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Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA
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Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA
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Dipannita Basu a , Yuxin Tian a , Patricia Hui a , Jayant Bhandari a , Rodney L. Johnson b , Ram K. Mishra a,∗
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Department of Psychiatry and Behavioural Neurosciences, McMaster University, 1200 Main St. W., Hamilton, ON, Canada L8N 3Z5 Department of Medicinal Chemistry, University of Minnesota, 308 Harvard Street SE, Minneapolis, MN 55455, USA
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Article history: Received 29 August 2014 Received in revised form 20 January 2015 Accepted 20 January 2015 Available online xxx
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Keywords: Allosteric drugs Schizophrenia Synapsin II Dopamine D2 receptor Antipsychotic drug
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Introduction
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The hallmark symptoms of schizophrenia include profound disturbances in thought, perception, cognition etc., which negatively impacts an individual’s quality of life. Current antipsychotic drugs are not effective in treating all symptoms of this disorder, and often cause severe movement and metabolic side effects. Consequently, there remains a strong impetus to develop safer and more efficacious therapeutics for patients, as well as elucidating the etiology of schizophrenia. Previous work in our lab has introduced a novel candidate for the treatment of this disease: the dopamine D2 receptor (D2R) allosteric modulator, 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA). We have previously shown that PAOPA, by selectively modulating D2R, can ameliorate schizophrenia-like symptoms in animal models, although the precise mechanism is presently not understood. Synapsin II is a presynaptic vesicular protein which has been strongly implicated in schizophrenia, as it is reduced in the prefrontal cortex of patients, and knockdown of this protein elicits schizophrenia-like phenotypes in animal models. Given the therapeutic effects of PAOPA and the role of synapsin II in schizophrenia, the objective of this study was to investigate the effect of chronic administration of PAOPA (45 days) on neuronal synapsin II protein expression in rodents. Immunoblot results revealed that the synapsin IIa, but not the IIb isoform, was increased in the dopaminergic regions of the striatum, nucleus accumbens, and medial prefrontal cortex. The results of this study implicate a role for modulation of synapsin II as a possible therapeutic mechanism of action for potential antipsychotic drug PAOPA. © 2015 Elsevier Inc. All rights reserved.
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Schizophrenia is a debilitating mental disorder which presents significant therapeutic challenges for the healthcare system. The etiology of schizophrenia is strongly associated with the dopaminergic system, with affected patients showing hyperdopaminergic
Abbreviations: cAMP, cyclic adenosine monophosphate; D2R, dopamine D2 receptor; ECL, enhanced chemiluminescence; ERK 1/2, extracellular signal-regulated kinase 1/2; GABA, ␥-amino butyric-acid; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; HRP, horseradish peroxidase; IP, intraperitoneal; MIF-1, melanocyte-inhibiting factor 1; mPFC, medial prefrontal cortex; NMDA, N-methyl-D-aspartate; NAc, nucleus accumbens; PAOPA, 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide; PBS, phosphate-buffered saline; PKA, protein kinase A; PLG, L-prolyl-l-leucyl glycinamide; PVDF, polyvinlidene; SDS, sodium dodecyl sulfate; SEM, standard error of mean; SNP, single nucleotide polymorphism. ∗ Corresponding author. Tel.: +1 905 525 9140 ext. 22396; fax: +1 905 522 8804. E-mail address: [email protected] (R.K. Mishra).
activity in the striatum and nucleus accumbens (NAc), and hypodopaminergic activity in cortical regions [23,27]. Furthermore, deficits in cortical glutamate activity have also been observed in patients with schizophrenia [14,43]. While the causality for these observations has yet to be identified, research has revealed deficits in multiple transcripts and proteins of presynaptic secretory machinery in this disorder [7,19,20]. Advancing our studies of these proteins can enhance our understanding of this disorder’s pathophysiology, and improve targeted treatment options. Synapsins are a neuron-specific family of presynaptic phosphoproteins, which have shown to play key roles in the mechanisms of synaptic function and neuronal exocytosis. These proteins have a range of effects in controlling neurite outgrowth and synaptogenesis, regulating neurotransmitter release, vesicle docking and maintenance of synaptic vesicle stores [7]. The effects of synapsins distinctly vary with the neurotransmitter system being studied. In glutamate and ␥-amino butyric-acid (GABA) neurons, synapsins act as positive modulators, whereby knockout of synapsins decrease the release of these neurotransmitters
http://dx.doi.org/10.1016/j.peptides.2015.01.004 0196-9781/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Basu D, et al. Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.01.004
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[4,24,25]. Conversely, knockout studies have shown that the release of catecholamines, including dopamine, is enhanced after synapsin function is lost [49]. Due to its role in controlling synaptic morphology and regulating neurotransmitter release, synapsins have become a focus of studies in understanding schizophrenia. Alternate splicing of three genes (SYN1–3) results in the expression of five different isoforms of the synapsin protein in humans [7]. Among the five different isoforms of synapsin proteins, the synapsin II subtype, composed of the synapsin IIa and IIb isoforms, has been of particular importance in the study of schizophrenia. Microarray analysis by Mirnics et al. [36] has demonstrated reductions in synapsin II in the prefrontal cortex of patients with schizophrenia. Also, studies conducted by Chen et al. [8,9] presented a positive association with select polymorphisms on the synapsin II gene, and increased susceptibility to schizophrenia. Another study of 37 pedigrees of Northern European descent found at least two single nucleotide polymorphisms (SNP) significantly associated with schizophrenia, most of which were in introns 5 and 6 of the synapsin II gene. In addition, one haplotype of the SNPs within these introns was significantly over-transmitted to affected offspring [40]. Similarly, results from our lab showed decreased levels of the synapsin II transcript in the dorsolateral prefrontal cortex of patients affected with schizophrenia [47]. The lifetime antipsychotic drug use in these studies has been shown to positively correlate with synapsin II expression, particular the synapsin IIa isoform, via interaction with dopamine D1 and D2 receptors (D2R) [10,26,41,47]. Finally, previous studies from our lab have also shown that knockdown of synapsin II globally [17,18] as well as specifically within the medial prefrontal cortical regions of animal models [16,19], induces schizophrenia-like behavioral abnormalities. Taken together, this evidence supports a significant role of the synapsin II protein within the etiology of schizophrenia. Our previous studies [3,21,28,30,42] have developed a wide range of ligands to modulate D2R, a target which is strongly implicated in the etiology of schizophrenia. These ligands have been designed based on the functional pharmacophore of the endogenous tripeptide l-prolyl-l-leucyl glycinamide (PLG, also known as melanocyte-inhibiting factor-1 [MIF-1]), and have been shown to modulate dopamine binding via interaction with an allosteric site on the D2R [34]. Allosteric modulators are proving to be a promising avenue for the development of more specific, effective and safer drugs [12,13,29]. The current study utilizes the potent allosteric MIF-1 peptidomimetic (3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]2-oxo-1-pyrrolidineacetamide, PAOPA) [50], which has shown to effectively prevent and reverse a range of schizophrenia-like behavioral abnormalities [2,15]. Characterization of the pharmacokinetic and toxicological properties of PAOPA have revealed this ligand to have metabolic properties comparable to current marketed antipsychotic drugs, without the movement, hematological and metabolic adverse events commonly associated with the use of these drugs [46]. When acutely administered, Tan et al. found that the half-life of PAOPA given orally to rats was 112 ± 12 min, and time to maximum concentration was 50 ± 10 min, which is shorter than most of the currently available antipsychotic drugs. Finally, it was found that the concentration of PAOPA was greatest in the striatum, frontal cortex, and hypothalamus, indicating that PAOPA can cross the blood–brain barrier [45]. The mechanism of action of PAOPA is currently being investigated. Characterization of PAOPA’s pharmacological properties has revealed that it modulates agonist binding to dopamine D2 and D4 receptors in a biphasic manner [48]. PAOPA has also shown to promote D2R internalization, and upregulate proteins involved in this process, revealing initial processes linked to its potential mechanism of therapeutic action [1]. However, the effect of PAOPA on the expression of the crucial presynaptic secretory proteins mentioned above is currently unknown. The objective of this
study was, therefore, to determine the in vivo effects of PAOPA on synapsin II protein expression levels to improve our understanding of its mechanism of action as a potential new antipsychotic drug.
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Materials and methods
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Animals
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Twelve adult male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 200–250 g were individually housed at the McMaster Central Animal Facility. Animals were maintained under constant temperature and humidity, with a 12:12 light/dark cycle, and ad libitum access to food and water. All experiments were approved by the McMaster Animal Research Ethics Board, and conformed to the guidelines of the Canadian Council on Animal Care (animal utilization protocol # 10-08-59). Drug regimen and tissue collection The dopamine D2 allosteric peptide PAOPA was synthesized in the laboratory of Dr. Rodney L. Johnson (University of Minnesota, MN) as described previously [50], and peptide solutions (1 mg/mL) were prepared fresh daily. All rats were randomly divided into two groups (n = 6/group) and injected via intraperitoneal (IP) route daily for 45 consecutive days. The drug-treated group received chronic treatment of PAOPA at a 1 mg/kg dose and the control group was treated with 0.9% saline at the same dose. One hour following the final drug administration, rats were anaesthetized with isoflurane and sacrificed by decapitation. Rat brains were removed. The striatum, cerebellum, NAc and medial prefrontal cortex (mPFC) were dissected over ice, and stored at −80◦ C until use. Immunoblot Collected rat striatum, NAc, mPFC and cerebellum were homogenized using a pestle in phosphate-buffered saline (PBS) with Complete Mini, EDTA-free protease inhibitor tablet (Hoffmann La-Roche, Mississauga, ON) and PhosStop phosphatase inhibitor cocktail tablet (Hoffmann La-Roche, Mississauga, ON). Fifteen micrograms of sample was resuspended in 2× sodium dodecyl sulfate (SDS) sample buffer, boiled for 10 min and separated by electrophoresis on 10% acrylamide gels, using protocols previously described by our lab [11]. Briefly, separated proteins were transferred to a 0.45 M polyvinylidene (PVDF) membrane and blocked for 1 h in 5% skim milk at room temperature. The membranes were then labeled overnight with 1:2500 polyclonal synapsin II antibody (Enzo Life Sciences, Ann Arbor, MI) followed by 1.5 h of 1:5000 antirabbit IgG horseradish peroxidase (HRP)-linked whole secondary antibody from donkey (Sigma Aldrich, St. Louis, MO). Protein bands were visualized using enhanced chemiluminescence (ECL) substrates. A housekeeping protein—glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—was used to normalize the changes in protein expression and was detected using anti-GAPDH antibody produced in mouse (1:10,000; Millipore, Billerica, MA). Data analysis All the statistical analyses were carried out using GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA). ImageJ 1.43M (National Institutes of Health) was used to quantify the intensity of protein bands. Student’s t-test was used to compare differences in protein expression between control and treated groups for immunoblot assays. A significant expression change was considered when p < 0.05. Data are presented as the percent change compared to control ± SEM.
Please cite this article in press as: Basu D, et al. Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.01.004
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Fig. 1. Effect of chronic PAOPA administration on expression of synapsin IIa and synapsin IIb. Quantified immunoblot showing chronic PAOPA treatment (1 mg/kg) to: (A) Significantly increase expression of synapsin IIa in treated rats by 48% in the striatum (*p = 0.0126); 119% in the nucleus accumbens (NAc) (*p = 0.0103); and 30% in the medial prefrontal cortex (mPFC) (*p = 0.0296); (B) not significantly change expression of synapsin IIb in treated rats in the striatum, NAc, and mPFC. Similarly, chronic PAOPA treatment did not significantly change expression of both synapsin IIa and IIb in the cerebellum. Data are shown with representative immunoblots, and expressed as a percentage of average control ± SEM where *p < 0.05 (Student’s t-test) and n = 6/group.
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Results Effect of PAOPA on the expression of synapsin IIa in the striatum, nucleus accumbens, and mPFC Synapsin expression is regulated by dopaminergic pathways which can be affected by long-term ligand modulation [10,41]. In this study, PAOPA was observed to have significant effects in modulating synapsin IIa expression within all three brain regions of treated animals, leading to an increase in levels of synapsin IIa by 48% in the striatum (*p = 0.0126), 119% in the nucleus accumbens (*p = 0.0103) and 30% in the mPFC (*p = 0.0296; Fig. 1A). Expression of the housekeeping protein, GAPDH, was also analyzed within the same samples to normalize the expression of synapsin IIa, and was shown not to change with PAOPA treatment (data not shown). Effect of PAOPA on the expression of synapsin IIb in the striatum, nucleus accumbens, and mPFC
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The synapsin IIb isoform has shown to be expressed and regulated differently than the synapsin IIa isoform in various brain regions [35]. Unlike the changes observed in synapsin IIa protein expression, quantitative analysis of synapsin IIb expression in PAOPA treated rats showed no elevation in protein expression in the striatum or mPFC, and a slight but non-significant increase in the NAc (Fig. 1B). Expression of the housekeeping protein, GAPDH, was again analyzed within the same samples to normalize the expression of synapsin IIb, and was shown not to change with PAOPA treatment (data not shown).
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Effect of PAOPA on the expression of synapsin II in the cerebellum
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To relate the modulatory effects of PAOPA observed within the striatum, NAc, and mPFC, to interactions with D2R, the cerebellum was used as a region of reference. The cerebellum is a region is considered to have a negligible density of D2R, and therefore used as a negative control to study D2-related effects [31,38,44]. Analysis of synapsin IIa and IIb showed no significant change in expression in the cerebellum (Fig. 1), indicating that PAOPA had no effect on levels of protein expression in this region of chronically treated rats. There was also no change in the expression of the housekeeping protein, GAPDH, which was used to normalize the results (data not shown).
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Discussion
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The presynaptic protein synapsin II has shown significant involvement in the etiology of schizophrenia, and previous studies
from our lab have linked modulation of this protein to activation of dopamine receptors [10,41]. The current study shows that a significant increase in synapsin IIa expression occurs in dopaminergic regions strongly implicated in schizophrenia, including the striatum, mPFC, and NAc, following chronic administration of PAOPA. These increases appeared to be isoform specific as synapsin IIb expression remained unchanged, as well as region specific, as expression of both isoforms was unchanged in the relatively nondopaminergic region of the cerebellum. Studies have shown the importance of synapsin IIa isoform, due to its ability to rescue the defects in synaptic depression and synaptic vesicle density observed in hippocampal neurons of triple synapsin knockout mice [24]. This phenotypic rescuing was not statistically achieved with reintroduction of any other synapsin isoform. Similarly, reintroduction of synapsin IIa also rescued the enhanced catecholamine exocytosis in triple synapsin knockout adrenal chromaffin cultured cells [49]. In this study, PAOPA enhanced expression specifically of the synapsin IIa isoform, and therefore, appears to be modulating the isoform with the more phenotypically robust rescuing effects. Regulation of synapsin II expression has previously been shown to occur by modulation of the dopamine D1 and D2 receptors [10,41]. In this current study, increases in synapsin IIa were observed in dopamine rich regions, including the striatum, NAc and mPFC. However, no such effect was observed in the cerebellum, which is often used as a negative control to attribute observed in vivo changes to D2R [31,38,44]. Previous in vitro and in vivo studies with PAOPA have shown its specificity in modulating D2 and D4 receptors, with no significant effects occurring at D1 or D3 receptors, or the ␣2-adrenergic receptor [48]. Additionally, within in vivo models, PAOPA can potentiate the effect of D2 agonists, such as apomorphine [6,37], and attenuate effects of the D2-specific antagonist, haloperidol [6]. These interactions have been shown to occur through an allosteric interaction with D2R, as PAOPA does not compete with radioligands binding to the D2 orthosteric site [34]. Therefore, the observed effects of PAOPA on the expression of synapsin IIa may most likely be occurring via its allosteric effects on D2R. Studies have shown opposing effects of dopamine D1, compared to D2 modulation, on regulating synapsin II expression. Stimulation of D1 receptor activity by agonists, or attenuation of D2 activity by antagonists, results in increased synapsin II levels [11,41]. The use of PAOPA, has previously been shown to promote internalization of the D2R, thereby decreasing overall availability of this receptor, similar to an antagonist, but with a more fine-tuned and subtle technique [1]. Reduced post-synaptic D2 activity caused by PAOPA-induced receptor internalization within the striatum, NAc and mPFC, is hypothesized to lead to decreased
Please cite this article in press as: Basu D, et al. Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.01.004
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inhibition of cyclic adenosine monophosphate (cAMP), causing increased protein kinase A (PKA) stimulation, which eventually results in Ap-2␣ transcription factor promoted enhancement of synapsin II transcription [41]. Enhanced expression of synapsin II has been shown to occur with the use of current antipsychotic drugs, and may reveal a convergent therapeutic point for their mechanisms of action. Initial studies by Chong et al. [10] have shown the typical antipsychotic drug, haloperidol, to increase levels of synapsin II maximally within the striatum, and to a lesser extent, within the mPFC and NAc of treated rats. Subsequent studies by Guest et al. [26] and Tan et al. [47] showed levels of synapsin II to correlate with antipsychotic drug usage, with atypical antipsychotic drugs showing an increased likelihood of affecting expression of this molecule. Increased synapsin II within the regions investigated here can have important consequences in normalizing the dysregulated neurotransmission repeatedly observed in the etiology of schizophrenia. Glutamate projections from the healthy cortex stimulate inhibitory GABA neurons in the basal ganglia system, which thereby reduces subsequent neurotransmitter release, including dopamine, from this region. In vivo SPECT imaging studies on clozapine-treated schizophrenia patients using the radiotracer [123 I]CNS-1261, an intrachannel NMDA receptor ligand, have found decreased binding in brain regions such as the thalamus and striatum compared to healthy subjects, adding some support to the NMDA hypofunction hypothesis of schizophrenia [39]. A decrease in cortical glutamate release would yield less stimulation of inhibitory GABA neurons, resulting in neuronal disinhibition, and increased dopamine release relayed back into the striatum and NAc [5,32,39,43]. PAOPA can attenuate this pathological state by increasing synapsin II levels within the prefrontal cortex, and normalizing glutamate release. Synapsin IIa, in particular, has been found to be specifically associated with transmission of glutamate-containing synaptic vesicles [24]. Therefore, PAOPA-mediated normalization of synapsin II levels can indicate restoration of glutamatergic signaling within the prefrontal cortex, which eventually cascades to restore normal dopamine release into the striatum and nucleus accumbens. Additionally, increased synapsin IIa levels in the striatum and NAc can act directly as a negative regulator of dopamine release [49], resulting in decreased dopamine release within these regions. The effects of increased expression of synapsin II, particularly the phenotypically more robust isoform synapsin IIa, can therefore, have therapeutic effects in normalizing neurotransmission imbalances observed in schizophrenia. Synapsin activity is often regulated by kinase mediated phosphorylation, and therefore, these proteins may act as downstream recipients of receptor activated kinase molecules [41]. Previous studies from our lab have demonstrated the downstream effect of PAOPA on kinases, such as extracellular signal-regulated kinase 1/2 (ERK 1/2) [1]. Therefore, expression of synapsin, as observed in our studies could also be a downstream modulatory result of ERK1/2, as has been shown to occur in previous studies [22,33]. In conclusion, PAOPA causes a significant increase in the expression levels of the presynaptic vesicular phosphoprotein synapsin IIa in the dopaminergic regions of the striatum, mPFC, and NAc. Synapsin IIa plays a dominant role in maintaining neurotransmitter reserve pools and regulating exocytosis, which can eventually affect synaptic architecture and plasticity. As a result of its physiological role, decreased expression of this crucial protein has been implicated in the etiology of schizophrenia, and may reveal a crucial therapeutic point for the improved treatment of this challenging disorder. Overall, understanding the effects of PAOPA on synapsin II not only advances the development of this modulator into an effective new antipsychotic drug, but also enhances our global understanding of the etiology of schizophrenia.
Acknowledgements We are grateful for this work to be funded by the Canadian Institutes of Health Research (CIHR) and the Ontario Mental Health Foundation (OMHF).
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Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA
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Dipannita Basu a , Yuxin Tian a , Patricia Hui a , Jayant Bhandari a , Rodney L. Johnson b , Ram K. Mishra a,∗
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Department of Psychiatry and Behavioural Neurosciences, McMaster University, 1200 Main St. W., Hamilton, ON, Canada L8N 3Z5 Department of Medicinal Chemistry, University of Minnesota, 308 Harvard Street SE, Minneapolis, MN 55455, USA
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Article history: Received 29 August 2014 Received in revised form 20 January 2015 Accepted 20 January 2015 Available online xxx
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Keywords: Allosteric drugs Schizophrenia Synapsin II Dopamine D2 receptor Antipsychotic drug
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Introduction
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The hallmark symptoms of schizophrenia include profound disturbances in thought, perception, cognition etc., which negatively impacts an individual’s quality of life. Current antipsychotic drugs are not effective in treating all symptoms of this disorder, and often cause severe movement and metabolic side effects. Consequently, there remains a strong impetus to develop safer and more efficacious therapeutics for patients, as well as elucidating the etiology of schizophrenia. Previous work in our lab has introduced a novel candidate for the treatment of this disease: the dopamine D2 receptor (D2R) allosteric modulator, 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA). We have previously shown that PAOPA, by selectively modulating D2R, can ameliorate schizophrenia-like symptoms in animal models, although the precise mechanism is presently not understood. Synapsin II is a presynaptic vesicular protein which has been strongly implicated in schizophrenia, as it is reduced in the prefrontal cortex of patients, and knockdown of this protein elicits schizophrenia-like phenotypes in animal models. Given the therapeutic effects of PAOPA and the role of synapsin II in schizophrenia, the objective of this study was to investigate the effect of chronic administration of PAOPA (45 days) on neuronal synapsin II protein expression in rodents. Immunoblot results revealed that the synapsin IIa, but not the IIb isoform, was increased in the dopaminergic regions of the striatum, nucleus accumbens, and medial prefrontal cortex. The results of this study implicate a role for modulation of synapsin II as a possible therapeutic mechanism of action for potential antipsychotic drug PAOPA. © 2015 Elsevier Inc. All rights reserved.
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Schizophrenia is a debilitating mental disorder which presents significant therapeutic challenges for the healthcare system. The etiology of schizophrenia is strongly associated with the dopaminergic system, with affected patients showing hyperdopaminergic
Abbreviations: cAMP, cyclic adenosine monophosphate; D2R, dopamine D2 receptor; ECL, enhanced chemiluminescence; ERK 1/2, extracellular signal-regulated kinase 1/2; GABA, ␥-amino butyric-acid; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; HRP, horseradish peroxidase; IP, intraperitoneal; MIF-1, melanocyte-inhibiting factor 1; mPFC, medial prefrontal cortex; NMDA, N-methyl-D-aspartate; NAc, nucleus accumbens; PAOPA, 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide; PBS, phosphate-buffered saline; PKA, protein kinase A; PLG, L-prolyl-l-leucyl glycinamide; PVDF, polyvinlidene; SDS, sodium dodecyl sulfate; SEM, standard error of mean; SNP, single nucleotide polymorphism. ∗ Corresponding author. Tel.: +1 905 525 9140 ext. 22396; fax: +1 905 522 8804. E-mail address: [email protected] (R.K. Mishra).
activity in the striatum and nucleus accumbens (NAc), and hypodopaminergic activity in cortical regions [23,27]. Furthermore, deficits in cortical glutamate activity have also been observed in patients with schizophrenia [14,43]. While the causality for these observations has yet to be identified, research has revealed deficits in multiple transcripts and proteins of presynaptic secretory machinery in this disorder [7,19,20]. Advancing our studies of these proteins can enhance our understanding of this disorder’s pathophysiology, and improve targeted treatment options. Synapsins are a neuron-specific family of presynaptic phosphoproteins, which have shown to play key roles in the mechanisms of synaptic function and neuronal exocytosis. These proteins have a range of effects in controlling neurite outgrowth and synaptogenesis, regulating neurotransmitter release, vesicle docking and maintenance of synaptic vesicle stores [7]. The effects of synapsins distinctly vary with the neurotransmitter system being studied. In glutamate and ␥-amino butyric-acid (GABA) neurons, synapsins act as positive modulators, whereby knockout of synapsins decrease the release of these neurotransmitters
http://dx.doi.org/10.1016/j.peptides.2015.01.004 0196-9781/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Basu D, et al. Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.01.004
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[4,24,25]. Conversely, knockout studies have shown that the release of catecholamines, including dopamine, is enhanced after synapsin function is lost [49]. Due to its role in controlling synaptic morphology and regulating neurotransmitter release, synapsins have become a focus of studies in understanding schizophrenia. Alternate splicing of three genes (SYN1–3) results in the expression of five different isoforms of the synapsin protein in humans [7]. Among the five different isoforms of synapsin proteins, the synapsin II subtype, composed of the synapsin IIa and IIb isoforms, has been of particular importance in the study of schizophrenia. Microarray analysis by Mirnics et al. [36] has demonstrated reductions in synapsin II in the prefrontal cortex of patients with schizophrenia. Also, studies conducted by Chen et al. [8,9] presented a positive association with select polymorphisms on the synapsin II gene, and increased susceptibility to schizophrenia. Another study of 37 pedigrees of Northern European descent found at least two single nucleotide polymorphisms (SNP) significantly associated with schizophrenia, most of which were in introns 5 and 6 of the synapsin II gene. In addition, one haplotype of the SNPs within these introns was significantly over-transmitted to affected offspring [40]. Similarly, results from our lab showed decreased levels of the synapsin II transcript in the dorsolateral prefrontal cortex of patients affected with schizophrenia [47]. The lifetime antipsychotic drug use in these studies has been shown to positively correlate with synapsin II expression, particular the synapsin IIa isoform, via interaction with dopamine D1 and D2 receptors (D2R) [10,26,41,47]. Finally, previous studies from our lab have also shown that knockdown of synapsin II globally [17,18] as well as specifically within the medial prefrontal cortical regions of animal models [16,19], induces schizophrenia-like behavioral abnormalities. Taken together, this evidence supports a significant role of the synapsin II protein within the etiology of schizophrenia. Our previous studies [3,21,28,30,42] have developed a wide range of ligands to modulate D2R, a target which is strongly implicated in the etiology of schizophrenia. These ligands have been designed based on the functional pharmacophore of the endogenous tripeptide l-prolyl-l-leucyl glycinamide (PLG, also known as melanocyte-inhibiting factor-1 [MIF-1]), and have been shown to modulate dopamine binding via interaction with an allosteric site on the D2R [34]. Allosteric modulators are proving to be a promising avenue for the development of more specific, effective and safer drugs [12,13,29]. The current study utilizes the potent allosteric MIF-1 peptidomimetic (3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]2-oxo-1-pyrrolidineacetamide, PAOPA) [50], which has shown to effectively prevent and reverse a range of schizophrenia-like behavioral abnormalities [2,15]. Characterization of the pharmacokinetic and toxicological properties of PAOPA have revealed this ligand to have metabolic properties comparable to current marketed antipsychotic drugs, without the movement, hematological and metabolic adverse events commonly associated with the use of these drugs [46]. When acutely administered, Tan et al. found that the half-life of PAOPA given orally to rats was 112 ± 12 min, and time to maximum concentration was 50 ± 10 min, which is shorter than most of the currently available antipsychotic drugs. Finally, it was found that the concentration of PAOPA was greatest in the striatum, frontal cortex, and hypothalamus, indicating that PAOPA can cross the blood–brain barrier [45]. The mechanism of action of PAOPA is currently being investigated. Characterization of PAOPA’s pharmacological properties has revealed that it modulates agonist binding to dopamine D2 and D4 receptors in a biphasic manner [48]. PAOPA has also shown to promote D2R internalization, and upregulate proteins involved in this process, revealing initial processes linked to its potential mechanism of therapeutic action [1]. However, the effect of PAOPA on the expression of the crucial presynaptic secretory proteins mentioned above is currently unknown. The objective of this
study was, therefore, to determine the in vivo effects of PAOPA on synapsin II protein expression levels to improve our understanding of its mechanism of action as a potential new antipsychotic drug.
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Materials and methods
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Animals
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Twelve adult male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 200–250 g were individually housed at the McMaster Central Animal Facility. Animals were maintained under constant temperature and humidity, with a 12:12 light/dark cycle, and ad libitum access to food and water. All experiments were approved by the McMaster Animal Research Ethics Board, and conformed to the guidelines of the Canadian Council on Animal Care (animal utilization protocol # 10-08-59). Drug regimen and tissue collection The dopamine D2 allosteric peptide PAOPA was synthesized in the laboratory of Dr. Rodney L. Johnson (University of Minnesota, MN) as described previously [50], and peptide solutions (1 mg/mL) were prepared fresh daily. All rats were randomly divided into two groups (n = 6/group) and injected via intraperitoneal (IP) route daily for 45 consecutive days. The drug-treated group received chronic treatment of PAOPA at a 1 mg/kg dose and the control group was treated with 0.9% saline at the same dose. One hour following the final drug administration, rats were anaesthetized with isoflurane and sacrificed by decapitation. Rat brains were removed. The striatum, cerebellum, NAc and medial prefrontal cortex (mPFC) were dissected over ice, and stored at −80◦ C until use. Immunoblot Collected rat striatum, NAc, mPFC and cerebellum were homogenized using a pestle in phosphate-buffered saline (PBS) with Complete Mini, EDTA-free protease inhibitor tablet (Hoffmann La-Roche, Mississauga, ON) and PhosStop phosphatase inhibitor cocktail tablet (Hoffmann La-Roche, Mississauga, ON). Fifteen micrograms of sample was resuspended in 2× sodium dodecyl sulfate (SDS) sample buffer, boiled for 10 min and separated by electrophoresis on 10% acrylamide gels, using protocols previously described by our lab [11]. Briefly, separated proteins were transferred to a 0.45 M polyvinylidene (PVDF) membrane and blocked for 1 h in 5% skim milk at room temperature. The membranes were then labeled overnight with 1:2500 polyclonal synapsin II antibody (Enzo Life Sciences, Ann Arbor, MI) followed by 1.5 h of 1:5000 antirabbit IgG horseradish peroxidase (HRP)-linked whole secondary antibody from donkey (Sigma Aldrich, St. Louis, MO). Protein bands were visualized using enhanced chemiluminescence (ECL) substrates. A housekeeping protein—glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—was used to normalize the changes in protein expression and was detected using anti-GAPDH antibody produced in mouse (1:10,000; Millipore, Billerica, MA). Data analysis All the statistical analyses were carried out using GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA). ImageJ 1.43M (National Institutes of Health) was used to quantify the intensity of protein bands. Student’s t-test was used to compare differences in protein expression between control and treated groups for immunoblot assays. A significant expression change was considered when p < 0.05. Data are presented as the percent change compared to control ± SEM.
Please cite this article in press as: Basu D, et al. Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.01.004
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Fig. 1. Effect of chronic PAOPA administration on expression of synapsin IIa and synapsin IIb. Quantified immunoblot showing chronic PAOPA treatment (1 mg/kg) to: (A) Significantly increase expression of synapsin IIa in treated rats by 48% in the striatum (*p = 0.0126); 119% in the nucleus accumbens (NAc) (*p = 0.0103); and 30% in the medial prefrontal cortex (mPFC) (*p = 0.0296); (B) not significantly change expression of synapsin IIb in treated rats in the striatum, NAc, and mPFC. Similarly, chronic PAOPA treatment did not significantly change expression of both synapsin IIa and IIb in the cerebellum. Data are shown with representative immunoblots, and expressed as a percentage of average control ± SEM where *p < 0.05 (Student’s t-test) and n = 6/group.
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Results Effect of PAOPA on the expression of synapsin IIa in the striatum, nucleus accumbens, and mPFC Synapsin expression is regulated by dopaminergic pathways which can be affected by long-term ligand modulation [10,41]. In this study, PAOPA was observed to have significant effects in modulating synapsin IIa expression within all three brain regions of treated animals, leading to an increase in levels of synapsin IIa by 48% in the striatum (*p = 0.0126), 119% in the nucleus accumbens (*p = 0.0103) and 30% in the mPFC (*p = 0.0296; Fig. 1A). Expression of the housekeeping protein, GAPDH, was also analyzed within the same samples to normalize the expression of synapsin IIa, and was shown not to change with PAOPA treatment (data not shown). Effect of PAOPA on the expression of synapsin IIb in the striatum, nucleus accumbens, and mPFC
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The synapsin IIb isoform has shown to be expressed and regulated differently than the synapsin IIa isoform in various brain regions [35]. Unlike the changes observed in synapsin IIa protein expression, quantitative analysis of synapsin IIb expression in PAOPA treated rats showed no elevation in protein expression in the striatum or mPFC, and a slight but non-significant increase in the NAc (Fig. 1B). Expression of the housekeeping protein, GAPDH, was again analyzed within the same samples to normalize the expression of synapsin IIb, and was shown not to change with PAOPA treatment (data not shown).
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Effect of PAOPA on the expression of synapsin II in the cerebellum
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To relate the modulatory effects of PAOPA observed within the striatum, NAc, and mPFC, to interactions with D2R, the cerebellum was used as a region of reference. The cerebellum is a region is considered to have a negligible density of D2R, and therefore used as a negative control to study D2-related effects [31,38,44]. Analysis of synapsin IIa and IIb showed no significant change in expression in the cerebellum (Fig. 1), indicating that PAOPA had no effect on levels of protein expression in this region of chronically treated rats. There was also no change in the expression of the housekeeping protein, GAPDH, which was used to normalize the results (data not shown).
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Discussion
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The presynaptic protein synapsin II has shown significant involvement in the etiology of schizophrenia, and previous studies
from our lab have linked modulation of this protein to activation of dopamine receptors [10,41]. The current study shows that a significant increase in synapsin IIa expression occurs in dopaminergic regions strongly implicated in schizophrenia, including the striatum, mPFC, and NAc, following chronic administration of PAOPA. These increases appeared to be isoform specific as synapsin IIb expression remained unchanged, as well as region specific, as expression of both isoforms was unchanged in the relatively nondopaminergic region of the cerebellum. Studies have shown the importance of synapsin IIa isoform, due to its ability to rescue the defects in synaptic depression and synaptic vesicle density observed in hippocampal neurons of triple synapsin knockout mice [24]. This phenotypic rescuing was not statistically achieved with reintroduction of any other synapsin isoform. Similarly, reintroduction of synapsin IIa also rescued the enhanced catecholamine exocytosis in triple synapsin knockout adrenal chromaffin cultured cells [49]. In this study, PAOPA enhanced expression specifically of the synapsin IIa isoform, and therefore, appears to be modulating the isoform with the more phenotypically robust rescuing effects. Regulation of synapsin II expression has previously been shown to occur by modulation of the dopamine D1 and D2 receptors [10,41]. In this current study, increases in synapsin IIa were observed in dopamine rich regions, including the striatum, NAc and mPFC. However, no such effect was observed in the cerebellum, which is often used as a negative control to attribute observed in vivo changes to D2R [31,38,44]. Previous in vitro and in vivo studies with PAOPA have shown its specificity in modulating D2 and D4 receptors, with no significant effects occurring at D1 or D3 receptors, or the ␣2-adrenergic receptor [48]. Additionally, within in vivo models, PAOPA can potentiate the effect of D2 agonists, such as apomorphine [6,37], and attenuate effects of the D2-specific antagonist, haloperidol [6]. These interactions have been shown to occur through an allosteric interaction with D2R, as PAOPA does not compete with radioligands binding to the D2 orthosteric site [34]. Therefore, the observed effects of PAOPA on the expression of synapsin IIa may most likely be occurring via its allosteric effects on D2R. Studies have shown opposing effects of dopamine D1, compared to D2 modulation, on regulating synapsin II expression. Stimulation of D1 receptor activity by agonists, or attenuation of D2 activity by antagonists, results in increased synapsin II levels [11,41]. The use of PAOPA, has previously been shown to promote internalization of the D2R, thereby decreasing overall availability of this receptor, similar to an antagonist, but with a more fine-tuned and subtle technique [1]. Reduced post-synaptic D2 activity caused by PAOPA-induced receptor internalization within the striatum, NAc and mPFC, is hypothesized to lead to decreased
Please cite this article in press as: Basu D, et al. Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.01.004
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inhibition of cyclic adenosine monophosphate (cAMP), causing increased protein kinase A (PKA) stimulation, which eventually results in Ap-2␣ transcription factor promoted enhancement of synapsin II transcription [41]. Enhanced expression of synapsin II has been shown to occur with the use of current antipsychotic drugs, and may reveal a convergent therapeutic point for their mechanisms of action. Initial studies by Chong et al. [10] have shown the typical antipsychotic drug, haloperidol, to increase levels of synapsin II maximally within the striatum, and to a lesser extent, within the mPFC and NAc of treated rats. Subsequent studies by Guest et al. [26] and Tan et al. [47] showed levels of synapsin II to correlate with antipsychotic drug usage, with atypical antipsychotic drugs showing an increased likelihood of affecting expression of this molecule. Increased synapsin II within the regions investigated here can have important consequences in normalizing the dysregulated neurotransmission repeatedly observed in the etiology of schizophrenia. Glutamate projections from the healthy cortex stimulate inhibitory GABA neurons in the basal ganglia system, which thereby reduces subsequent neurotransmitter release, including dopamine, from this region. In vivo SPECT imaging studies on clozapine-treated schizophrenia patients using the radiotracer [123 I]CNS-1261, an intrachannel NMDA receptor ligand, have found decreased binding in brain regions such as the thalamus and striatum compared to healthy subjects, adding some support to the NMDA hypofunction hypothesis of schizophrenia [39]. A decrease in cortical glutamate release would yield less stimulation of inhibitory GABA neurons, resulting in neuronal disinhibition, and increased dopamine release relayed back into the striatum and NAc [5,32,39,43]. PAOPA can attenuate this pathological state by increasing synapsin II levels within the prefrontal cortex, and normalizing glutamate release. Synapsin IIa, in particular, has been found to be specifically associated with transmission of glutamate-containing synaptic vesicles [24]. Therefore, PAOPA-mediated normalization of synapsin II levels can indicate restoration of glutamatergic signaling within the prefrontal cortex, which eventually cascades to restore normal dopamine release into the striatum and nucleus accumbens. Additionally, increased synapsin IIa levels in the striatum and NAc can act directly as a negative regulator of dopamine release [49], resulting in decreased dopamine release within these regions. The effects of increased expression of synapsin II, particularly the phenotypically more robust isoform synapsin IIa, can therefore, have therapeutic effects in normalizing neurotransmission imbalances observed in schizophrenia. Synapsin activity is often regulated by kinase mediated phosphorylation, and therefore, these proteins may act as downstream recipients of receptor activated kinase molecules [41]. Previous studies from our lab have demonstrated the downstream effect of PAOPA on kinases, such as extracellular signal-regulated kinase 1/2 (ERK 1/2) [1]. Therefore, expression of synapsin, as observed in our studies could also be a downstream modulatory result of ERK1/2, as has been shown to occur in previous studies [22,33]. In conclusion, PAOPA causes a significant increase in the expression levels of the presynaptic vesicular phosphoprotein synapsin IIa in the dopaminergic regions of the striatum, mPFC, and NAc. Synapsin IIa plays a dominant role in maintaining neurotransmitter reserve pools and regulating exocytosis, which can eventually affect synaptic architecture and plasticity. As a result of its physiological role, decreased expression of this crucial protein has been implicated in the etiology of schizophrenia, and may reveal a crucial therapeutic point for the improved treatment of this challenging disorder. Overall, understanding the effects of PAOPA on synapsin II not only advances the development of this modulator into an effective new antipsychotic drug, but also enhances our global understanding of the etiology of schizophrenia.
Acknowledgements We are grateful for this work to be funded by the Canadian Institutes of Health Research (CIHR) and the Ontario Mental Health Foundation (OMHF).
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