widely used in the treatment of bipolar disorder, but despite its proven therapeutic efficacy, the molecular mechanisms of action are not fully understood. The present study was undertaken to explore lithium effects of the MEK/ERK cascade of protein kinases in astrocytes and neurons. In asynchronously proliferating rat cortical astrocytes, lithium decreased time- and dose-dependently the phosphorylation of MEK and ERK, with 1 mM concentrations achieving 60 and 50% inhibition of ERK and MEK, respectively, after a 7-day exposure. Lithium also inhibited [3H]thymidine incorporation into DNA and induced a G2/M cell cycle arrest. In serum-deprived, quiescent astrocytes, pre-exposure to lithium resulted in the inhibition of cell cycle re-entry as stimulated by the mitogen endothelin-1: under this experimental setting, lithium did not affect the rapid, peak phosphorylation of MEK taking place after 3-5 min, but was effective in inhibiting the long-term, sustained phosphorylation of MEK. Lithium inhibition of the astrocyte MEK/ERK pathway was independent of inositol depletion. Further, compound SB216763 inhibited Tau phosphorylation at Ser396 and stabilized cytosolic beta-catenin, consistent with the inhibition of glycogen synthase kinase-3 beta (GSK-3 beta), but failed to reproduce lithium effects on MEK and ERK phosphorylation and cell cycle arrest. In cerebellar granule neurons, millimolar concentrations of lithium enhanced MEK and ERK phosphorylation in a concentration-dependent manner, again through an inositol and GSK-3 beta independent mechanism. These opposing effects in astrocytes and neurons make lithium treatment a promising strategy to favour neural repair and reduce reactive gliosis after traumatic injury.     

Involvement of altered cytoskeletal protein phosphorylation in aluminum-induced CNS dysfunction

In the present investigation, we have studied the effects of aluminum (10 mg/kg of body weight per day, i.p.) and desferrioxamine (6 mg/kg of body weight per day, i.p.) alone and in combination for 4 weeks on the regulation of phosphorylation of neuronal proteins. A marked decrease in the biological activity of calmodulin was observed after aluminum treatment; however, in combination with desferrioxamine, a reversal in the levels of calmodulin, in terms of nmol cAMP hydrolyzed/min/mg protein, was observed. Exogenous addition of calmodulin had an inhibitory effect on calmodulin-mediated synaptosomal protein phosphorylation in aluminum-exposed animals. An almost complete reversal of this inhibition was observed following coexposure to aluminum and desferrioxamine. Cyclic AMP-dependent synaptosomal protein phosphorylation was, however, stimulated following aluminum exposure. Coadministration of desferrioxamine along with aluminum was found to mitigate the neurotoxic effect of aluminum. © 1997 John Wiley & Sons, Inc.     

Change of synaptic membrane lipid composition and fluidity by chronic administration of lithium

The effect of chronic administration of lithium salts on the lipid composition and physical properties of the synaptosomal plasma membrane was examined in rat brain. The effect of lithium treatment has been studied on the fluorescence polarization of synaptosomal plasma membrane and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from lithium-treated animals. Altered DPH polarization was due to a decrease in the order parameter of the probe. The lithium-treatment also changed the fluorescence of 1-anilino-8-naphthalene sulfonate (ANS), a probe that binds to the polar head group of the phospholipids and to proteins on the membrane surface. Synaptic plasma membranes from treated rats presented no significant changes on the cholesterol-to-phospholipid ratio, although the phospholipid class distribution was altered and the membrane phospholipid unsaturation increased. In summary, the neural plasma membranes became disorder after chronic lithium administration at therapeutic levels. This structural change may be due to changes in plasma membrane phospholipid distribution and to the degree of unsaturation of phospholipid fatty acids.     

Chronic Stress and Lithium Treatments Alter Hippocampal Glutamate Uptake and Release in the Rat and Potentiate Necrotic Cellular Death After Oxygen and Glucose Deprivation

This study was undertaken to evaluate the effects of chronic variate stress and lithium treatment on glutamatergic activity and neuronal vulnerability of rat hippocampus. Male Wistar rats were simultaneously treated with lithium and submitted to a chronic variate stress protocol during 40?days, and afterwards the hippocampal glutamatergic uptake and release, measured in slices and synaptosomes, were evaluated. We observed an increased synaptosomal [(3)H]glutamate uptake and an increase in [(3)H]glutamate stimulated release in hippocampus of lithium-treated rats. Chronic stress increased basal [(3)H]glutamate release by synaptosomes, and decreased [(3)H]glutamate uptake in hippocampal slices. When evaluating cellular vulnerability, both stress and lithium increased cellular death after oxygen and glucose deprivation (OGD). We suggest that the manipulation of glutamatergic activity induced by stress may be in part responsible for the neuroendangerment observed after stress exposure, and that, in spite of the described neuroprotective effects of lithium, it increased the neuronal vulnerability after OGD.     

Effect of Digestion with Phospholipase A2 on Endogenous Protein Phosphorylation in Particulate Fractions from Rat Brain Synaptosomes

The endogenous phosphorylation of synapsin 1 in cyclic AMP-containing media was greatly decreased by digestion of synaptic vesicles and synaptosomal membranes with phospholipase A2, suggesting that the system is functionally dependent on the membrane structure. Treatment of the synaptic vesicle fraction with phospholipase A2 also caused a small but significant inhibition of the Ca2+/calmodulin-dependent phosphorylation of the same protein. The Ca2+/calmodulin-dependent phosphorylation of other major acceptors, and the basal phosphorylation of a 52-kD acceptor enriched in the vesicle fraction, remained unchanged after cleavage of the membrane phospholipids with phospholipase A2. The significance of the selective effect of phospholipase A2 treatment on endogenous membrane phosphorylation is discussed.     

Involvement of the role of Chk1 in lithium-induced G2/M phase cell cycle arrest in hepatocellular carcinoma cells

Lithium, a therapeutic agent for bipolar disorder, can induce G2/M arrest in various cells, but the mechanism is unclear. In this article, we demonstrated that lithium arrested hepatocellular carcinoma cell SMMC-7721 at G2/M checkpoint by inducing the phosphorylation of cdc2 (Tyr-15). This effect was p53 independent and not concerned with the inhibition of glycogen synthase kinase-3 and inositol monophosphatase, two well-documented targets of lithium. Checkpoint kinase 1 (Chk1), a critical enzyme in DNA damage-induced G2/M arrest, was at least partially responsible for the lithium action. The lithium-induced phosphorylation of cdc2 and G2/M arrest was abrogated largely by SB218078, a potent Chk1 inhibitor, as well as by Chk1 siRNA or the over-expression of kinase dead Chk1. Furthermore, lithium-induced cdc25C phosphorylation in 7721 cells and in vitro kinase assay showed that the activity of Chk1 was enhanced after lithium treatment. Interestingly, the increase of Chk1 activity by lithium may be independent of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) kinase. This is because no elevated phosphorylation on Chk1 (Ser-317 and Ser-345) was observed after lithium treatment. Moreover, caffeine, a known ATM/ATR kinase inhibitor, relieved the phosphorylation of cdc2 (Tyr-15) by hydroxyurea, but not that by lithium. Our study's results revealed the role of Chk1 in lithium-induced G2/M arrest. Given that Chk1 has been proposed to be a novel tumor suppressor, we suggest that the effect of lithium on Chk1 and cell cycle is useful in tumor prevention and therapy.     

Determination of Changes in the Phosphorylation State of the Neuron-Specific Protein Kinase C Substrate B-50 (GAP43) by Quantitative Immunoprecipitation

To determine changes in the degree of phosphorylation of the protein kinase C substrate B-50 in vivo, a quantitative immunoprecipitation assay for B-50 (GAP43, F1, pp46) was developed. B-50 was phosphorylated in intact hippocampal slices with 32Pi or in synaptosomal plasma membranes with [gamma-32P]ATP. Phosphorylated B-50 was immunoprecipitated from slice homogenates or synaptosomal plasma membranes using polyclonal anti-B-50 antiserum. Proteins in the immunoprecipitate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the incorporation of 32P into B-50 was quantified by densitometric scanning of the autoradiogram. Only a single 48-kilodalton phosphoband was detectable in the immunoprecipitate, but this band was absent when preimmune serum was used. The B-50 immunoprecipitation assay was quantitative under the following condition chosen, as (1) recovery of purified 32P-labelled B-50 added to slice homogenates or synaptosomal plasma membranes was greater than 95%; and (2) modulation of B-50 phosphorylation in synaptosomal plasma membranes with adrenocorticotrophic hormone, polymyxin B, or purified protein kinase C in the presence of phorbol diester resulted in EC50 values identical to those obtained without immunoprecipitation. With this immunoprecipitation assay we found that treatment of hippocampal slices with 4 beta-phorbol 12,13-dibutyrate stimulated B-50 phosphorylation, whereas 4 alpha-phorbol 12,13-didecanoate was inactive. Thus, we conclude that the B-50 immunoprecipitation assay is suitable to monitor changes in B-50 phosphorylation in intact neuronal tissue.     

Altered phosphorylation and localization of the A-type channel, Kv4.2 in status epilepticus

Extracelluar signal-regulated kinase (ERK) pathway activation has been demonstrated following convulsant stimulation; however, little is known about the molecular targets of ERK in seizure models. Recently, it has been shown that ERK phosphorylates Kv4.2 channels leading to down-regulation of channel function, and substantially alters dendritic excitability. In the kainate model of status epilepticus (SE), we investigated whether ERK phosphorylates Kv4.2 and whether the changes in Kv4.2 were evident at a synaptosomal level during SE. Western blotting was performed on rat hippocampal whole cell, membrane, synaptosomal, and surface biotinylated extracts following systemic kainate using an antibody generated against the Kv4.2 ERK sites and for Kv4.2, ERK, and phospho-ERK. ERK activation was associated with an increase in Kv4.2 phosphorylation during behavioral SE. During SE, ERK activation and Kv4.2 phosphorylation were evident at the whole cell and synaptosomal levels. In addition, while whole-cell preparations revealed no alterations in total Kv4.2 levels, a decrease in synaptosomal and surface expression of Kv4.2 was evident after prolonged SE. These results demonstrate ERK pathway coupling to Kv4.2 phosphorylation. The finding of decreased Kv4.2 levels in hippocampal synaptosomes and surface membranes suggest additional mechanisms for decreasing the dendritic A-current, which could lead to altered intrinsic membrane excitability during SE.     

Lithium-induced inhibition of Src tyrosine kinase in rat cerebral cortical neurons: a role in neuroprotection against N-methyl-D-aspartate receptor-mediated excitotoxicity

The neuroprotective effects of lithium, a mood stabilizer, against glutamate-induced excitotoxicity in rat cortical neurons were associated with a decrease in Tyr1472 phosphorylation of the N-methyl-D-aspartate (NMDA) receptor NR2B subunit and a loss of receptor activity. Since this receptor tyrosine phosphorylation is mediated by the Src-family tyrosine kinases, we investigated the effects of lithium on the Src kinase activity. Levels of phosphorylated Src kinase at Tyr416, an index of Src activation, were reduced after treatment with LiCl (1 mM) for more than 3 days. Protein levels of Src-family kinases such as Src, Fyn, and Yes were unchanged by lithium treatment. The activities of cytosolic protein tyrosine kinase and protein phosphatase were also unchanged by lithium treatment, indicating the selectivity and the modulation. Moreover, the levels of postsynaptic densities (PSD) and SynGAP, the scaffolding proteins of the NMDA receptor complex, were unaltered by lithium. A Src kinase inhibitor, SU6656, and an NR2B antagonist, ifenprodil, partially blocked glutamate excitotoxicity. Our results suggest that lithium-induced inactivation of Src kinase contributes to this drug-induced NMDA receptor inhibition and neuroprotection against excitotoxicity.     

Autophosphorylation of chick brain synaptic polypeptides. Phosphorylation of proteins during incubation of intact synaptosomes with 32PO4

Chick brain synaptosomes incorporated phosphate into proteins when incubated in physiological buffer containing energy sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that three synaptosomal polypeptides were significantly phosphorylated after 15 sec incubation while at least fifteen polypeptides were active kinase substrates after 15 min incubation. Labeled synaptosomes were hypotonically lysed and separated by centrifugation into soluble, membrane, and mitochondrial fractions. Every fraction exhibited significant phosphate incorporation. Electrophoresis revealed that each fraction had several unique phosphorylated polypeptides and a distinctive phosphorylation pattern. The same polypeptides appear to be labeled whether MgATP was added to synaptic plasma membranes or synaptic plasma membranes were isolated after synaptosomal autophosphorylation.     

Calcium-dependent phosphorylation of specific synaptosomal fraction proteins: Possible role of phosphoproteins in mediating neurotransmitter release

Calcium ions caused a marked increase in the level of endogenous phosphorylation of specific proteins from synaptosomal fractions prepared from rat cerebral cortex. The levels of phosphorylation of these specific proteins were dependent upon the presence of calcium and regulated by small changes in the concenrration of calcium ions. The effect of calcium was independent of ATP concentration over a wide range of concentrations. The results are compatible with the hypothesis that some of the effects of calcium on synaptic transmission might be mediated by the effect of calcium on the phosphorylation of specific synaptosomal proteins.     

Regional and Subcellular Localization of Li+ and Other Cations in the Rat Brain Following Long-Term Lithium Administration

Abstract: Rats were given LiCl in their diet (40 mmol/kg dry weight) for at least 3 months to elucidate the regional and subcellular localization of Li⁺ in the brain as well as the effect of chronic lithium administration on the distribution of other cations. At steady-state the mean concentrations of Li⁺ were 0.66 mmol/kg wet weight in the whole brain and 0.52 mM in plasma. The tissue/plasma concentration ratio exceeded unity in all anatomical regions. No region showed excessive accumulation of Li⁺. Whole brain or regional contents of Na⁺ or K⁺ were unaffected by lithium treatment. Subcellular Li⁺ localization was demonstrated in nuclear, crude mitochondrial, and microsomal fractions of whole brain homogenate. Subfractionation of the crude mitochondrial fraction revealed energy-independent intrasynaptosomal and intramitochondrial Li⁺ and K⁺ localization at 0–4°C. Li⁺ administered in vivo disappeared within 10 min from synaptosomes incubated at 37°C. Li⁺ added in vitro at 1 mM attained a synaptosomal steady-state concentration within 30 min at 37°C. In control rats, synaptosomal concentrations and synaptosomal/medium concentration gradients of cations paralleled their respective in vivo concentrations and gradients. Lithium treatment caused synaptosomal depletion of K⁺ and Mg²⁺ and hence probably partial membrane depolarization. Addition of 1 mM Li⁺ in vitro also caused synaptosomal Mg²⁺ depletion. The results indicate that Li⁺ is “accumulated” in brain sediments and synaptosomes following its long-term treatment. The estimated intracellular and intrasynaptosomal Li⁺ concentrations are lower than predicted by passive distribution according to the Nernst equation, evidencing active extrusion of Li⁺.     

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain.

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg2+ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.     

Inhibition of Ca-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 μM, morin and rutin had similar effects at concentrations of about 200 μM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca²⁺-transport ATPase from synaptosomal vesicles: quercetin > morin > rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Synaptic membrane proteins as substrates for cyclic AMP-stimulated protein phosphorylation in various regions of rat brain

Synaptosomal plasma membranes from mammalian brain contain protein kinase activity which phosphorylates endogenous membrane proteins and is stimulated by cyclic AMP. Using polyacrylamide gel electrophoresis it was shown that at least ten proteins in the synaptosomal plasma membrane fraction could be phosphorylated by endogenous cyclic AMP-stimulated protein kinase activity. The number of proteins whose phosphorylation was stimulated by cyclic AMP was strongly influenced by the pH and Mg²⁺ concentration used in the phosphorylation reaction. A complex pattern of cyclic AMP-stimulated protein phosphorylation was obtained only with synaptosomal plasma membranes and a crude microsomal fraction. Mitochondrial and myelin fractions exhibited no cyclic AMP-stimulated protein kinase activity. Investigation of the distribution of substrates for cyclic AMP-stimulated phosphorylation among various brain regions failed to reveal any regional differences.     

Dephosphin, a 96,000 Da substrate of protein kinase C in synaptosomal cytosol, is phosphorylated in intact synaptosomes

A 96,000 dalton phosphoprotein, called dephosphin, is phosphorylated in intact synaptosomes from rat brain and is rapidly dephosphorylated upon depolarisation-dependent calcium entry. A 96,000 dalton phosphoprotein is also a substrate of protein kinase C in synaptosomal cytosol, and the aim of the study was to determine whether the two proteins may be the same. Dephosphin in intact synaptosomes and the 96,000 dalton protein kinase C substrate comigrated on polyacrylamide gels. Both phosphoproteins had identical phosphopeptide maps after digestion with V8 protease. Both phosphoproteins ran on isoelectric focussing gels with a pI of 6.3-6.7 and focussed as a series of 5-6 spots. Both proteins were phosphorylated exclusively on serine. Both proteins could be resolved into a doublet on longer polyacrylamide gels. The two subunits were of 96 and 93 kDa in both phosphorylation conditions and had dissimilar phosphopeptide maps. However, phosphopeptide maps of either the 96 or 93 kDa subunits were identical in intact synaptosomes compared with synaptosomal cytosol. These results show that a phosphoprotein phosphorylated in intact synaptosomes and a 96,000 dalton protein kinase C substrate from rat brain synaptosomal cytosol are the same, and raise the possibility that protein kinase C is the protein kinase responsible for dephosphin phosphorylation in intact synaptosomes.     

Blood–Brain Barrier Protein and Phosphorylation and Dephosphorylation

Capillaries in vertebrate brain have unique permeability properties that make up the blood-brain barrier (BBB). Although it is known that capillaries are innervated by nerve endings of intracerebral origin and that brain capillary function is likely acutely regulated by neuronal inputs, the possible mechanisms of neuronal regulation of capillary function are at present unknown. One possible mode of regulation is via the phosphorylation of brain capillary proteins. The present studies characterize, for the first time, the major phosphoproteins in the bovine brain capillary using both intact bovine brain capillaries and plasma membrane fractions from bovine brain capillaries. The patterns of endogenous phosphorylation of capillary proteins are compared to similar patterns obtained with synaptosomal (P2) fractions from bovine brain. The major findings of this study are: (a) The activity of protein phosphorylation in brain capillaries is localized almost exclusively to the capillary plasma membrane, and is nearly comparable to the activity of protein phosphorylation in synaptosomal membranes. (b) A major phosphoprotein doublet in the capillary fraction comigrates on a sodium dodecyl sulfate gel with a major phosphoprotein doublet of approximate molecular weight of 80K in the synaptosomal fraction, and the latter is presumed to be synapsin I; in dephosphorylation assays the synaptosomal 80K phosphoprotein doublet is not subject to measurable dephosphorylation, whereas the capillary 80K doublet is subject to rapid dephosphorylation, and is essentially completely dephosphorylated within 5 s at 0 degrees C. (c) A prominent triplet of phosphoproteins with molecular weight of 50-55K is present in the capillary fraction, and is not present in the synaptosomal fraction; thus, this 50-55K triplet of phosphoproteins appears specific for brain capillaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of adult type Sept5 (CDCrel-1) by cyclin-dependent kinase 5 inhibits interaction with syntaxin-1

Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.     

Lithium inhibits a late step in agrin-induced AChR aggregation

Agrin activates an intracellular signaling pathway to induce the formation of postsynaptic specializations on muscle fibers. In myotubes in culture, this pathway has been shown to include autophosphorylation of the muscle-specific kinase MuSK, activation of Src-family kinases, tyrosine phosphorylation of the acetylcholine receptor (AChR) beta subunit, a decrease in receptor detergent extractability, and the accumulation of AChRs into high-density aggregates. Here we report that treating chick myotubes with lithium prevented any detectable agrin-induced change in AChR distribution without affecting the number of AChRs or the agrin-induced change in AChR tyrosine phosphorylation and detergent extractability. Lithium treatment also increased the rate at which AChR aggregates disappeared when agrin was removed. The effects of lithium developed slowly over the course of approximately 12 h. Thus, sensitivity to lithium identifies a late step in the agrin signaling pathway, after agrin-induced MuSK and AChR phosphorylation, that is necessary for the recruitment of AChRs into visible aggregates.