Regulation Of Glycogen Content In Astrocytes Via Cav-1/PTEN/AKT/GSK-3β Pathway By Three Anti-bipolar Drugs

Introduction:As an essential component of central nervous system（CNS）,astrocytes have various important biological functions.Astrocytes support synaptic transmission and regulate neurotransmitter balance in the brain.Glycogen,which stores energy in the brain,is mainly distributed in astrocytes,and the role of glycogen is not limited to the emergency supply of energy substances.Glycogen synthesis and glycogenolysis,is indispensable for learning.Bipolar disorder（BD）is a severe chronic mood disorder characterized by episodes of mania and alternating or intertwining episodes of depression.With the development of the disease,many patients suffer from memory and cognitive dysfunction,BD brings a huge burden to the community.Astrocytes are closely related to bipolar disorder.Astroglia undergo complex pathological changes in neurological disorders.In major depression and in bipolar disorder astrocytes show signs of atrophy and their number decreases which may affect brain homeostasis and underlie deregulation of neurotransmission.Three classical anti-bipolar disorder drugs,lithium（Li⁺）,carbamazepine（CBZ）and valproic acid（VPA）are widely used in the treatment of bipolar disorder.Of note,classic anti-bipolar drugs have to be administered for a couple of weeks before their therapeutic action becomes manifest.In studies of mechanisms（s）of action for anti-bipolar drugs it is therefore important to analyse effects that are not immediate but only appear after chronic treatment.The three drugs differ in their chemical structures and in acute,so exploring the synergy of the three drugs is of great importance for the treatment of bipolar disorder.In recent years,we have extensively studied shared chronic effects of these drugs on astrocytes.These include regulation of gene expressions of phospholipase c PLA_2,Glu K2 kainite receptor and canonic transient receptor potential TRPC1 channel,decrease of astrocytic glutamate release as well as increase in cytosolic p H.Among these three drugs,Li⁺is the only one that has been established as an inhibitor of glycogen synthase kinase 3 or GSK-3.The involvement GSK-3 in bipolar disorder is well documented.The GSK-3 is an enzyme,which has been initially discovered as a deactivator of glycogen synthase（GS）that converts glucose to glycogen.GSK-3 exists in two isoforms,GSK-3α and GSK-3β.High levels of GSK-3β are detected in the central nervous system（CNS）.The GSK-3βis an important component of several signalling pathways,including insulin/insulin-like growth factor（IGF-1）signalling,neurotrophic factor signalling,and the Wnt signalling.Caveolin-1（Cav-1）binds directly to phosphatase and tensin homolog（PTEN）,an important component of the cave.PTEN inhibits the PI3K/AKT pathway through PI3K dephosphorylation;the serine-threonine kinase AKT phosphorylates GSK-3βand down-regulates its activity.Recently,we have demonstrated that chronic treatment of astrocytes with selective serotonin reuptake inhibitor（SSRI）fluoxetine induced,in a concentration-dependent manner,bi-phasic regulation of glycogen content through Cav-1/PTEN/AKT/GSK-3βpathway.We hypothesised that increase of GSK-3βphosphorylation and decrease of its activity via Cav-1/PTEN/AKT pathway may be one of the shared effects of three anti-bipolar drugs in astroglial cells.In the present study,we used primary astroglial cultures to investigate（i）acute effects all three anti-bipolar drugs on phosphorylation of AKT and ERK_1/2;（ii）expression of Cav-1 m RNA and protein in cultures chronically treated with the three anti-bipoar drugs;（iii）effects of chronic treatment with three anti-bipoar drugs on membrane content of PTEN,and phosphorylation of AKT and GSK-3β;（iv）effects of chronic treatment with three anti-bipoar drugs on astroglial glycogen content.Methods:1.Primary culture of astrocyte were isolated from neopallia of the cerebral hemispheres of the newborn CD-1 mice.Mature astrocytes were obtained after almost2 weeks culture.2.Membrane protein lysate was used to extract astrocyte membrane protein after administration.3.The Lowery method was used to determine the protein concentration of protein samples collected using protein lysates.4.To determine the acute effect of the three drugs after treating with CBZ（25μM、50μM）,Li₂CO₃（0.5 m M、1 m M）and VPA（0.1 m M、1 m M）for 20 min,1 hour and 2 hours,measuring the phosphorylation of AKT and ERK_1/2 in astrocytes by Western blotting;to determine the chronic effect of the three drugs after 2 weeks treatment,measuring the protein content of Cav-1,membrance content of PTEN,the phosphorylation of AKT and GSK-3βin astrocytes by Western blotting.5.Astrocytes were treated with CBZ（50μM）,Li₂CO₃（1m M）and VPA（1 m M）for 2 weeks before measuring its gene expression of Cav-1 by RT-PCR.6.The glycogen content in astrocytes were measured by NADPH colorimetric method after treating with CBZ（25μM、50μM）,Li₂CO₃（0.5 m M、1 m M）and VPA（0.1 m M、1 m M）for 2 weeks.7.The results were analysed with one-way ANOVA by SPSS22.0 software.P<0.05 indicates the statically significant difference.Results:1.Acute effects of three anti-bipolar disorder drugs.After acute-treatment of cultured astrocytes with CBZ（25μM and 50μM）,Li₂CO₃（0.5 m M、1 m M）and VPA（0.1 m M and 1 m M）for 20 min,1 hour and 2 hours,the AKT and ERK_1/2phosphorylation levels were measured over time.CBZ at 50μM,Li₂CO₃at 1 m M and VPA at 1 m M significantly increased the phosphorylation levels of AKT after 20minutes treatment of the three drugs,whereas ERK_1/2 phosphorylation levels were significantly increased in both concentrations of the three drugs.No significant changes in AKT and ERK_1/2 phosphorylation were observed after 1 h of drug treatment.2.Chronic effects of three anti-bipolar disorder drugs.1)Effect on the expression of Cav-1 m RNA and protein in astrocytes.After chronic-treatment of cultured astrocytes with CBZ（50μM）,Li₂CO₃（1 m M）and VPA（1 m M）for 2 weeks,the expression of Cav-1 m RNA was significantly decreased.After chronic-treatment of cultured astrocytes with CBZ（25μM and 50μM）,Li₂CO₃（0.5 m M and 1 m M）and VPA（0.1m M and 1 m M）for 2 weeks,both CBZ and Li₂CO₃at two concentrations and VPA at 1m M induced a significant down-regulation of Cav-1 protein expression,however VPA at 0.1 m M had no significant effect.2)Effect of MEK inhibitors.U0126 at 10μM had no effect on the down-regulation of Cav-1 induced by CBZ,Li₂CO₃ or VPA.3)Effect on membrane PTEN content in astrocytes.After chronic-treatment of cultured astrocytes with CBZ（25μM and 50μM）,Li₂CO₃（0.5 m M and 1 m M）and VPA（0.1m M and 1 m M）for 2 weeks,both CBZ and Li₂CO₃ at two concentrations and VPA at 1m M induced a significant down-regulation of membrane PTEN content,however,VPA at 0.1 m M had no significant effect.4)Effect on the phosphorylation of AKT and GSK-3βin astrocytes.After chronic-treatment of cultured astrocytes with CBZ（25μM and 50μM）,Li₂CO₃（0.5 m M and 1 m M）and VPA（0.1 m M and 1 m M）for 2 weeks,both CBZ and Li₂CO₃at two concentrations and VPA at 1 m M up-regulated AKT and GSK-3βphosphorylation,however,VPA at 0.1 m M had no significant effect.5)PI3K inhibitors can inhibit the changes of GSK-3βphosphorylation induced by carbamazepine,while MEK inhibitors have no significant effect.6)Effect on glycogen content in astrocytes.After chronic-treatment of cultured astrocytes with CBZ（25μM and 50μM）,Li₂CO₃（0.5 m M and 1 m M）and VPA（0.1 m M and 1 m M）for 2 weeks,CBZ at two concentrations,Li₂CO₃at 1 m M and VPA at 1 m M significantly increased glycogen content in astrocytes.Li₂CO₃at 0.5 m M and VPA at 0.1 m M had no significant effect.Discussion:Increased GSK-3βactivity is generally accepted to contribute to pathophysiology of major depression and bipolar.The researchers found that GSK-3βactivity was enhanced in both animal models of mood disorder and patients.When the ratio of phosphorylated GSK-3βto total GSK-3βis decreased,GSK-3βactivity is increased.Li⁺can directly or indirectly inhibit the activity of GSK-3βthrough competition with Mg²⁺and GSK-3βphosphorylation.The indirect inhibition of Li⁺requires AKT involvement.Similarly,the researchers found that VPA has a similar effect with Li⁺,in SH-SY5Y human neuroblastoma VPA can exert some neuroprotective effects via the AKT/GSK-3βsignaling pathway,however,effect of CBZ on this pathway was found.In the present study,we first report the acute effects shared by all three anti-bipolar drugs.All three drugs can down-regulate phosphorylation of AKT at 20 minutes and up-regulate ERK_1/2 phosphorylation at the same time.However,this effect disappeared after 1 hour drug treatment.In addition,MEK inhibitors were found to have no effect on changes in Cav-1 expression induced by the chronic effects of the three drugs.These all show that the acute effects of AKT and ERK_1/2phosphorylation changes are not involved in the chronic effects.Secondly,we found that chronic treatment with anti-bipolar drugs decreased Cav-1 protein expression,in turn,decreased membrane content of PTEN,activated the PI3K/AKT pathway and increased the phosphorylation of AKT.At the same time,we also observed that GSK-3βphosphorylation level increased,activity decreased.The regulation of GSK-3βphosphorylation and activity modulates the activity of glycogen synthetase,which,in turn,affects glycogen content in astrocytes,which is essential for learning and memory.In addition,Cav-1 is an important signaling molecule based on previous studies and the results of this experiment,that may play key roles in pathogenesis of a numbers of CNS diseases and regulation of its gene expression may be responsible for both therapeutic and side effects of anti-depressants.Conclusion:The increased activity of GSK-3βis closely related to the pathophysiological development of mood disorders.In the present study,we found for the first time,that chronic treatment with all three anti-bipolar disorder drugs down-regulate the activity of GSK-3βvia Cav-1/PTEN/PI3K/AKT signaling pathway,thus up-regulate the glycogen content in astrocytes.The discovery of new targets of the three drugs in astrocytes has important implications for further revealing their mechanism of action.