Introduction: Due to the heterogeneity of psychiatric illnesses and overlapping mechanisms, patients with psychosis are differentially responsive to pharmaceutical drugs. In addition to having therapeutic effects for schizophrenia and bipolar disorder, antipsychotics and mood stabilizers have many clinical applications and are used unconventionally due to their direct and indirect effects on neurotransmitters. Synapsins, a family of neuronal phosphoproteins, play a key regulatory role in neurotransmitter release at synapses. In this study, we investigated the effects of mood stabilizers, lithium, and valproic acid on synapsin gene expression in the rat brain. Methods: Intraperitoneal injections of saline, lithium, and valproic acid were administered to male Sprague Dawley rats twice daily for 14 d, corresponding to their treatment group. Following decapitation and brain tissue isolation, mRNA was extracted from various brain regions including the hippocampus, striatum, prefrontal cortex, and frontal cortex. Results: Biochemical analysis revealed that lithium significantly increased gene expression of synapsin I in the striatum, synapsin IIa in the hippocampus and prefrontal cortex, and synapsin IIb in the hippocampus and striatum. Valproic acid significantly increased synapsin IIa in the hippocampus and prefrontal cortex, as well as synapsin IIb in the hippocampus and striatum. Conclusion: These significant changes in synapsin I and II expression may implicate a common transcription factor, early growth response 1, in its mechanistic pathway. Overall, these results elucidate mechanisms through which lithium and valproic acid act on downstream targets compared with antipsychotics and provide deeper insight on the involvement of synaptic proteins in treating neuropsychiatric illnesses. Keywords: lithium, valproic acid, synapsin, bipolar disorder Introduction Bipolar disorder (BD) is a severely disabling psychiatric illness are intense, persistent feelings of despair and hopelessness last- characterized by the occurrence of both manic and depres- ing over a 2-week period (APA, 2013). Mood fluctuations in BD sive episodes. The Diagnostic and Statistical Manual of Mental patients can be severe enough to result in hospitalization to pre- Disorders, fifth edition (DSM-V) describes a manic episode as a vent harm to oneself or others. A higher risk of suicidal behavior distinct period of abnormally and persistently elevated, expan- is also observed in individuals with BD compared with individuals sive, or irritable mood lasting at least 1 week. Depressive episodes with other psychiatric conditions or the healthy population (Song Received: October 21, 2017; Revised: February 21, 2018; Accepted: March 26, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 616 provided the original work is properly cited. For commercial re-use, please contact email@example.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/616/4956861 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Joshi et al. | 617 Significance Statement Lithium and valproic acid (VPA) are the most commonly prescribed drugs for the treatment of bipolar disorder. However, the exact mechanisms through which these drugs exert their effects are unknown. The aim of this study was to investigate the effects of lithium and VPA on synapsin phosphoproteins in various regions of the rat brain. Synapsins play a key role in regulat- ing synapse formation, synaptic signalling, and synaptic plasticity. In this study, rats were injected with saline, lithium, or VPA twice daily for 14 days. Several brain regions, including the hippocampus, striatum, prefrontal cortex, and frontal cortex, were analyzed for the expression of different synapsin isoforms including synapsin I, IIa, and IIb. Lithium and VPA significantly altered synapsin I, IIa, and IIb expression in different regions. The results of this study will increase our understanding of the biochem- ical mechanisms through which lithium and VPA exert their therapeutic effects. et al., 2017). Furthermore, patients may find it difficult to return to III is often expressed during the developmental stages of neurons their day-to-day lifestyle including work and social interactions and synapses. The N terminus is highly conserved amongst all due to the nature and prognosis of the disease. In Europe from synapsins, and the C terminus consists of many variable domains 1999 to 2009, 70% of patients with BD were underemployed in found in different isoforms. The transcription complexity as well Germany, and 63% to 67% of patients were unemployed in Italy as distinguishing features of the synapsins may suggest key dif- (Fajutrao et al., 2009). BD was ranked in the top 20 causes of dis- ferences in functionality that are yet to be completely elucidated. ability among all medical conditions worldwide by a recent global Although the structural and functional relationship of synapsins burden of disease study by the World Health Organization (WHO) has not been specifically established, many studies focus on the (Vos et al., 2012). A greater understanding of the pathophysiology functional role of synapsins in neurotransmitter signalling (Song of BD may contribute to alleviating risks associated with the dis- and Augustine, 2015). Neurotransmitters, including GABA, glutam- order and improving quality of life for patients. ate, and dopamine, have been linked and associated with various Lithium and valproic acid (VPA) are the most commonly pre- isoforms of synapsins. These phosphoproteins are known to tether scribed mood stabilizers for patients with BD. Lithium is the only glutamatergic vesicles within the reserve pool and regulate the size treatment that has been shown to be effective in preventing of the reserve pool (Gitler et al., 2004). In triple knockout mice, gluta- mania and depression. It is a monovalent cation with powerful matergic synaptic depression is specifically rescued by the addition antiinflammatory, antioxidative properties (Patel and Frey, 2015). of synapsin IIa (Gitler et al., 2008). Synapsins regulate a late step in Lithium decreases suicidal risk in patients and increases the vol- GABAergic synaptic vesicle trafficking that precedes the fusion of ume of brain regions associated with emotional regulation, includ- GABAergic synaptic vesicle exocytosis (Gitler et al., 2004). However, ing the amygdala, hippocampus, and prefrontal cortex (Cipriani the specific isoform regulating this vesicle trafficking is unclear et al., 2013; Malhi, et al., 2013). Although the mechanisms by which (Song and Augustine, 2015). Synapsin I knockout mice and synapsin lithium exerts its effects are still unclear, lithium has been shown III knockout mice display reduced basal inhibitory synaptic trans- to directly inhibit glycogen synthase kinase 3B (GSK3B) through mission, while synapsin II knockout mice display increased inhibi- 2+ competition with Mg binding as well as indirectly inhibit GSK3B tory transmission (Feng et al., 2002 Baldelli et ; al., 2007Medrihan ; through the activation of Wnt signalling pathways (Di Daniel et al., 2013). Lack of all synapsin isoforms increases catecholamine et al., 2005; Chiu et al., 2013; Lazzara and Kim, 2015). Interestingly, release in chromaffin cells and is only rescued by synapsin IIa, lithium has been shown to suppress glutamate excitotoxicity both exhibiting its function as a negative regulator of catecholamine in vitro and in vivo (Chalecka-Franaszek et al., 1999). release (Villanueva et al., 2006). Synapsin IIa appears to have oppos- Comparatively, VPA is an anticonvulsant drug used in the ite effects on glutamate release compared with catecholamine maintenance treatment of BD. Although an anticonvulsant drug, release. It increases glutamate release but decreases the release of VPA was originally proposed to treat BD due to its effect on the catecholamines (Song and Augustine, 2015). A triple knockout of enhancement of GABAergic activity and its direct effects on synapsins increases dopamine release from presynaptic terminals enzymes involved in GABA metabolism (Gould et al., 2004). It is but does not affect serotonin release (Kile et al., 2010). However, it an established histone deacetylase inhibitor that has been shown is suggested that negative regulation of dopamine release is medi- to be highly effective in attenuating manic episodes observed ated by synapsin IIa, as the same phenotype is observed in synap- in BD. Similarly to lithium, VPA indirectly activates Wnt signal- sin IIa knockout mice (Kile et al., 2010). Other isoforms implicated ling pathways, but it also robustly induces the activation of the in dopaminergic and GABAergic synaptic vesicle regulation remain mitogen associated protein kinase pathway (Chiu et al., 2013; unclear. Expression of synapsins in bipolar disorder (BD) has previ- Lazzara and Kim, 2015). However, VPA does not show the same ously been characterized, and studies have shown decreased lev- effects in reducing suicidal ideations and behaviors compared els of synapsins in the medial prefrontal cortex and hippocampus with lithium. Although both lithium and VPA play a role in several of patients with BD (Tan et al., 2014 . A ) recent study investigating interconnected pathways, the exact mechanisms underlying the DNA modifications showed a change in CpG methylation pattern functionality of these drugs and their effects on various neuro- of synapsin genes in patients with BD (Cruceanu et al., 2016). The transmission pathways still remain to be further investigated. objective of this study was to investigate the effect of mood stabiliz- Synapsins are among the first neuronal phosphoproteins iden- ers lithium and VPA on synapsin expression to address the mecha- tified that play a key role in synapse regulation, including neuro- nisms involved in the action of mood stabilizers. transmitter release, vesicle maintenance, and synaptic plasticity (Hilfiker et al., 2005; Song and Augustine, 2015). Their functions Materials and Methods are mediated by phosphorylation on various sites by many differ - ent kinases (Jovanovic et al., 1996Hosaka et ; al., 1999; Cesca et al., Animal Handling/Drug Administration 2010). The synapsin family consists of 3 functional genes, synapsin I, II, and III, and each subtype has several isoforms. Synapsin I and Twenty-two male Sprague Dawley rats (Charles Rivers II are more commonly found in mature synapses, while synapsin Laboratories), obtained at weights 300 to 350 g, were housed Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/616/4956861 by Ed 'DeepDyve' Gillespie user on 21 June 2018 618 | International Journal of Neuropsychopharmacology, 2018 individually in the central animal facility at McMaster University, plates (25 µL total reaction). All cDNA samples were amplified Hamilton. Animals were maintained under a reversed 12-:12- using MX3000P (Stratagene) cycler for 40 cycles. PCR amplifica- hour light cycle in a room with controlled temperature (22°C) tions began with heat activation of 95°C for 5 minutes and cycle and humidity (50% ± 5%). Animals were allowed to habituate to conditions were standard for all the primers with 95°C for 10 their homeroom for 1 week prior to animal handling and had seconds and 60°C for 30 seconds, as the annealing and exten- free access to food and water. All animal procedures adhered to sion steps were combined with the SYBR Green PCR kit. Negative the policies outlined by the Canadian Council on Animal Care controls for every plate consisted of No Reverse Transcriptase and McMaster University’s Animal Research and Ethics Board (containing DNase treated RNA without reverse transcript- (AUP 14-08-28). ase, made while synthesizing cDNA) and No Template Control Both lithium and VPA were obtained from Sigma Aldrich. (using nuclease free water in the reaction instead of cDNA). Weights of all animals were monitored daily to ensure the PCR specificity was confirmed when melting curve analysis of absence of large fluctuations in weight and proper dosage of the amplified product produced a single peak for each product. drugs for all animals. All rats were randomly divided into 3 All reactions were performed in duplicates, and Ct values were treatment groups—control (saline treated, = 6), n lithium (n= 8), limited to a variability of ±0.5. For analysis purposes, average and VPA (n = 8)—and were injected via i.p. route twice daily for of the duplicates was used. DNA was extracted from the PCR 2 consecutive weeks. Stock solutions of lithium (47.5 mg/mL) amplicons, and the amplicon was then subjected to electrophor - and VPA (200 mg/mL) were made using sterile saline. Saline, esis on a 1.5% agarose gel after having been resuspended in a lithium, and VPA were administered to each treatment group solution with 6x loading dye and nuclease free water. The sin- respectively in a volume of 1 mL/kg. Rats were anaesthetized gle band of interest was verified and excised for each gene. The with isoflurane and killed by decapitation 5 hours after the final amplicon was extracted using the Freeze N’ Squeeze Columns injection, which was consistent with the half-life of lithium in (BioRad) as per the manufacturer’s protocol. rodents (Wood et al. 1986). Rat brain regions, including cortex, striatum, PFC, and hippocampus, were dissected over ice and Sequencing Analysis of Synapsins and Internal stored at −80°C until use. Control Confirmed Amplicon Sequences Amplicons were run on a 1.5% agarose gel alongside a nega- Plasma Collection and Dosage Evaluation tive control from the same plate (supplementary Figure 1). Blood was collected in BD vacutainer SST blood collection tubes Sequencing analysis confirmed the nucleotide sequence of coated with ethylenediaminetetraacetic acid immediately fol- the amplicons. Synapsin I had an amplicon length of 181 bp lowing decapitation. Samples were inverted 5 times and stored (GenBank: X04655.1). Synapsin IIa had an amplicon length of at room temperature for 30 minutes. The plasma was isolated 183 bp (NCBI Ref: NM_001034020.1). Synapsin IIb had an ampli- from whole blood by centrifugation of tubes at 3000 rpm for 10 con length of 184 bp (NCBI Ref: NM_019159.1). GAPDH had an minutes without a stopping break on the Eppendorf 5810R cen- amplicon length of 118 bp (NCBI Ref: XM_017593963.1). trifuge. Plasma was stored at -80°C until processing. Lithium and VPA plasma levels were analyzed at St. Joseph’s Healthcare Statistical Analysis Hamilton (Charlton Campus) using the Easylyte machine GraphPad Prism 6 was used for all statistical analyses. Relative (Medica Vendo Cypress Diagnostics Inc.) to ensure that levels fell within the therapeutic range. gene expression was compared using the delta-delta Ct method as described by Schmittgen and Livak (2008) using the equa- -((Ct-Gene of interest – Ct-Housekeeping) – (Ct-Avg Ctrl Gene of interest – Ct-Avg Ctrl tion ΔΔCt = 2 RNA Extraction/cDNA Synthesis Housekeeping)) . Raw Ct values, as well as an absolute quantification Total RNA was extracted using TRIzol from Ambion by Life method with standard curve, were used to ensure no differences Technologies as per the manufacturer’s protocol (catalog in the housekeeping gene GAPDH. Two-way ANOVA with Tukey’s no. 15596018). Using 1 µg of total RNA, DNAse treatment was posthoc was used to compare differences between groups in conducted using the supplier’s protocol. Following DNAse treat- gene expression. One-way ANOVA with Tukey’s posthoc test was ment, cDNA was synthesized using an identical amount of RNA used to analyze weight changes. Outliers were removed via the (QuantaBio qScript cDNA SuperMix) as per the manufacturer’s ROUT method developed by GraphPad using the default and protocol. conservative ROUT coefficient of 1% (Motulsky and Brown, 2006). Significance was established as P < .05. Real-Time PCR Results The mRNA expression of synapsins was assessed via real-time PCR using the QuantaFast SYBR Green PCR kit from Qiagen. Lithium and VPA Concentrations in Plasma The primers used for synapsin I, IIa, and IIb and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (internal control) are Blood plasma drug levels for both lithium and VPA were evalu- as follows: Synapsin I Fwd: GTG TCA GGG AAC TGG AAG ACC, ated for all rats. Therapeutic concentrations for lithium range Synapsin I Rev: AGG AGC CCA CCA CCT CAA TA, Synapsin IIa between 0.6 and 1.2 mmol/L and therapeutic concentrations for Fwd: ACT GCC ACC TTC TTC CTC, Synapsin IIa Rev: GAC TTG VPA range between 50 and 100 µM/mL (Sproule, 2002; Löscher, TTG AGC TGT GGG, Synapsin IIb Fwd: TCA GCA AGA TGA ACC 2007; Zanni et al., 2017). Control rats showed undetectable AGC, Synapsin IIb Rev: GGA CCT ACT GCA ATG CC, GAPDH Fwd: lithium and VPA levels. Lithium-treated rats showed lithium CAA CTC CCT CAA GAT TGT CAG CAA, and GAPDH Rev: GGC ATG levels between 0.3 and 0.5 mmol/L and VPA levels <50 µM/mL GAC TGT GGT CAT GA. All primers were used at a final concen- (Figure 1A). VPA-treated rats showed lithium levels <0.2 mmol/L tration of 1 µM. Reagents and concentrations corresponded to and VPA levels ranging from 51 to 100 µM/mL (Figure 1B). Rat the protocol as per the SYBR Green PCR kit (Qiagen) for 96-well 2 (circled on Figure 1) in the VPA group had a toxic plasma Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/616/4956861 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Joshi et al. | 619 Figure 1. Blood plasma concentrations of lithium and valproic acid (VPA) treatment drugs. Plasma concentrations of lithium and VPA measured 5 hours after adminis- tration in (A) lithium-treated rats and (B) VPA-treated rats. Lower limit of the therapeutic concentration range is shown by red dotted line. Rat 2 in the VPA-treated rats (circled in red) showed symptoms of toxicity including very high plasma concentrations and therefore was not used in further analyses. concentration of 336 µM/mL along with behavioral and physio- logical symptoms showing toxicity (red feces, little movement, no interest in food). Therefore, that rat was excluded from all further analyses. Both Lithium and VPA Increase Synapsin I Gene Expression in the Striatum To ascertain whether mood stabilizers affect gene expression of synapsins in the brain, we quantified the expression of synap- sin I in brain regions of rats following treatment with the mood stabilizers. There was a significant effect of lithium and VPA treatment on synapsin I expression (F(DFn, DFd): F(2, 72) = 3.619, P = .0318), and region (F(DFn, DFd): F(3, 72)= 8.035, P = .0001); an interaction between the two was not significant (F(DFn, DFd): F(6, 72) = 2.094, P = .0643). Tukey’s posthoc analysis revealed a sig- Figure 2. Lithium and valproic acid (VPA) increase synapsin I gene expression. nificant increase of synapsin I in the striatum by lithium treat- Synapsin I gene mRNA expression increased post lithium and VPA treatment ment (P= .0096) compared to saline treatment. There were no (P = .0318). Specifically, a significant increase in the striatum (Str) by lithium significant changes in other regions (Figure 2). treatment was observed through Tukey’s test. **P < .01. Mood Stabilizers Increase Expression of Synapsin IIa in Rat Brain Regions In order to determine whether mood stabilizers such as lithium and VPA have selective effects on synapsins, we also quantified the gene expression for synapsin IIa and IIb. A significant effect of treatment was observed on synapsin IIa mRNA expression (F(2,71) = 11.99, P < .0001) and region (F(3,71) = 3.284, P = .0257); an interaction between the two was not significant (F(6,71) = 0.9864, P = .4411). Tukey’s post hoc analysis revealed that there was a sig- nificant increase of synapsin IIa in the hippocampus and PFC following lithium treatment ( = P .0250, .0157 respectively), com- pared to saline treatment. There was also a significant increase in the hippocampus and PFC upon VPA treatment (P = .0142, .0019, respectively) compared to saline treatment. There were no significant changes in the other brain regions (Figure 3). Figure 3. Lithium and valproic acid (VPA) increase synapsin IIa gene expression. Synapsin IIa gene mRNA expression increased post lithium and VPA treatment Mood Stabilizers Increase Expression of Synapsin (P < .0001). A significant increase in gene expression was observed in the hippo- IIb in Rat Brain Regions campus (Hippo) and prefrontal cortex (PFC) by lithium and VPA treatment indi- vidually. *P < .05, **P < .01. There was a significant effect of treatment on synapsin IIb mRNA expression (F(2,72) = 14.02, P < .0001) and region (F(3,72) = 8.562, P < .0001); an interaction between the two was also significant hippocampus and striatum upon lithium treatment (P = .0014, (F(6, 72) = 2.35, P = .0396). Tukey’s posthoc analysis revealed that .0260, respectively) compared with saline treatment. Similarly, synapsin IIb mRNA expression significantly increased in the VPA treatment also significantly increased synapsin IIb Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/616/4956861 by Ed 'DeepDyve' Gillespie user on 21 June 2018 620 | International Journal of Neuropsychopharmacology, 2018 expression in the hippocampus and striatum (P < .0001, .0463, was observed (F(3,76) = 56.21, P < .0001), indicating differential respectively). There were no significant changes in other regions expression amongst regions. (Figure 4). Inhibition of Weight Gain by Lithium and VPA No Changes in Expression of the Housekeeping Rats were weighed daily prior to the morning i.p. injections for Gene, GAPDH, by Mood Stabilizers Lithium and VPA the 14-day treatment period. Weight gain was calculated by sub- tracting the weight before the first treatment from the weight Two-way ANOVA and posthoc analysis indicated no significant changes in raw cycle threshold (Ct) values of GAPDH upon treat- before the final injection. Weight gained during the 14-day treat- ment period was analyzed (supplementary Figure 2A). One- ment with lithium and VPA (F(2,72) = 1.68, P = .1936) (Figure 5A). A significant effect with respect to region (F(3,72)= 64.73, way ANOVA and Tukey’s posthoc analysis showed a significant effect of treatment on weight gain (P < .01). Lithium and VPA P < .0001) was detected; however, this was just indicative of different levels of GAPDH in different regions of the rat brain. significantly inhibited the weight increase observed in the con- trol group (P = .0095,P = .0014, respectively). Averaged weights Posthoc analysis revealed no significant difference comparing lithium and VPA-treated rats to saline-treated rats in the cor - through time were also graphed (supplementary Figure 2B). tex (P= .9856, .9624), hippocampus (P = .7371, .8893), striatum (P = .2315, .6249), and PFC (P = .9918, .3871). Raw Ct values were Discussion used for this analysis as GAPDH was intended to be used as the internal control. Absolute quantification of raw Ct values In the present study, we describe the effects of lithium and VPA, using a standard curve also showed no significant changes with two commonly prescribed mood stabilizers, on synapsin I and respect to treatment or interaction (F(2,76)= 2.844, P = .0644; F(6, II expression in different rat brain regions. There was an overall 76) = 0.7629, P = .6013) (Figure 5B). A significant effect of region effect of treatment on synapsin I, IIa, and IIb mRNA expression. Specifically, treatment with lithium resulted in a robust increase of synapsin I in the striatum. Both lithium and VPA caused a significant increase in synapsin IIa in the PFC and hippocam- pus, and synapsin IIb in the striatum and hippocampus. In vivo results from this study are consistent with the in vitro results of lithium treatment in lymphoblastoid cell lines from patients with bipolar disorder (Cruceanu et al., 2012). The mechanisms involved in upregulating synapsin gene expression may involve the transcription factor sites on pro- moter regions for synapsin I and II. There are some similarities and differences in these transcription factor sites on the pro- moter regions for synapsin I and II genes. Both synapsin I and II contain early growth response factor 1 (EGR-1) as a common transcription factor site, whereas activating protein-2 (AP2 α α) and polyoma enhancer activator 3 (PEA-3) are absent on the syn- apsin I promoter. Similarly, neural-restrictive silencing element (NRSE) and cAMP response element (CRE) are specifically local- ized to the synapsin I promoter (Chong et al., 2002 Skob ; lenick Figure 4. Lithium and valproic acid (VPA) increase synapsin IIb gene expression. et al., 2010). To summarize, synapsin I regulation can involve Synapsin IIb gene mRNA expression increased post lithium and VPA treatment EGR1, NRSE, and CRE transcription factors, while synapsin II (P < .0001). A significant increase in gene expression was observed in the hippo- regulation can involve EGR1, AP2,α and PEA-3 transcription fac- campus (Hippo) and striatum (Str) by lithium and VPA treatment individually. *P < .05, **P < .01, ****P < .0001. tors solely based on their promoter regions. Previous studies Figure 5. Lithium and valproic acid (VPA) do not change GAPDH expression. GAPDH gene expression did not change in brain regions, including striatum (Str), PFC, hippocampus (Hippo), and cortex (Cor) of rats treated with lithium and VPA by (A) analysis of raw cycle threshold (Ct) values or by (B) absolute analysis using a stand- ard curve. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/616/4956861 by Ed 'DeepDyve' Gillespie user on 21 June 2018 Joshi et al. | 621 from our laboratory have established the role of AP2 trαanscrip- effect of lithium and VPA on synapsins I, IIa, and IIb. There was a tion factor in increasing synapsin IIa and IIb expression with the region-specific increase by lithium of synapsin I in the striatum, treatment of antipsychotic drugs such as haloperidol, a dopa- synapsin IIa in the hippocampus and PFC, and synapsin Iib in mine D2 receptor antagonist (Chong et al., 2002 2006 , ; Skoblenick the hippocampus and striatum. These findings may provide fur - et al., 2010). Dopaminergic agents regulate synapsin II involv- ther insight on the specific roles of synapsins in the therapeutic ing the AP2a transcription factor through cyclic AMP (cAMP)- effects of these mood stabilizers. Overall, these findings help to mediated mechanisms. However, lithium and VPA likely involve elucidate the mechanisms through which lithium and VPA con- EGR-1, which is common to both synapsin I and II promoters, trast in downstream targets from antipsychotics, and provide to upregulate gene expression of both phosphoproteins. In this deeper insight on the involvement of synapsins in psychiatric regard, other studies have shown increases in EGR-1 expression illnesses such as bipolar disorder. by lithium in various brain regions (Lamprecht and Dudai, 1995) and by VPA in PC12 cells (Almutawaa et al., 2014). These find- Supplementary Material ings are consistent with the hypothesis that the EGR-1 transcrip- tion factor is involved in the increase in synapsin I and II gene Supplementary data are available at International Journal of expression by mood stabilizers. Future directions may involve Neuropsychopharmacology online. assessing the role of EGR-1 in the mechanistic pathway involv- ing upregulation of synapsins by lithium and VPA. Additionally, it Funding has been reported that treatments with mood stabilizers induce epigenetic changes in rodents and humans (Ookubo et al, 2013; This work was supported by the Canadian Institute of Health Lee et al., 2015; Cruceanu et al., 2016). Postmortem studies on Research (grant no. 126004). human brains of patients with BD revealed epigenetic changes of the synapsin gene (Cruceanu et al., 2016). This may be another Acknowledgments mechanism through which synapsin gene expression is altered by these mood stabilizers. However, the mechanisms by which The authors are grateful to Dr. Benicio Frey, Ashley Bernardo, mood stabilizers alter synapsin I and II expression may not be Dima Malkawi, Khaled Nawar, William Brett McIntyre, and all limited to the ones hypothesized here. Furthermore, there was the members of the Mishra laboratory for their help and support no common effect on all brain regions, as synapsin I and II were throughout the project. altered differently in the various brain regions. This finding may suggest that transcriptional regulation varies with different Statement of Interest brain regions and that mood stabilizers act at various levels in the expression of synapsins. None. In this study, we have shown that lithium and VPA change the expression of synapsin I and II mRNA levels in the brain. References Synaptic proteins play key regulatory roles in the cell involv- ing the maintenance of synapses and neurotransmitters. Both Almutawaa W, Kang NH, Pan Y, Niles LP (2014) Induction synapsin I and II have also been widely implicated in mood of neurotrophic and differentiation factors in neural disorders as well as neurodegenerative disorders such as stem cells by valproic acid. Basic Clin Pharmacol Toxicol Huntington’s disease and Alzheimer’s disease (Bernardo et al., 115:216–221. 2017). The upregulation of synapsins by lithium and VPA may American Psychiatric Association (2013) Diagnostic and statis- point to their effects on synaptic plasticity in the brain, which tical manual of mental disorders, DSM-IV-TR. Washington, DC: have been extensively researched (Monti et al., 2009Gr ; ay and American Psychiatric Association. McEwen, 2013). Some limitations of our study include the treat- Baldelli P, Fassio A, Valtorta F, Benfenati F (2007) Lack of synapsin ment regimens and subjects chosen. In a clinical setting, lithium I reduces the readily releasable pool of synaptic vesicles at and VPA are often given in combination to patients with BD for a central inhibitory synapses. J Neurosci 27:13520–13531. period of time that extends much beyond 2 weeks. In our study, Bernardo A, Prashar S, Molinaro L, Mishra RK (2017) Synapsin II. these drugs were administered individually to healthy rats as In: Encycopedia of signaling molecules (Choi S, ed), pp1–11. a longer and combination treatment of drugs would have been New York: Springer. toxic for the rats. Additionally, as no current rodent models of Cesca F, Baldelli P, Valtorta F, Benfenati F (2010) The synapsins: BD have been well established, these treatments were given to key actors of synapse function and plasticity. Prog Neurobiol healthy, drug naïve rats. To see the effects of combination treat- 91:313–348. ments, cellular in vitro models can be used. Lastly, the lithium Chalecka-Franaszek E, Chuang DM (1999) Lithium activates the plasma levels detected were just below the therapeutic range. serine/threonine kinase akt-1 and suppresses glutamate- Therefore, although significant differences were detected upon induced inhibition of akt-1 activity in neurons. Proc Natl treatment with lithium in both synapsin I and II, the level of Acad Sci USA 96:8745–8750. significance may be different or other effects of lithium may not Chiu CT, Wang Z, Hunsberger JG, Chuang DM (2013) Therapeutic be observed. An explanation for the observed plasma concentra- potential of mood stabilizers lithium and valproic acid: tions is that levels were measured approximately 5 hours after beyond bipolar disorder. Pharmacol Rev 65:105–142. the final injection, and the half-life of lithium in rats is approxi- Chong VZ, Young LT, Mishra RK (2002) Cdna array reveals dif- mately 6 hours (Wood et al., 1986). Thus, the plasma levels may ferential gene expression following chronic neuroleptic be lower than the therapeutic range due to the time frame administration: implications of synapsin II in haloperidol between blood collection and drug administration. treatment. J Neurochem 82:1533–1539. In conclusion, this study was one of the first to examine Chong VZ, Skoblenick K, Morin F, Xu Y, Mishra RK (2006) the effects of mood stabilizers, lithium and VPA, on the class Dopamine-D1 and -D2 receptors differentially regulate syn- of synapsin phosphoproteins in vivo. There was a significant apsin II expression in the rat brain. Neuroscience 138:587–599. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/616/4956861 by Ed 'DeepDyve' Gillespie user on 21 June 2018 622 | International Journal of Neuropsychopharmacology, 2018 Cipriani A, Hawton K, Stockton S, Geddes JR (2013) Lithium in the Malhi GS, Tanious M, Das P, Coulston CM, Berk M (2013) Potential prevention of suicide in mood disorders: updated systematic mechanisms of action of lithium in bipolar disorder. Current review and meta-analysis. Bmj 346:f3646. understanding. CNS Drugs 27:135–153. Cruceanu C, Alda M, Grof P, Rouleau GA, Turecki G (2012) Synapsin Medrihan L, Cesca F, Raimondi A, Lignani G, Baldelli P, Benfenati II is involved in the molecular pathway of lithium treatment F (2013) Synapsin II desynchronizes neurotransmitter release in bipolar disorder. PLoS One 7:e32680. at inhibitory synapses by interacting with presynaptic cal- Cruceanu C, Kutsarova E, Chen ES, Checknita DR, Nagy C, Lopez cium channels. Nat Commun 4:1512. JP, Alda M, Rouleau GA, Turecki G (2016) DNA hypomethyla- Monti B, Polazzi E, Contestabile A (2009) Biochemical, molecular tion of synapsin II cpg islands associates with increased gene and epigenetic mechanisms of valproic acid neuroprotection. expression in bipolar disorder and major depression. BMC Curr Mol Pharmacol 2:95–109. Psychiatry 16:286. Motulsky HJ, Brown RE (2006) Detecting outliers when fitting Di Daniel E, Mudge AW, Maycox PR (2005) Comparative analysis data with nonlinear regression - a new method based on of the effects of four mood stabilizers in SH-SY5Y cells and in robust nonlinear regression and the false discovery rate. BMC primary neurons. Bipolar Disord 7:33–41. Bioinformatics 7:123. Fajutrao L, Locklear J, Priaulx J, Heyes A (2009) A systematic Ookubo M, Kanai H, Aoki H, Yamada N (2013) Antidepressants review of the evidence of the burden of bipolar disorder in and mood stabilizers effects on histone deacetylase expres- europe. Clin Pract Epidemiol Ment Health 5:3. sion in C57BL/6 mice: brain region specific changes. J Feng J, Chi P, Blanpied TA, Xu Y, Magarinos AM, Ferreira A, Psychiatr Res 47:1204–1214. Takahashi RH, Kao HT, McEwen BS, Ryan TA, Augustine GJ, Patel JP, Frey BN (2015) Disruption in the blood-brain barrier: the Greengard P (2002) Regulation of neurotransmitter release by missing link between brain and body inflammation in bipolar synapsin III. J Neurosci 22:4372–4380. disorder? Neural Plast 2015:708306. Gitler D, Takagishi Y, Feng J, Ren Y, Rodriguiz RM, Wetsel WC, Purcell SM, Wray NR, Stone JL, Visscher PM, O’Donovan MC, Greengard P, Augustine GJ (2004) Different presynaptic roles Sullivan PF, Sklar P (2009) Common polygenic variation con- of synapsins at excitatory and inhibitory synapses. J Neurosci tributes to risk of schizophrenia and bipolar disorder. Nature 24:11368–11380. 460:748–752. Gitler D, Cheng Q, Greengard P, Augustine GJ (2008) Synapsin iia Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by controls the reserve pool of glutamatergic synaptic vesicles. J the comparative C(T) method. Nat Protoc 3:1101–1108. Neurosci 28:10835–10843. Skoblenick KJ, Argintaru N, Xu Y, Dyck BA, Basu D, Tan ML, Gould TD, Quiroz JA, Singh J, Zarate CA, Manji HK (2004) Emerging Mazurek MF, Mishra RK (2010) Role of AP-2alpha transcrip- experimental therapeutics for bipolar disorder: insights from tion factor in the regulation of synapsin II gene expression the molecular and cellular actions of current mood stabiliz- by dopamine D1 and D2 receptors. J Mol Neurosci 41:267–277. ers. Mol Psychiatry 9:734–755. Song J, Sjölander A, Joas E, Bergen SE, Runeson B, Larsson H, Gray JD, McEwen BS (2013) Lithium’s role in neural plasticity and Landén M, Lichtenstein P (2017) Suicidal behavior during lith- its implications for mood disorders. Acta Psychiatr Scand ium and valproate treatment: a within-individual 8-year pro- 128:347–361. spective study of 50,000 patients with bipolar disorder. Am J Hilfiker S, Benfenati F, Doussau F, Nairn AC, Czernik AJ, Augustine Psychiatry 174:795–802. GJ, Greengard P (2005) Structural domains involved in the Song SH, Augustine GJ (2015) Synapsin isoforms and synaptic regulation of transmitter release by synapsins. J Neurosci vesicle trafficking. Mol Cells 38:936–940. 25:2658–2669. Sproule B (2002) Lithium in bipolar disorder: can drug con- Hosaka M, Hammer RE, Südhof TC (1999) A phospho-switch centrations predict therapeutic effect? Clin Pharmacokinet controls the dynamic association of synapsins with synaptic 41:639–660. vesicles. Neuron 24:377–387. Tan ML, Dyck BA, Gabriele J, Daya RP, Thomas N, Sookram C, Jovanovic JN, Benfenati F, Siow YL, Sihra TS, Sanghera JS, Pelech Basu D, Ferro MA, Chong VZ, Mishra RK (2014) Synapsin SL, Greengard P, Czernik AJ (1996) Neurotrophins stimulate II gene expression in the dorsolateral prefrontal cortex of phosphorylation of synapsin I by MAP kinase and regu- brain specimens from patients with schizophrenia and bipo- late synapsin I-actin interactions. Proc Natl Acad Sci USA lar disorder: effect of lifetime intake of antipsychotic drugs. 93:3679–3683. Pharmacogenomics J 14:63–69. Kile BM, Guillot TS, Venton BJ, Wetsel WC, Augustine GJ, Vawter MP, Thatcher L, Usen N, Hyde TM, Kleinman JE, Freed WJ Wightman RM (2010) Synapsins differentially control dopa- (2002) Reduction of synapsin in the hippocampus of patients with mine and serotonin release. J Neurosci 30:9762–9770. bipolar disorder and schizophrenia. Mol Psychiatry 7:571–578. Lamprecht R, Dudai Y (1995) Differential modulation of brain Villanueva M, Thornley K, Augustine GJ, Wightman RM (2006) immediate early genes by intraperitoneal licl. Neuroreport Synapsin II negatively regulates catecholamine release. Brain 7:289–293. Cell Biol 35:125–136. Lazzara CA, Kim YH (2015) Potential application of lithium in Vos T, et al. (2012) Years lived with disability (YLDs) for 1160 Parkinson’s and other neurodegenerative diseases. Front sequelae of 289 diseases and injuries 1990-2010: a systematic Neurosci 9:403. analysis for the global burden of disease study 2010. Lancet Lee RS, Pirooznia M, Guintivano J, Ly M, Ewald ER, Tamashiro 380:2163–2196. KL, Gould TD, Moran TH, Potash JB (2015) Search for common Wood AJ, Goodwin GM, De Souza R, Green AR (1986) The pharma- targets of lithium and valproic acid identifies novel epigen- cokinetic profile of lithium in rat and mouse; an important etic effects of lithium on the rat leptin receptor gene. Transl factor in psychopharmacological investigation of the drug. Psychiatry 5:e600. Neuropharmacology 25:1285–1288. Löscher W (2007) The pharmacokinetics of antiepileptic drugs in Zanni G, Michno W, Di Martino E, Tjärnlund-Wolf A, Pettersson rats: consequences for maintaining effective drug levels dur - J, Mason CE, Hellspong G, Blomgren K, Hanrieder J (2017) ing prolonged drug administration in rat models of epilepsy. Lithium accumulates in neurogenic brain regions as revealed Epilepsia 48:1245–1258. by high resolution ion imaging. Sci Rep 7:40726. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/6/616/4956861 by Ed 'DeepDyve' Gillespie user on 21 June 2018
International Journal of Neuropsychopharmacology – Oxford University Press
Published: Mar 30, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera