Oxidative stress regulates DTNBPI dysbindin 1 expression and degradation via a pest sequence in it s c terminus

Oxidative Stress Regulates DTNBP1/Dysbindin-1 Expression and Degradation via a PEST Sequence in its C-terminus YAP MEI YI ALICIA (B.Sc. (Hons), NUS) SUPERVISOR: ASSOCIATE PROFESSOR LO YEW LONG CO-SUPERVISOR: ASSOCIATE PROFESSOR ONG WEI YI A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2013 Declaration DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _______________________ YAP MEI YI ALICIA 19 August 2013 I Acknowledgements ACKNOWLEDGEMENTS I would like to offer my deepest appreciation to my supervisor, Associate Professor Lo Yew Long, Department of Anatomy, National University of Singapore, for his utmost support throughout the course of my project; and to my co-supervisor Associate Professor Ong Wei Yi, Department of Anatomy, National University of Singapore, for his patient guidance and encouragement throughout my entire candidature. His patience, constructive criticisms and kind understanding have played a major role in the accomplishment of this thesis. I would also like to extend my utmost gratitude to my mentors; Jinatta Jittiwat, Kazuhiro Tanaka and Tang Yan for imparting invaluable techniques that are essential in this study and without them this thesis would have remained a dream. To my fellow seniors; Chia Wan Jie, Kim Ji Hyun, Ma May Thu, Ng Pei Ern Mary, and Poh Kay Wee, I am deeply grateful for your timely advice and never ending encouragement that assisted me in overcoming obstacles faced. My thanks and appreciation also goes to Ms Ang Lye Geck, Carolyne and Mdm Dilijit Kour D/O Bachan Singh for their secretarial assistance. To my peers; Chew Wee Siong, Ee Sze Min and Loke Sau Yeen, and juniors; Chan Vee Nee, Shalini D/O Suku Maran, Tan Siew Hon Charlene, and Tan Wee Shan Joey, a big thank you for standing by me during the completion of this thesis. Last but not least, to my dad and mom; Winson and Cathryn, and sister; Gloria, thank you for always believing that I could achieve greater heights and to my beloved; Jonathan, for his endless support and understanding. II Table of Contents TABLE OF CONTENTS CONTENTS PAGE Declaration Page I Acknowledgements II Table of Contents III Summary VI List of Figures VII Abbreviations IX CHAPTER 1 – INTRODUCTION 1 1.1 Schizophrenia 2 1.2 Role of genetics in schizophrenia 3 1.2.1 Role of dysbindin-1 in schizophrenia 5 1.2.1.1 Dysbindin and its protein family 5 1.2.1.2 Dysbindin and its functions 6 1.3 Role of environment in schizophrenia 14 1.4 Role of oxidative stress in schizophrenia 16 1.4.1 Kainic acid-mediated excitotoxicity 18 1.5 PEST sequence as a protein degradation signal peptide 20 CHAPTER 2 – HYPOTHESIS AND AIMS 21 CHAPTER 3 – CHANGES IN DYSBINDIN-1 CORE PROMOTER ACTIVITY AFTER OXIDATIVE STRESS 3.1 Introduction 23 3.2 Materials and Methods 26 24 3.2.1 Cells and Constructs 26 3.2.2 Dual-Luciferase® Reporter Assay 28 3.2.3 Effect of oxidative stress on dysbindin-1A core promoter 29 III Table of Contents activity 3.2.4 Effect of WP631 on dysbindin-1A core promoter activity 3.3 Results 3.3.1 Effect of oxidative stress on dysbindin-1A core promoter 29 30 30 activity 3.4 Discussion 33 CHAPTER 4 – ROLE OF OXIDATIVE STRESS AND PEST SEQUENCE ON DYSBINDIN-1 EXPRESSION IN VITRO 36 4.1 Introduction 37 4.2 Materials and Methods 40 4.2.1 Cells and Constructs 40 4.2.2 Effect of oxidative stress on dysbindin-1A expression 42 4.2.3 Effect of the proteasome inhibitor on dysbindin-1A 42 expression 4.2.4 Effect of the PEST sequence of dysbindin-1A on protein 43 expression after oxidative stress 4.2.5 Western blot analyses 4.3 Results 4.3.1 Effect of oxidative stress and oxygen free radicals on 43 46 46 dysbindin-1A expression 4.3.2 Effect of proteasome inhibitor and the PEST sequence on 48 dysbindin-1A expression 4.3.3 Effect of the PEST sequence of dysbindin-1A on protein 49 expression after oxidative stress 4.4 Discussion 52 CHAPTER 5 – ROLE OF KAINATE EXCITOTOXICITY ON DYSBINDIN-1 EXPRESSION IN VIVO 56 5.1 Introduction 57 5.2 Materials and Methods 60 IV Table of Contents 5.2.1 Kainate injections 60 5.2.2 Immunohistochemistry 60 5.2.3 Real time RT-PCR analyses 62 5.2.4 Western blot analyses 63 5.3 Results 5.3.1 Effect of oxidative stress on dysbindin-1 localization in the 64 64 hippocampal formation 5.3.2 Effect of oxidative stress on dysbindin-1 mRNA and protein 68 expression in vivo 5.4 Discussion 71 CHAPTER 6 - CONCLUSION 74 CHAPTER 7 - REFERENCES 79 V Summary SUMMARY Variation in the gene encoding dysbindin-1, dystrobrevin binding protein 1 (DTNBP1), has been associated with schizophrenia. Dysbindin-1 protein levels are reduced in several brain areas including the hippocampus in affected individuals. However, this may not be related to decrease DTNBP1 mRNA expression. Increasing number of studies has shown that oxidative stress resulting from the production of reactive oxygen species and nitrogen reactive species is an etiological factor in schizophrenia. Therefore, we tested whether oxidative stress modulates DTNBP1 mRNA expression. Using DTNBP1 transcription reporter, we found that oxidative stress induced DTNBP1 mRNA expression and this induction was abolished by a putative Sp1 inhibitor, WP631. Intriguingly, oxidative stress and free radicals induced degradation of the dysbindin-1 protein, as confirmed by treatment with the free radical scavenger, PBN, the proteasome inhibitor, MG132, and by monitoring protein turnover of a truncated dysbindin-1 protein, devoid of PEST domain. Excitotoxic injury and oxidative stress, triggered by intracerebroventricular kainate injections, resulted in increased number of dysbindin-1 expressing neurons in the dentate gyrus and CA1, but decreased number of neurons in CA3 of the hippocampus, at 1 day post-injection. Together, these findings suggest that, while oxidative stress increases DTNBP1 transcription, it strongly promotes dysbindin-1 protein degradation, leading to the reported loss of dysbindin-1 protein in the brain of schizophrenia patients. VI List of Figures LIST OF FIGURES FIGURE PAGE CHAPTER 1 Figure 1.2.1.2 Schematic structure of dysbindin-1 isoforms in human. Figure 1.4.1 6 Schematic diagram of KA-mediated neuronal cell death pathway. 18 Figure 3.2.1 Partial genomic sequence of the promoter sequence of dysbindin-1A. 26 Figure 3.3.1 Fold change in firefly:renilla luciferase activity of SH-SY5Y cells after 24 h. 30 Figure 4.3.1 Analysis of untransfected SH-SY5Y cells treated with or without PBN 24 h after 500 µM H2O2 treatment. 46 Figure 4.3.2 Analysis of SH-SY5Y cells treated with or without MG132 after 24 h. 48 Figure 4.3.3 Western blot analysis of SH-SY5Y cells transfected with dysbindin-1A, or without its PEST sequence or vector control. 49 CHAPTER 3 CHAPTER 4 CHAPTER 5 Figure 5.3.1.1 Dysbindin-1 immunoreactivity in the HF of rats post 1 day KA injection. Figure 5.3.1.2 Dysbindin-1 immunoreactivity in the HF of rats 2 weeks post KA injection. Figure 5.3.1.3 Number of positive dysbindin-1 labelled neurons in the rat HF, 1 day and 2 weeks 64 65 66 VII List of Figures after KA or saline treatment ipsilateral to injection. Figure 5.3.2.1 Dysbindin-1 expression in the rat HF 1 day after KA treatment. Figure 5.3.2.2 Dysbindin-1 expression in the rat HF 2 weeks after KA treatment. 68 70 VIII Abbreviations ABBREVIATIONS AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ARG Apoptosis response gene BLOC-1 Biogenesis of lysosome-related organelles complex 1 BMD Becker muscular dystrophy Ca2+ Calcium CAT Catalase CCD Coiled coil domain Cdk1 Cyclin-dependent kinase 1 COMT Catechol-O-methyltransferase CTR C-terminus region DAB 3,3-diaminobenzidine tetrahydrochloride DG Dentate gyrus DGC Dystrophin glycoprotein complex DISC Disrupted-in-schizophrenia DMD Duchenne muscular dystrophy DNA Deoxyribonucleic acid DTNBP1 Dysbindin-1 ER Endoplasmic reticulum erbB-4 Tyrosine-protein kinase receptor Fe2+ Iron GFP Green fluorescent protein IX Abbreviations GPx Glutathione peroxidise GSH Glutathione H2O2 Hydrogen peroxide Hax-1 HCLS1-associated protein X-1 HF Hippocampal formation HSF2 Heat shock transcription factor 2 IκBα Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor KA Kainic acid LB Lysogeny broth LROs Lysosome-related organelles Mdx Dystrophin-null MG132 Proteasome inhibitor mM Millimolar non-NMDA Non- N-methyl-D-aspartic acid NRG1 Neuregulin-1 NTR N-terminus region PBN Phenyl- N- tert-butylnitrone PBS Phosphate-buffered saline PCR Polymerase chain reaction PEST Proline-Glutamate-Serine-Threonine PKB/Akt Protein kinase B PRODH Proline dehydrogenase PUFAs Polyunsaturated fatty acids X Abbreviations PVDF Polyvinylidene difluoride RNA Ribonucleic acid RNS Reactive nitrogen species ROS Reactive oxygen species RT-PCR Reverse transcription polymerase chain reaction SCF Stem cell growth factor Sdy Dysbindin-null siRNA Small interfering ribonucleic acid SNPs Single nucleotide polymorphisms SOD Superoxide dismutase Sp1 Specificity protein 1 TBARS Thiobarbituric reactive substances TBS Tris-buffered saline TRIM32 Tripartite motif-containing protein 32 XI Chapter 1 Introduction SECTION I INTRODUCTION 1 Chapter 1 Introduction 1.1 Schizophrenia Schizophrenia is a severe and complex mental disorder (Mueser and McGurk, 2004; Lindenmayer et al., 2007) with an estimated lifetime prevalence of 0.72% (McGrath et al., 2008). That means about 50 million people alive today are or will be affected with this disorder in their lifetime. Symptoms of schizophrenia are evident usually in late adolescence and early adulthood, with an incidence equal among sexes, though reports have shown that females tend to display its symptoms earlier than males, and with a less severe form of schizophrenia (Angermeyer et al., 1990). Despite males and females having equal chances of suffering from schizophrenia, its occurrence differs across the world, within each country and even one’s household (Kirkbride et al., 2007). This affliction is expressed in three core features: (1) positive symptoms such as disorganized speech, hallucinations, and delusions (Andreasen et al., 1995; Lindenmayer et al., 2007), (2) negative symptoms including absence of motivation, inability to experience pleasure, and poverty of speech (Andreasen et al., 1995; Lindenmayer et al., 2007; Mäkinen et al., 2008), and (3) cognitive deficits such as impaired working memory, reduced executive function, and conceptual disorganization (Green et al., 2000; Sharma and Antonova, 2003; Lesh et al., 2011). In view of these symptoms, schizophrenia patients could potentially face difficulties in their daily lives which include, work, school, parenting, and dealings with interpersonal relationships (Mueser and McGurk, 2004). Current drug treatments often ameliorate the positive symptoms, but have little effect on the negative symptoms (Mäkinen et al., 2008; Miyamoto et al., 2 Chapter 1 Introduction 2012) or cognitive deficits (Fumagalli et al., 2009; Hill et al., 2010; Tcheremissine et al., 2012), both of which are more debilitating than the positive features of the disorder (Milev et al., 2005; Kurtz, 2006; Tabares-Seisdedos et al., 2008). None of the attempts to develop effective treatments for these features of schizophrenia over the last decade has succeeded (Hill et al., 2010; Miyamoto et al., 2012). Considering the detrimental effects of schizophrenia and a slow progress in effective treatments, it is pertinent to further investigate factors that could contribute to a lower schizophrenia-susceptibility rate possibly through in vivo and in vitro studies to aid the understanding of its etiology and pathogenesis of schizophrenia. 1.2 Role of genetics in schizophrenia Taking into account the severity of schizophrenia in the human population, progressive studies are being carried out to search for the exact cause(s) of schizophrenia. Evidence has shown that genetic and environmental factors may add to the risk for the onset of schizophrenia (Tsuang, 2000; Sullivan, 2005; van Os et al., 2008). Studies have highlighted that the former could have a larger impact than the latter on the susceptibility of an individual to schizophrenia (Kendler et al., 1994). Schizophrenia is highly heritable and evolves from a particular group of genes, which determines an individual’s genetic vulnerability. It has been shown that individuals who have a first degree relative or a monozygotic twin with the disease run the greatest risk for developing schizophrenia at 6.5% and 40%, respectively (Picchioni and Murray, 2007). 3 Chapter 1 Introduction Picchioni et al. (2007) also suggest that the onset of schizophrenia could involve many genes, each contributing to a small effect. The known and putative candidate genes of schizophrenia include dysbindin-1 (DTNBP1) (Blake et al., 1999), catechol-O-methyltransferase (COMT) (Shifman et al., 2002), disruptedin-schizophrenia (DISC) (Blackwood et al., 2001), erbB-4 (a receptor tyrosineprotein kinase) (Sastry and Sita Ratna, 2004), neuregulin-1 (NRG1) (Stefansson et al., 2002), and proline dehydrogenase (PRODH) (Li et al., 2004b). However, similar to many other complex diseases, genes that predispose to schizophrenia are elusive and are non-exhaustive. Although there seems to be an increase in the number of possible schizophrenia susceptibility genes, DTNBP1 remains to be the more widely accepted candidate gene of schizophrenia (Allen et al., 2008; Sun et al., 2008). This is mainly due to its discovery as the first schizophrenia susceptibility gene (Straub et al., 2002; Williams et al., 2005), and together with many converging evidences supporting its potential role in psychosis and cognition (Barch, 2005; Fallgatter et al., 2006; Suchankova et al., 2009). 4 Chapter 1 Introduction 1.2.1 Role of dysbindin-1 in schizophrenia 1.2.1.1 Dysbindin and its protein family The dysbindin family is made up of 3 different members, dysbindin-1, dysbindin-2, and dysbindin-3, and are found to be expressed in many species but are more commonly studied in humans (Talbot et al., 2009). There are 8 human dysbindin transcripts (i.e. dysbindin-1A, -1B, -1C, -2A, -2B, -2C, -3A, and -3B) as reported in the National Center for Biotechnology Information (NCBI) database. Dysbindin-2A, a 261 amino acid protein, is one of the three dysbindin-2 isoforms. This full length isoform of dysbindin-2 is the only dysbindin isoform known to possess a signal peptide and is postulated to be a precursor of a secretory protein (Brunig et al., 2002). Dysbindin-2B is similar to its full length isoform, except for its truncated N-terminus region (NTR). This isoform was discovered to be an apoptosis response gene (ARG) that was stimulated upon the inactivation of a stem cell growth factor (SCF) (Haenggi and Fritschy, 2006). Since a reduction or inhibition of programmed cell death has resulted in abnormal neuronal development (Rapaport et al., 1991), dysbindin-2B, an ARG, could be involved in the normal development of the nervous system. Dysbindin-2C is relatively similar to its 2B isoform, with the former having a shorter C-terminus region (CTR). Its exact function has yet to be elucidated but studies have shown that it is a protein secreted independent of the endoplasmic reticulum (ER) and Golgi complex (Kumagai et al., 2001). Dysbindin-3A is a 176 amino acid protein, one of the two isoforms identified in dysbindin-3. Till date, no other dysbindin-3A isoform has been found in other species except in humans. Dysbindin-3B is a 20 5 Chapter 1 Introduction amino acid protein shorter than its 3A isoform (Talbot et al., 2009). Similar to dysbindin-2C, both dysbindin-3A and -3B are found to be proteins secreted in a non-classical manner, independent of the ER and Golgi complex (Talbot et al., 2009). The main structural difference that distinguishes dysbindin-1 from the rest of its members is the presence of the coiled coil domain (CCD) which will be described later in this section. Of greater interest, dysbindin-1 unlike its other members has shown to be significantly associated with the pathogenesis of schizophrenia. Hence, dysbindin-1 will be the focus of this thesis. 1.2.1.2 Dysbindin-1 and its functions Figure 1.2.1.2 Schematic diagram of dysbindin-1 isoforms in human. Dysbindin-1 isoforms are characterized by 3 main regions: 1) C-terminus region (CTR), 2) Coiled coil domain (CCD), and 3) N-terminus region (NTR). Dysbindin-1A is the full length dysbindin isoform, while dysbindin-1B and dysbindin-1C are exactly like its full length isoform except for a truncated CTR which lacks its PEST domain (blue region), or the NTR, respectively [Adapted from (Talbot et al., 2009)]. Dysbindin-1 is first discovered as a protein binding partner of dystrobrevin, a dystrophin-related protein (Benson et al., 2001). Studies have found mutations in the gene expressing dystrophin as the cause of Duchenne and Becker 6 Chapter 1 Introduction muscular dystrophy (DMD and BMD, respectively) (Blake et al., 1999). A loss and reduced level of dystrophin were reported in patients with DMD and BMD, respectively (Burdick et al., 2006). Dystrophin is a major component of the dystrophin glycoprotein complex (DGC), which is essential in the maintenance of muscle membrane integrity and modulation of extracellular signals to the cytoskeleton (Nian et al., 2007; Luciano et al., 2009). DGCs found in the muscle fibers play an integral role in providing structural support and relaying important signals, while DGCs present in the brain may be involved in neurotransmission between GABAnergic (Brunig et al., 2002) and glutamatergic neurons (Haenggi and Fritschy, 2006). Moreover, in a dystrophin-null (mdx) mouse model, longterm memory and learning abilities were impaired (Vaillend et al., 2004). Therefore, further studies on the interactions of brain DCGs with their component proteins such as dystrobrevins could shed light and probably account for the learning deficit observed in 18-63% of DMD and 3-25% of BMD patients (Rapaport et al., 1991; Kumagai et al., 2001; Talbot et al., 2009). Dystrobrevin is one of the major interacting protein partners of DGC and there are two main dystrobrevins, the α-isoform which is commonly found in muscles, and the βisoform which is present in the brain. Since β-dystrobrevins are expressed in nerve cells, unlike α-dystrobrevins which are usually found in muscle cells, studies on the potential binding partners of β-dystrobrevin could explain its association to cognitive deficit observed in patients with this disorder. In 1999, dysbindin-1 was first discovered as a novel β-dystrobrevin binding partner via the yeast two-hybrid screening of a mouse cDNA library 7 Chapter 1 Introduction (Blake et al., 1999). Concurrently, Straub et al. (2002) have identified many single nucleotide polymorphisms (SNPs) and risk haplotypes in different regions along DTNBP1 that were significantly correlated to schizophrenia. Interest on DTNBP1 grew as it was found to be the first schizophrenia-susceptibility gene via positional cloning (Straub et al., 2002). Genome-wide association studies and bioinformatics analysis have also concluded DTNBP1 as the most promising schizophrenia candidate gene (Allen et al., 2008; Sun et al., 2008). Schizophrenia is highly heritable (Owen et al., 2002; Gejman et al., 2010), and studies on its genetic risk factors can provide important clues to its causes and cellular abnormalities. Among the many proposed genetic risk factors in schizophrenia (Sun et al., 2008; Gejman et al., 2010) are SNPs or multi-SNP haplotypes of the dysbindin-1 gene, DTNBP1. While association of these variants with schizophrenia have not met the high level of significance (p < 10-8) required in large-scale, genome-wide association studies, they have been substantiated in 21 studies on smaller, less heterogeneous populations in Asia, Europe, and the U.S. (Talbot et al., 2009; Maher et al., 2010; Rethelyi et al., 2010; Voisey et al., 2010; Fatjo-Vilas et al., 2011). One or more DTNBP1 risk SNPs are associated with the severity of negative symptoms (Fanous et al., 2005; DeRosse et al., 2006; Wirgenes et al., 2009) and cognitive deficits (Burdick et al., 2006; Burdick et al., 2007; Donohoe et al., 2007; Zinkstok et al., 2007; Fatjo-Vilas et al., 2011) in schizophrenia. These risk SNPs are more evident in a specific group of schizophrenia cases distinguished by earlier onset in adulthood and more prominent cognitive deficits and both negative and positive symptoms (Wessman 8 Chapter 1 Introduction et al., 2009). Moreover, studies have also found that individuals who possess SNPs in DTNBP1 associated with schizophrenia but do not display obvious symptoms of schizophrenia, exhibit cognitive deficit such as working memory and attention impairment (Burdick et al., 2006; Luciano et al., 2009). Genetic variation in DTNBP1 is thus associated with schizophrenia in diverse populations and with features of the disorder for which we lack adequate treatments. How DTNBP1 risk variants affect the protein encoded is unclear (Tang et al., 2009; Dwyer et al., 2010), but it is known that based on postmortem analysis on the brains of schizophrenia patients, dysbindin-1 gene and protein expression are reduced compared to its matched-paired controls (Weickert et al., 2008; Tang et al., 2009). Specifically, levels of dysbindin-1 are reduced in synaptic tissue of the dorsolateral prefrontal cortex, auditory association cortices, and hippocampal formation (HF) in 67-93% of the schizophrenia cases studied to date (Talbot et al., 2004; Tang et al., 2009; Talbot et al., 2011). Given convincing evidence showing strong correlation between dysbindin-1 and schizophrenia, further studies and analysis on the factors that affect dysbindin-1 expression could potentially provide important clues to the pathogenesis and pathophysiology of schizophrenia. The DTNBP1 gene which translates into the dysbindin-1 protein is found at the chromosome locus 6p22.3 in humans and 17 in rats. It is relatively abundant in the body, including the brain (Talbot et al., 2004). There are three major transcripts namely dysbindin-1A, -1B, -1C (Figure 1.2.1.2). Dysbindin-1A is known to be the full length isoform, a 351 amino acid protein expressed in 9 Chapter 1 Introduction humans and 352 amino acid protein expressed in rats. Dysbindin-1B is similar to its full length isoform except for a truncated CTR and is a 303 amino acid protein found in humans but not expressed in rats. Dysbindin-1C on the other hand is an isoform that lacks a NTR, and is a 270 amino acid protein. It is detected in humans but its protein length in rats could not be determined as there is a lack of information on this dysbindin paralog (Talbot et al., 2009). Numerous serine and threonine kinases sites such as protein kinase B (PKB/Akt) and cyclin-dependent kinase 1 (Cdk1) are found in the NTR of dysbindin-1A and -1B (Talbot et al., 2009). Though its exact function has yet to be elucidated, phosphorylation of these sites in the NTR could affect protein-protein binding in the CCD (Talbot et al., 2009). The CCD is a region made up of many seven-residue repeats with each repeat consisting of alternate hydrophilic and hydrophobic residues, forming alpha helices which are able to bind and interact with other proteins with CCD (Lupas, 1996; Lupas and Gruber, 2005). It is thus hypothesized that dysbindin-1 is likely to form interactions with its binding partners at its CCD and thus eliciting its functions (Talbot et al., 2009). Of greater interest, the PEST domain (i.e. blue region in Figure 1.2.1.2) present in the CTR is a hydrophilic motif that acts as a target for degradation upon phosphorylation (Rechsteiner and Rogers, 1996; Singh et al., 2006) (please refer to Section 1.5 for more details on the PEST domain). Dysbindin-1 is widely expressed in the brain, specifically in the axon fibers of the corpus callosum, specific group of axon terminals such as the mossy-fiber terminal of the hippocampus and cerebellum, and neuropil of the hippocampus, 10 Chapter 1 Introduction neocortex and substantia nigra (Benson et al., 2001). The key and potential functions of dysbindin-1 are believed to be mediated by the different binding partners it is associated with. Ring finger protein 151 (RNF151), a known binding partner of dysbindin-1, is found to be located in the spermatids and is postulated to be involved in spermatogenesis (Nian et al., 2007). Interaction between dysbindin-1 and RNF151 is found to induce the formation of acrosome (Nian et al., 2007), which is an organelle found at the tip of sperm containing digestive enzymes, allowing the fusion between a sperm and ovum (Green, 1978). The presence of putative binding factors of dysbindin-1 (e.g. transcription factor IIIB, isoform 3 and cyclin A2), transcription factor binding sites (i.e. Sp1 and NF-1) found in the promoter region of dysbindin-1, and levels of DTNBP1 gene and protein peaking during cell proliferation in prenatal events suggest its vital role in cell development (Talbot et al., 2009). Besides its role in cell development, the presence of Sp1 (specificity protein 1) transcription factor binding sites in dysbindin-1 promoter also suggests a neuroprotective role involved. Cultured cerebrocortical neurons which overexpress full length Sp1 were found to be more resistant to hydrogen peroxide induced-oxidative stress (Ryu et al., 2003). Similarly, despite being deprived from serum, cell viability in cultured cerebrocortical neurons which overexpressed dysbindin-1 was increased and decreased when the cells were treated with an siRNA inhibitor against dysbindin1 (Numakawa et al., 2004). Taken together, dysbindin-1 may be involved in cell proliferation and development, and also regulate the population of neurons due to its anti-apoptotic effect as observed in cultured cerebrocortical neurons. Since 11 Chapter 1 Introduction dysbindin has a significant role in neuronal growth and proliferation, individuals who are carriers of the DTNBP1 risk SNPs may be deficient in normal neuronal development, and hence may account for the smaller brain volume observed in them as compared to non-carriers (Narr et al., 2009). The main function of dysbindin-1 may be modulated by biogenesis of lysosome-related organelles complex 1 (BLOC-1), which is a multimer consisting of different proteins (in addition to dysbindin-1); BLOC-1 subunit-1 (BLOS-1), BLOS-2, BLOS-3, cappuccino, muted, pallidin, and snapin (Li et al., 2004c; Starcevic and Dell'Angelica, 2004). BLOC-1 is primarily involved in trafficking proteins to lysosome-related organelles (LROs) which are essential in its maturation and function (Setty et al., 2007). BLOC-1 binds to other protein complexes, such as AP-3, an adaptor protein assembly which recognizes proteins with a specific signal peptide, and delivers them to their target LROs (Bonifacino and Glick, 2004). Evidence has shown that the BLOC-1-AP-3 complex delivers proteins to LROs present in non-neuronal cells (e.g. melanocytes), and neurons (i.e. nerve terminals and axons) (Bonifacino and Glick, 2004; Newell-Litwa et al., 2007; Setty et al., 2007). Reduced levels of these complexes have reported abnormalities in the formation of synaptic vesicles (Newell-Litwa et al., 2009), and the expression of neurotransmitter receptors on cell surfaces (Iizuka et al., 2007). These abnormalities could induce neurobehavioral hallmarks of schizophrenia seen in mouse and Drosophila models which display similar phenotypes observed in schizophrenia patients (Bhardwaj et al., 2009; Cheli et al., 2010; Papaleo et al., 2012). For example, 12 Chapter 1 Introduction when placed in a new environment, dysbindin-1 deficient (sdy) mice did not habituate, unlike matched controls (Hattori et al., 2008; Bhardwaj et al., 2009). Habituation is a process of repeated exposure to the same non-threatening stimulus that usually results in decreased response, and this adaptive response reflects memory of past events (Bhardwaj et al., 2009). The absence of this response in sdy mice proposes that the loss of dysbindin-1 could lead to cognitive deficits affecting declarative and recognition memory, characteristics similar to those observed in schizophrenia patients (Cirillo and Seidman, 2003; Pelletier et al., 2005). Taken together, this suggests that dysbindin-1 plays a significant role in cognitive functioning and memory (Owen et al., 2004). Of specific interest, dependent on dose and in the absence of Ca2+ influx, dysbindin-1 also plays an essential role in intracellular and intercellular signalling, modulation of presynaptic retrograde and homeostatic neurotransmission (Dickman and Davis, 2009). Moreover, based on electron microscopy, dysbindin1 is found to be localized in axon terminals and dendrites of hippocampal neurons (Talbot et al., 2009), and sdy mice which have loss of dysbindin-1 expression showed decrease in the reserve pool of synaptic vesicles (Chen et al., 2008). In addition, cultured neurons with knockdown of dysbindin-1 showed reduced glutamate release (Numakawa et al., 2004). Loss of dysbindin-1 may therefore result in decreased communication between glutamatergic neurons that may lead to the affliction of schizophrenia expressed by the onset of its symptoms (Cherlyn et al., 2010). These reductions in dysbindin-1 may be a potential cause in the negative symptoms and cognitive deficits in schizophrenia 13 Chapter 1 Introduction since such behaviors are observed in sdy mice with loss of DTNBP1 (Talbot, 2009). Many studies on the dorsolateral prefrontal cortex in schizophrenia patients have concluded that reduced DTNBP1 transcription is not a cause of the reduction in dysbindin-1 (Weickert et al., 2008; Tang et al., 2009; Fung et al., 2011). In addition, despite many positive large-scale association studies in countries such as China (Shi and Liu, 2003), and England (Datta, 2003) yielding positive reports, negative studies have also been reported in the U.K. (Sanders et al., 2008; Sullivan et al., 2008). These latter reports have found no significant association to schizophrenia (Van Den Bogaert et al., 2003; van den Oord et al., 2003). In a separate study, the frequency of high-risk dysbindin-1 haplotypes (018%) as observed in the schizophrenia population was much lower than the frequency of dysbindin-1 reduction (73-93%) as seen in schizophrenia patients (Van Den Bogaert et al., 2003; van den Oord et al., 2003). Taken together, these discrepancies further suggest that genetics alone may not fully account for the susceptibility of schizophrenia and its increased susceptibility is probably due to a synergy among genetic and environmental factors. 1.3 Role of environment in schizophrenia Since a reduced DTNBP1 transcription could not fully account for the reduction in dysbindin-1 protein expression reported in schizophrenia cases, this highlights the imperative role of environment in the pathogenesis of schizophrenia. The course of schizophrenia could be enlightened by the stress14 Chapter 1 Introduction vulnerability model as proposed by Mueser and McGurk (Mueser and McGurk, 2004). This model attributes the cause of schizophrenia to psychobiological vulnerability that is usually predetermined by genetic and environmental conditions early in life and once the vulnerability has been ascertained, the onset and the course of the illness are largely dependent on the dynamics of biological and psychosocial factors. Biological factors such as medication and substance abuse are of great importance as they affect the onset of schizophrenia. Though medication may alleviate the severity of symptoms, substance abuse may increase the chance of relapses. Additionally, psychosocial factors such as stress and social support also play a pertinent role in the course of schizophrenia (Mueser and McGurk, 2004). Similarly, epidemiological studies also found that environmental risk factors such as early childhood malnourishment (Brown and Susser, 2008), drug dependence (Sullivan, 2005), medical conditions (e.g. obesity and hypoxia) (Jeste et al., 1996; Mittal et al., 2008) and brain trauma (Morgan and Fisher, 2007; Do et al., 2009) are associated with the pathogenesis of schizophrenia. Interestingly, environmental stressors as mentioned above (e.g. malnourishment, infection, stress and trauma) are known to induce oxidative stress (Do et al., 2009) and an increasing number of studies suggest that oxidative stress is of great relevance to schizophrenia (Do et al., 2009; Bitanihirwe and Woo, 2011; Yao and Keshavan, 2011). Taken together, dysbindin-1 reductions might be due to oxidative stress resulting from elevated reactive oxygen and reactive nitrogen species (ROS and RON) and/or 15 Chapter 1 Introduction diminished antioxidant activities collectively called the antioxidant defense system (Yao and Keshavan, 2011). 1.4 Role of oxidative stress in schizophrenia The brain is particularly susceptible to the generation of ROS (i.e. H2O2, O2-, OH-, and .OH) and RNS (i.e. .NO and ONOO-) as it is metabolically active and contains a considerable amount of polyunsaturated fatty acids (PUFAs) that are highly vulnerable to peroxidation and redox-free metals (Mahadik et al., 2001; Andersen, 2004; Valko et al., 2007). Moreover, in certain parts of the human brain, iron (Fe2+) levels are elevated and ascorbate levels are usually high. When the body is under oxidative stress which is inducible by conditions such as stroke or aging, the presence of Fe2+ and ascorbate may be potent oxidants to the brain membranous layer (Zaleska et al., 1989). However, the presence of free radicals is not always detrimental as it is found to be involved in a number of physiological functions including intracellular signalling and meiosis (Wood et al., 2009). Moreover, the body possess natural defense mechanisms which utilize enzymes such as superoxide dismutase (SOD) (which converts superoxide radicals to hydrogen peroxide), catalase (CAT) (which converts hydrogen peroxide to water and oxygen) and glutathione peroxidise (GPx) (which converts hydrogen peroxide into water) to regulate the amount of ROS found in the body. Nonenzymatic pathways which utilize glutathione, uric acid, and dietary vitamins such as Vitamin A, C and E are also present to regulate the amount of ROS generated in the body (Mahadik et al., 2001). 16 Chapter 1 Introduction However, excessive production of free radicals for the body’s intrinsic natural antioxidant system to cope would eventually lead to oxidative stress. This could inevitably lead to oxidative cell injury which is commonly characterized by the peroxidation of PUFAs, proteins, and DNA. Based on the findings of many (though not all) studies, levels or indices of oxidative stress are increased, while antioxidant defenses are decreased in the serum and plasma of schizophrenia cases (Gysin et al., 2007; Zhang et al., 2010; Ciobica et al., 2011; Li et al., 2011; Yao and Keshavan, 2011). The same imbalance of oxidants and antioxidant defenses has also been reported in the cerebrospinal fluid and brain tissue (Do et al., 2009; Ciobica et al., 2011; Gawryluk et al., 2011; Yao and Keshavan, 2011). Levels of serum and plasma levels of oxidative stress markers are positively correlated with negative symptoms (Medina-Hernandez et al., 2007; Pazvantoglu et al., 2009), while levels of antioxidant glutathione (GSH) in the prefrontal cortex are inversely correlated with those symptoms (Matsuzawa et al., 2008). Moreover, GSH in schizophrenia cases is reduced in the prefrontal cortex (Do et al., 2009; Gawryluk et al., 2011). Rodents with GSH deficits share a number of features with animal models of schizophrenia, including dysbindin-1 mutant (sdy) mice (Talbot, 2009; Talbot et al., 2012), specifically reduced prepulse inhibition, decreased parvalbumin in fast spiking interneurons, NMDA receptor hypofunction, and reduced long term potentiation (LTP) (Dean et al., 2009; Do et al., 2009). Indeed, oxidative dysfunction of parvalbumin interneurons during development has been proposed as a causal factor in schizophrenia (Behrens and Sejnowski, 2009; Do et al., 2009; Powell et al., 2012). 17 Chapter 1 Introduction Taken together, besides genetic factors, oxidative stress has shown to play an important role in the pathogenesis of schizophrenia. Hence in this thesis, the effects of oxidative stress on dysbindin-1 expression were studied in vitro and in vivo by treating human neuroblastoma cells with hydrogen peroxide (H2O2), a reactive oxygen species, and using kainic acid (KA) in rats, respectively. 1.4.1 Kainic acid-mediated excitotoxicity Figure 1.4.1. Schematic diagram of KA-mediated neuronal cell death pathway. (1) Binding of KA to Ca2+ AMPA/KA receptors leads to Ca2+ influx; (2) activation of Ca2+ - dependent enzymes 2+ and production of ROS; (3) excessive Ca and ROS would lead to the opening of the 18 Chapter 1 Introduction mitochondrion permeability transition pore; (4) release of mitochondrion factors such as cytochrome-c and apoptotic-inducing factor (AIF) (5) triggering apoptosome complex formation and caspase-3 pathway activation; (6) leading to nuclear condensation and eventually neuronal cell death. On the other hand, Ca2+ influx may lead to excessive ROS production, causing ATP decrease, mitochondria damage, protein, lipids and DNA oxidation and ultimately neuronal cell death [Adapted from (Wang et al., 2005a)]. KA is a glutamate analogue that acts as an agonist for non- N-methyl-Daspartic acid (non-NMDA) receptors such as α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor and KA receptors (Bleakman and Lodge, 1998). The administration of KA has shown to increase ROS and RNS which not only lead to mitochondrion dysfunction but also trigger apoptosis in neurons in different parts of the brain (i.e. CA1, CA3 and hilus of the dentate gyrus) (Wang et al., 2005a). These events are elicited by an influx of calcium (Ca2+) upon the binding of KA to KA receptors (Sun and Chen, 1998) (Figure 1.4.1). Taken together, KA is an established experimental model in inducing seizures and selective neuronal damage in susceptible limbic structures, particularly in the CA3 of the hippocampus (Schwob et al., 1980; Ben-Ari, 1985). Since KA can induce oxidative stress and neurodegeneration in vivo (Wang et al., 2005a), this study aims to use this glutamate analogue to investigate the changes in dysbindin-1 mRNA and protein expression in response to oxidative stress in rats. The hippocampus would be emphasized, since the former has been long established as one of the brain regions commonly affected in schizophrenia (Jeste and Lohr, 1989; Roberts, 1990). 19 Chapter 1 Introduction 1.5 PEST sequence as a protein degradation signal peptide A structural feature of two major dysbindin-1 isoforms in the brain (dysbindin-1A and -1C) suggests why it may be degraded by oxidative stress. These isoforms contain a C-terminus PEST (Proline-Glutamate-Serine-Threonine) sequence with many predicted phosphorylation sites, including one for casein kinase II (Talbot et al., 2009). It seems the larger the number of proline (P), glutamate (E), serine (S), and threonine (T) residues, the lower the hydrophobic index and, greater probability of the sequence acting as a proteolytic signal. Phosphorylation of predicted kinases sites in the PEST sequence may elicit a change in conformation which is recognizable by proteasome, causing the rapid degradation of its protein (Rechsteiner and Rogers, 1996; Garcia-Alai et al., 2006). For example, oxidative stress-induced degradation of a protein, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκBα) is mediated largely by casein kinase II phosphorylation of its C-terminus PEST sequence (Schoonbroodt et al., 2000). Interestingly, Locke et al. also found a binding site for E3 ubiquitin ligase, tripartite motif-containing protein 32 (TRIM32) which may promote its degradation in the C-terminus of dysbindin-1 (Locke et al., 2009). Therefore, a mutation in this region may minimize or prevent the reduction of dysbindin-1 which is characteristic in schizophrenia cases (Talbot et al., 2004; Weickert et al., 2008). Together, this suggests in response to oxidative stress, the PEST sequence is important in the regulation of dysbindin-1 present in the brain. 20 Chapter 2 Hypothesis and Aims CHAPTER 2 HYPOTHESIS AND AIMS 21 Chapter 2 Hypothesis and Aims The present study tests the hypothesis that oxidative stress can affect levels of dysbindin-1 expression in the brain via its core promoter, or the protein’s PEST domain. Cultured SH-SY5Y neuroblastoma cells were used to determine if the putative core promoter sequence of the dysbindin-1 gene (DTNBP1) of Liao and Chen (Liao and Chen, 2004) is involved in the regulation of dysbindin-1A upon oxidative stress. SH-SY5Y human neuroblastoma cells that stably overexpress dysbindin-1A or dysbindin-1A without its PEST sequence were also used to determine the effects of oxidative stress, the proteasome inhibitor and the PEST sequence of dysbindin-1 on protein expression. The effect of the potent glutamate analog, kainic acid (KA), on hippocampal dysbindin-1 expression was also elucidated. KA induces excitotoxicity in hippocampal neurons and since many converging evidences have shown that this is associated with oxidative stress and lipid peroxidation (Ong et al., 2000; Wang et al., 2005b; Sanganahalli et al., 2006), analyses of dysbindin-1 expression after KA might provide insights into effects of oxidative stress on dysbindin-1 expression in vivo. 22 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress CHAPTER 3 CHANGES IN DYSBINDIN-1 CORE PROMOTER ACTIVITY AFTER OXIDATIVE STRESS 23 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress 3.1 Introduction Several studies have found significant correlations between schizophrenia and haplotypes of SNPs in specific genes following the discovery of DTNBP1 as a schizophrenia-susceptibility gene (Cloninger, 2002; O'Donovan et al., 2003; Owen et al., 2004). Though most of the SNPs associated with increased schizophrenia risk were mostly found in the introns (Riley and Kendler, 2006; Duan et al., 2007), SNPs were also found in the promoter region of DTNBP1. Specifically, Pedrosa et al. have identified a putative promoter site on chromosome 6p22.3 that encodes dysbindin-1 (Pedrosa et al., 2009). As shown in many studies, this finding is crucial as this specific region formerly mentioned contains a SNP site associated with schizophrenia (Numakawa et al., 2004; Williams et al., 2004). Converging evidences have also shown high levels of lipid peroxidation product such as thiobarbituric reactive substances (TBARS) and SOD in schizophrenia patients as compared to controls (D'Angelo and Bresolin, 2006). Abnormal SOD, GPx and CAT levels were also observed in the blood and plasma samples from schizophrenia patients (Herken et al., 2001; Zhang et al., 2006). Taken together, this suggests that the promoter of dysbindin-1 could be involved in its regulation under oxidative stress. Dysbindin-1 expression has been widely studied in postmortem brains of schizophrenia cases (Talbot et al., 2004; Weickert et al., 2004; Weickert et al., 2008); however, the effects of oxidative stress which have shown an association to schizophrenia on dysbindin-1 promoter have not been clearly studied. Therefore, to bridge this gap of knowledge, this chapter aims to understand the 24 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress activity of dysbindin-1 promoter under oxidative stress. Cultured SH-SY5Y neuroblastoma cells were used to determine if the putative core promoter sequence of DTNBP1 of Liao and Chen (Liao and Chen, 2004) is involved in the regulation of dysbindin-1A upon oxidative stress. The Dual-Luciferase® Reporter Assay System was used in this study to elucidate the activity of dysbindin-1 core promoter activity in response to oxidative stress induced by H2O2. The luciferase assay is a technique commonly used for the understanding of many aspects in cell biology, and is a reliable tool commonly used to study specific cloned promoter sequence activity in vitro in cell lines (Greer and Szalay, 2002; Massoud et al., 2007; de Almeida et al., 2011). Moreover, translated protein in this system is readily available without the need to undergo posttranslational modification (Ow et al., 1986; de Wet et al., 1987), allowing rapid quantification with minimal confounding variables. This reporter system is sensitive and its quantification method has very minimal interference from endogenous expression of host cells (Solberg and Krauss, 2013). Measurement results have also shown to be reliable, accurate and reproducible (McNabb et al., 2005; Solberg and Krauss, 2013). Together, this suggests that the dual luciferase reporter assay is a sensitive and accurate system to investigate the promoter activity of dysbindin-1. 25 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress 3.2 Materials and Methods 3.2.1 Cells and Constructs Figure 3.2.1 Partial genomic sequence of the promoter sequence of dysbindin-1A. Underlined sequences show the forward and reverse primers used, while the sequences in bold show the putative dysbindin-1A core promoter sequence proposed by Liao and Chen (2004). A transient cell line was generated to study the effects of oxidative stress on dysbindin-1 core promoter activity. A 630-nt promoter fragment with flanking XhoI and HindIII sites including the predicted core promoter of dysbindin-1A was isolated using a forward CAGTCTCGAGAGGACTGGGGATGTCACTCA-3’) primer and reverse (5’primer (5’- GTACAAGCTTAACCCAGCCTTCTCCAAG-3’) using rat genomic DNA (Clontech, CA, USA) as the template (Figure 3.2.1). Sequences underlined in the forward and reverse primers are restriction sites (i.e. XhoI and HindIII respectively). Reverse transcription conditions were: 95oC for 30 s, 40 cycles of 95oC for 30 s, 65oC for 30 s and 72oC for 30 s. The amplification process was completed with 72oC for 2 min. PCR product was resolved in 1% agarose gel at 100 V in 0.5 X TAE buffer that contained 0.5 µg/ml of ethidium bromide. 500bp DNA Ladder (Promega, CA, USA) was also loaded. After electrophoresis, the gel was 26 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress observed under UV light, and bands that corresponded to the putative core promoter (630bp) were excised and purified using the QIAquick Gel Extraction Kit (Qiagen, CA, USA) according to the manufacturer’s protocol. The purified DNA and vector were individually digested. 1 µg of DNA or vector, 5 µl of 10X Digestion Buffer and 2.5 µl of XhoI (Promega, #R6161) and 2.5 µl of HindIII (Promega, #R6041) was pipetted into a PCR tube and was brought to a total volume of 50 µl with nuclease-free water. Reaction was carried out in a PCR thermocycler and conditions were as follow: 37oC for 1 h and 65oC for 20 min. After linearization, both DNA and vector contained sticky ends that were generated by XhoI and HindIII. Ligation of the linearized dysbindin-1A promoter DNA sequence into the vector pGL4.10 was completed via the LigaFast™ Rapid DNA Ligation System (Promega) according to the manufacturer’s instructions. 10 µl of ligation product was pipetted into 50 µl of chemically competent E. coli cells (Subcloning Efficiency™ DH5α™ competent cells, Invitrogen, CA, USA) and mixed gently. This mixture was incubated on ice for 30 min, 42oC water bath for 20 s and immediately chilled on ice for 2 min. 1 ml of Lysogeny Broth (LB) medium (10 g of tryptone, 5 g of yeast extract and 10 g NaCl in 1 L of distilled water and autoclaved) was aseptically added into the vial and centrifuged at 225 rpm, 37oC for 1 h. 200 µl of the purified transformation mix was added to a LB agar plate that contained 50 µg/ml of ampicillin and incubated overnight at 37oC. Colonies were selected and added to 5 ml of LB medium containing 50 µg/ml of ampicillin. The suspension was centrifuged at 225 rpm at 37oC overnight. Ampicillin-resistant DNA plasmids were extracted using the QIAprep Spin 27 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress Miniprep Kit (Qiagen, #27104). SH-SY5Y cells (CRL-2266™, ATCC) were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Invitrogen). Samples with a density of 1.0 X 105 cells/well were seeded into a 24-well plate and incubated at 37oC for 1 day. The DNA sequence was verified via reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing before transfection was carried out. 3.2.2 Dual-Luciferase® Reporter Assay The Dual-Luciferase® Reporter Assay system was used to study dysbindin-1 core promoter activity in response to oxidative stress. In this assay, two individual reporter enzymes were expressed simultaneously in a single system. One reporter enzyme (i.e. the firefly luciferase) measures the activity of the dysbindin-1A core promoter while the other provided an internal control and in this study, was shown by the ratio of firefly luciferase (vector containing dysbindin-1A core promoter) to renilla luciferase (reporter control). The verified DTNBP-1A XhoI/HindIII construct and pGL4.74 expression control vector were co-transfected into SH-SY5Y cells using Lipofectamine® LTX with Plus™ (Invitrogen) according to the manufacturer’s protocol. To minimize the possibly of trans effect between promoter elements, a small amount of control vector (ratio 50:1 for vector:co-reporter vector) was added during co-transfection. 28 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress 3.2.3 Effect of oxidative stress on dysbindin-1A core promoter activity SH-SY5Y cells were co-transfected with the verified DTNBP-1A XhoI/HindIII construct and pGL4.74 expression control vector as mentioned above, and divided into 4 groups: 1) Treatment with vehicle, 2) Treatment with 50 µM H2O2, 3) Treatment with 200 µM H2O2, 4) Treatment with 500 µM H2O2. Cells were treated for 24 h and luciferase luminescence values were determined using the Dual-Luciferase® Reporter Assay (Promega) according to the manufacturer's instructions. The mean ratio of firefly:renilla luminescence values and standard errors were calculated and any possible significant differences were analyzed by 1 way ANOVA with Tukey’s multiple comparison post-hoc test (n=5 in each group). P < 0.05 was considered significant. 3.2.4 Effect of WP631 on dysbindin-1A core promoter activity SH-SY5Y cells were co-transfected with the DTNBP-1A XhoI/HindIII construct and pGL4.74 expression control vector as mentioned above and divided into 4 groups: 1) Treatment with vehicle, 2) Treatment with 500 nM WP631, 3) Treatment with 500 µM H2O2, 4) Treatment with 500 µM H2O2 and 500 nM WP631 dihydrochloride. The experiment was repeated with 1 µM WP631. Cells were treated for 24 h and luminescence values were determined. The mean ratio of firefly:renilla luminescence values and standard errors were calculated and any possible significant differences were analyzed by 1 way ANOVA with Tukey’s multiple comparison post-hoc test (n=5 in each group). P < 0.05 was considered significant. 29 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress 3.3 Results Fold Change in Firefly:Renilla Luciferase Activity 3.3.1 Effect of oxidative stress on dysbindin-1A core promoter activity 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 # * 0 Fold change in Firefly:Renilla Luciferase Activity A 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 50 200 H2O2 Concentration/ µM * 500 # * ^ # * * Control B * 500nM WP631 H2O2 H2O2 +500nM WP631 30 Fold Change in Firefly:Renilla Luciferase Activity Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress 2.0 1.8 # 1.6 * 1.4 1.2 ^ 1.0 0.8 # 0.4 0.2 0.0 Control C * * 0.6 1µM WP631 H2O2 H2O2 +1µM WP631 Figure 3.3.1. Fold change in firefly:renilla luciferase activity of SH-SY5Y cells after 24 h. A: Firefly luciferase activity of SH-SY5Y cells co-transfected with the predicted core promoter of DTNBP-1A XhoI/HindIII construct and pGL4.74 expression control vector and treated with 0 µM, 50 µM, 200 µM or 500 µM of H2O2. B: Firefly luciferase activity of SH-SY5Y cells co-transfected with the predicted core promoter of DTNBP-1A XhoI/HindIII construct and pGL4.74 expression control vector and treated with: 1) vehicle control, 2) 500 nM WP631 dihydrochloride, 3) 500 µM H2O2, 4) 500 µM H2O2 and 500 nM WP631 dihydrochloride. C: Firefly luciferase activity of SHSY5Y cells co-transfected with the predicted core promoter of DTNBP-1A XhoI/HindIII construct and pGL4.74 expression control vector and treated with: 1) vehicle control, 2) 1 µM WP631 dihydrochloride, 3) 500 µM H2O2, 4) 500 µM H2O2 and 1 µM WP631 dihydrochloride. The mean +SE are shown. * indicates significant difference (P < 0.05) compared to control. # indicates significant difference compared to SH-SY5Y cells treated with WP631. ^ indicates significant difference compared to SH-SY5Y cells treated with 500 µM H2O2. SH-SY5Y cells that were transiently transfected with DTNBP-1A XhoI/HindIII construct and pGL4.74 expression control vector, and treated with different H2O2 concentrations, showed 1.22-fold, 1.38-fold, 1.47-fold increase in the ratios of firefly:renilla luciferase activity after treatment with 50 µM, 200 µM and 500 µM H2O2 respectively as compared to vehicle, indicating activation of dysbindin-1A core promoter as a result of oxidative stress (Figure 3.3.1A). In contrast, firefly and renilla luciferase expressing cells treated with 500 nM or 1 µM of WP631 dihydrochloride prior to H2O2 treatment showed reduced 31 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress luminescence to 1.17-fold and to 0.60-fold respectively, compared to SH-SY5Y cells treated with H2O2 only (Figures 3.3.1B and C). The results suggest that oxidative stress induces transcription of DTNBP-1A via an effect on its core promoter. 32 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress 3.4 Discussion The present study was carried out to elucidate the effect of oxidative stress on dysbindin-1 promoter activity. In vitro studies were first carried out. SHSY5Y cells were transiently transfected with the dysbindin-1A core promoter, and activity of the dysbindin-1 promoter after H2O2 treatment was analyzed. Increased luciferase activity after treatment with H2O2 indicates increased promoter activity, and suggests that the core promoter of dysbindin-1 is involved in the upregulation of dysbindin-1A expression after oxidative stress. The exact mechanism for this effect is unknown, although oxidative stress has been found to affect gene expression (Okamoto et al., 2002; Liu et al., 2004; Zhao and Meng, 2005). Some studies have shown that the activation of the specificity protein 1 (Sp1) which is a cis-acting regulator of dysbindin-1A may account for an increase in dysbindin-1A expression (Ryu et al., 2003; Iwanaga et al., 2006; Lee et al., 2006). Sp1 is found to stimulate the activation of promoter sequences involved in the transcription of a variety of genes (Li et al., 2004a) that modulates cell growth (Karlseder et al., 1996), embryogenesis (Zhao and Meng, 2005), NMDA subunit 1 (Okamoto et al., 2002; Liu et al., 2004), and adaptive strategies induced by oxidative stress (Ryu et al., 2003; Lee et al., 2006). For example, Sp1 is capable of binding to E2F1; a gene promoter and in the presence of different cellular conditions, the latter would elicit a wide array of cellular processes such as DNA repair, cell growth, and apoptosis. In the face of serum deprivation of embryonic fibroblasts that were transfected with dysbindin-1A, E2F1 was found to be 33 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress ectopically expressed. This resulted in a 10-fold increase in dysbindin-1 mRNA expression (Iwanaga et al., 2006). Therefore, the significant increase in dysbindin-1A mRNA as observed in this study could be due to the activation of Sp1 under oxidative stress. The increase in luciferase activity was then abolished by WP631 dihydrochloride. This is a DNA intercalator which inhibits transcription and could act as a direct competitor of the Sp1 transcription factor (Martin et al., 1999; Portugal et al., 2001; Gaidarova and Jimenez, 2002; Mansilla and Portugal, 2008). Studies have showed that WP631 has a higher specificity and potency towards Sp1-DNA complexes as compared to other Sp1 inhibitors such as daunorubicin (Villamarin et al., 2003), doxorubicin (Mansilla et al., 2007) and mitoxantrone (Gaidarova and Jimenez, 2002). WP631 is also able to elicit high inhibitory efficiency at nanomolar concentrations in vitro and in vivo (Portugal et al., 2001; Gaidarova and Jimenez, 2002; Villamarin et al., 2002). An online programme analysis (Prestridge, 1995) of the core dysbindin promoter has identified 7 out of 17 transcription factor binding sites were for Sp1. Hence, the significant decrease in luciferase activity observed in transfected cells treated with WP631 is likely due to Sp1 inhibition. Taken together, results suggest Sp1 binding sites present in dysbindin-1 core promoter may be involved in the upregulation of dysbindin-1 under oxidative stress. No doubt this present study highlighted the possible role of Sp1 binding site in the upregulation of dysbindin-1 upon oxidative stress, it has its limitations. Besides Sp1, another transcription factor, nuclear factor 1 (Gronostajski, 2000; 34 Chapter 3 Changes in Dysbindin-1 core promoter activity after oxidative stress Plachez et al., 2008), which is also unambiguously found in the core promoter of dysbindin-1 may regulate dysbindin-1 expression. Moreover, WP631 has been reported to affect other transcription function such as smad (Botella et al., 2001; Li et al., 2008) and nuclear factor (NF)- κB (Ashikawa et al., 2004), or the transactivation of Tat (Kutsch et al., 2004). Hence, present studies carried out could only suggest that Sp1 is involved in modulating dysbindin-1 expression upon oxidative stress. To validate and propose Sp1 as an activator of dysbindin1, further studies such as (1) mutagenesis of Sp1 binding site, (2) Sp1 knockdown experiment by siRNA, and (3) chromatin immunoprecipitation assay or electrophoresis mobility shift assay to confirm the binding of Sp1 to the dysbindin-1 promoter region, are needed to be addressed and carried out. 35 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro CHAPTER 4 ROLE OF OXIDATIVE STRESS AND PEST SEQUENCE ON DYSBINDIN-1 EXPRESSION IN VITRO 36 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro 4.1 Introduction In the previous chapter, the dysbindin-1 promoter activity was studied and results have shown (1) dysbindin-1 promoter activity was upregulated after H2O2 treatment, and (2) this significant increase was reduced in cells treated with H2O2 and WP631, a Sp1 inhibitor. These findings suggest Sp1 transcription binding sites found in the core promoter of dysbindin-1 could be involved in the regulation dysbindin-1 expression upon oxidative stress. Following the study on dysbindin-1 gene expression in the previous chapter, the level of dysbindin-1 protein expression will be investigated in this chapter. Many studies have associated ROS and free radicals to the progression and development of neurodegenerative diseases such as schizophrenia and Alzheimer’s disease (AD) (Butterfield and Kanski, 2001; Rao and Balachandran, 2002). Antioxidants that are able to reverse the adverse effects of free radicals are known to be potential neuroprotective agents (Floyd, 1999; Fang et al., 2002; Moosmann and Behl, 2002). In vitro experiments have also shown promising results involving antioxidants increasing neuronal viability, cell viability and reducing effects of oxidative damage (Brewer, 1997; Ricart and Fiszman, 2001). A H2O2 scavenger, phenyl- N- tert-butylnitrone (PBN) was used in this study to examine the effects of H2O2 induced oxidative stress on dysbindin-1 expression. PBN is found to display promising neuroprotective properties against free radicals in vitro and in vivo (Yue et al., 1992; Barth et al., 1996; Floyd and Hensley, 2002). PBN as a part of the family of nitrones can elicit its antioxidant role by converting free radicals with a reactive oxygen or carbon to nitroxide 37 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro radical species that are biochemically less reactive and more stable than its original state (Goldstein and Lestage, 2000). In addition, the effect of proteasome inhibitor and PEST sequence on dysbindin-1 protein expression under oxidative stress was also examined in this chapter. Ubiquitination is a protein posttranslational modification widely studied for the degradation of cytoplasmic and nuclear proteins by the proteasome (Jentsch, 1992; Yaron et al., 1998). Proteins targeted for degradation are recognized and labelled with ubiquitin moieties by E2-conjugating or/and E3ligating enzymes. These protein-ubiquitin complexes are then recognized by proteasome and degraded. Sequences that are rich in proline (P), glutamate (E), serine (S), and threonine (T) residues (i.e. PEST sequences) are found to act as a degradation signal (Roth et al., 1998; Shumway et al., 1999; Martinez et al., 2003) and is present in the C-terminus of dysbindin-1 (Talbot et al., 2009). The larger the number of amino acid residues as mentioned above, the lower the hydrophobic index and, greater probability the sequence acting as a proteolytic signal. Using the PEST-Find algorithm, this probability is translated and reflected as a score. A general consensus has been reached and protein sequences with a score above 5 are regarded highly susceptible to degradation (Kakkar et al., 1999; Singh et al., 2006). The PEST score for dysbindin-1A in rat is 10.48, consistent with the value reported by Talbot et al (Talbot et al., 2009). Together, with the discovery of a binding site for E3 ubiquitin ligase, TRIM32 in the C-terminus of dysbindin-1 (Locke et al., 2009), this protein could be regulated by proteasome and its PEST sequence. In this chapter, the effect of 38 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro PEST sequence under oxidative stress was investigated using SH-SY5Y human neuroblastoma cells that stably overexpress dysbindin-1A or dysbindin-1A without its PEST sequence. In addition, MG132, a proteasome inhibitor was used to further determine the effect of proteasome on dysbindin-1 protein degradation. 39 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro 4.2 Materials and Methods 4.2.1 Cells and Constructs Stable cell lines were generated to elucidate a possible effect of the PEST sequence of dysbindin-1 on protein expression. Total RNA was extracted from the hippocampus of normal rats, and the mRNA reverse transcribed to cDNA. A forward primer (5’-CAGTCTCGAGCGGATGCTGGAGACCCTGCGCGAG-3’) and reverse primer (5’-GTACGAATTCTTAAATGTCCTGAGTTGAGTC-3’) were used for the reverse transcription of full-length dysbindin-1A. A separate set of forward (5’-CAGTCTCGAGCGGATGCTGGAGACCCTGCGCGAG-3’) and reverse (5'GTACGAATTCTTACTTTCTGTCAGTGTTTAA-3') primers were used for the transcription of a truncated form of dysbindin-1A without its PEST sequence. Sequences underlined in the forward and reverse primers are restriction sites (i.e. XhoI and EcoRI respectively). Reverse transcription conditions were: 95oC for 30 s, 40 cycles of 95oC for 30 s, 59oC for 30 s and 72oC for 30 s. The amplification process was completed with 72oC for 2 min. PCR product was resolved in 1% agarose gel at 100 V in 0.5 X TAE buffer that contained 0.5 µg/ml of ethidium bromide. 1kb DNA Ladder (Promega) was also loaded. After electrophoresis, the gel was observed under UV light, and bands that corresponded to dysbindin-1A with or without its PEST sequence were excised and purified using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s protocol. The pIRES2-EGFP vector (Clontech) was used to construct a recombinant vector made by cloning dysbindin-1A with or without PEST sequence DNA into the XhoI/EcoRI sites found in the multiple cloning site (MCS) of pIRES2-EGFP. After 40 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro linearization, both DNA and vector contained sticky ends that were generated by XhoI and EcoRI. Ligation of the linearized dysbindin-1A DNA into the pIRES2EGFP was completed via the LigaFast™ Rapid DNA Ligation System (Promega) according to the manufacturer’s instructions. 10 µl of ligation product was pipetted into 50 µl of chemically competent E. coli cells (Subcloning Efficiency™ DH5α™ competent cells, Invitrogen) and mixed gently. This mixture was incubated on ice for 30 min, 42oC water bath for 20 s and immediately chilled on ice for 2 min. 1 ml of Lysogeny Broth (LB) medium (10 g of tryptone, 5 g of yeast extract and 10 g NaCl in 1 L of distilled water and autoclaved) was aseptically added into the vial and centrifuged at 225 rpm, 37oC for 1 h. 200 µl of the purified transformation mix was added to a LB agar plate that contained 30 µg/ml of kanamycin and incubated overnight at 37oC. Colonies were selected and added to 5 ml of LB medium containing 30 µg/ml of kanamycin. The suspension was centrifuged at 225 rpm at 37oC overnight. Kanamycin-resistant DNA plasmids were extracted using the QIAprep Spin Miniprep Kit (Qiagen, #27104). SH-SY5Y cells (CRL-2266™, ATCC) were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Invitrogen). Samples with a density of 1.0 X 105 cells/well were seeded into a 24-well plate and incubated at 37oC for 1 day. Transfections were carried out using Lipofectamine™ LTX and PLUS™ Reagents (Invitrogen) according to the manufacturer’s instructions. After 24 h transfection, cells were subcultured in a 6-well plate and 1 mg/ml of the selective reagent G418 (Invitrogen) was added. The growth medium (containing 1 mg/ml 41 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro of G418) was changed every 2 days for 10 days. Subsequently, 1 cell was plated into each 96 well via flow cytometry (Mo-Flo Legacy; Beckman-Coulter, FL, USA) to establish multiple clones. Cells were then subcultured into larger wells (i.e. 24well plate and 6-well plate) to obtain a stable cell line. 4.2.2 Effect of oxidative stress on dysbindin-1A expression SH-SY5Y human neuroblastoma cells were cultured in 100 mm2 dishes, and divided into 4 groups: 1) Treatment with vehicle, 2) Treatment with 500 µM H2O2 for 24 h, 3) Treatment with 500 µM H2O2 and free radical scavenger, PBN (Sigma, MO, USA). SH-SY5Y cells were pretreated with 1 µM PBN for 45 min, followed by 500 µM H2O2 for 24 h, and 4) Treatment with PBN. Total proteins were extracted and analyzed by Western blot as described below. The means and standard errors were calculated and any possible significant differences were analyzed by 1 way ANOVA with Tukey’s multiple comparison post-hoc test (n=4 in each group). P < 0.05 was considered significant. 4.2.3 Effect of proteasome inhibitor on dysbindin-1A expression The effect of proteasome inhibitor and PEST sequence on the expression of dysbindin-1A was examined. A total of 4 treatment groups were analyzed: 1) Treatment of SH-SY5Y cells stably overexpressing dysbindin-1A with vehicle, 2) Treatment of SH-SY5Y cells stably overexpressing dysbindin-1A with 0.5 µM MG132, 3) Treatment of SH-SY5Y cells stably overexpressing dysbindin-1A without its PEST sequence with vehicle, and 4) Treatment of SH-SY5Y cells 42 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro stably overexpressing dysbindin-1A without its PEST sequence with 0.5 µM MG132. Cells were treated for 24 h and harvested. Protein was extracted and the level of dysbindin-1A protein was analyzed by Western blot as mentioned below. The means and standard errors were calculated and any possible significant differences were analyzed by 1 way ANOVA with Tukey’s multiple comparison post-hoc test (n=3 in each group). P < 0.05 was considered significant. 4.2.4 Effect of the PEST sequence of dysbindin-1A on protein expression after oxidative stress SH-SY5Y cells stably overexpressing dysbindin-1A, dysbindin-1A without its PEST sequence, or control vector were used. After reaching 80% confluence, cells were treated with 0 µM, 50 µM, or 500 µM of H2O2. Dilutions were freshly prepared from a 30% H2O2 stock. Treatment was carried out for 24 h and proteins were extracted and analyzed by Western blot analysis as mentioned below. The means and standard errors were calculated and any possible significant differences were analyzed by 1 way ANOVA with Tukey’s multiple comparison post-hoc test (n=4 in each group). P < 0.05 was considered significant. 4.2.5 Western Blot analyses Proteins were extracted using the Mammalian Protein Extraction Reagent (M-PER, #78501, ThermoScientific, CA, USA) according to the manufacturer’s instructions, and the level of dysbindin-1A protein was analyzed by Western blot. 43 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro Using a 10% SDS polyacrylamide gel, 15 µg of proteins were separated via their molecular weight and electric charge and electrotransferred via the semi-dry transfer method (#170-3940, Bio-Rad Laboratories, CA, USA) onto a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). The PVDF membrane was then incubated with 5% non-fat milk for 1 h at room temperature to block non-specific binding sites. Thereafter, it was probed with a rabbit polyclonal antibody to the N-terminus of dysbindin-1 (diluted 1:250 in Tris-buffered saline [TBS]) (#sc-67171, Santa Cruz Biotechnology, CA, USA) overnight at 4°C. Following an overnight incubation, excess unbound primary antibody was washed with 0.1% Tween-20 in TBS, 6 times at 5 min interval with gentle agitation. The membrane was then incubated with a secondary antibody, horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (Amersham Pharmacia Biotech) to enhance its signal for 1 h at room temperature. Excess unbound secondary antibody was removed via 6 washes of 0.1% Tween-20 in TBS at 5 min interval with gentle agitation. Protein visualization was then carried using an enhanced chemiluminescence kit (Pierce, IL, USA) according to the manufacturer’s instructions. After exposure, the blot was stripped of antibodies and re-probed with a mouse monoclonal antibody to β-actin as a loading control (diluted 1:10000 in TBS at 4°C, Sigma). To test for transfection efficiency, the membrane was re-probed with an antibody to the coexpressed green fluorescent protein (GFP) (diluted 1:10000 in TBS at 4°C, Novus Biologicals, Cambridge, UK). Densitometric analysis of the bands in Western blots was performed using GelPro software (Media Cybernetics, MD, 44 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro USA) and any possible significant differences in the normalized mean densities were analyzed using 1 way ANOVA with Tukey’s multiple comparison post-hoc test. P < 0.05 was considered significant. 45 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro 4.3 Results 4.3.1 Effect of oxidative stress and oxygen free radicals on dysbindin-1A expression 1.2 # 1.0 0.8 * 0.6 0.4 0.2 0.0 Control B H2O2 H2O2 +PBN Dysbindin-1 Normalized to β-Actin Dysbindin-1 Normalized to β-Actin 1.4 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Control PBN Figure 4.3.1. Analysis of untransfected SH-SY5Y cells treated with or without PBN 24 h after 500 µM H2O2 treatment. A: Western blots showing: 1) Untreated SH-SY5Y cells (lanes 1-4), 2) SH-SY5Y cells treated with 500 µM H2O2 (lanes 5-8), 3) SH-SY5Y cells treated with the free radical scavenger, PBN, 45 min prior to treatment with 500 µM H2O2 (lanes 9-12), 4) Untreated SH-SY5Y cells (lanes 13-16), and 5) SH-SY5Y cells treated with the free radical scavenger, PBN for 45 min (lanes 17-20). B: Dysbindin-1 normalized to β-actin. Decrease in dysbindin-1 protein was detected 1 day after 500 µM H2O2 treatment. In contrast, dysbindin-1 protein levels were not affected if cells were pre-treated with PBN for 45 min prior to H2O2 treatment, indicating the role of reactive oxygen species in dysbindin-1 degradation. No significant difference was observed between untreated SH-SY5Y cells and SH-SY5Y cells treated with PBN only. The mean +SE are 46 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro shown. * indicates significant difference (P < 0.05) compared to control. # indicates significant difference compared to SH-SY5Y cells treated with 500 µM H2O2. The affinity purified rabbit polyclonal antibody that recognized the Nterminus (1-90 amino acid of the N-terminus of the human dysbindin origin) of dysbindin isoforms detected a single band at 50 kDa (Figure 4.3.1A). This band size of dysbindin-1A is higher than the predicted molecular weight of 40 kDa (Talbot et al., 2004). Recent reports have depicted dysbindin-1A with a higher molecular weight that ranges from 48-50 kDa. This could be due to the highly acidic nature of the C-terminus of dysbindin-1A (Talbot et al., 2004) or posttranslational modifications such as ubiquitination and/or phosphorylation (Talbot et al., 2004; Locke et al., 2009). A single band detected also dismisses the possibility of degradative product formed from improper handling of tissue lysate for Western blot. A significant decrease to 0.66-fold in dysbindin-1A protein expression was detected in SH-SY5Y human neuroblastoma cells after treatment with 500 µM H2O2, compared to vehicle (Figures 4.3.1A and B). This decrease was absent in SH-SY5Y cells that were pre-incubated with the free radical scavenger PBN prior to H2O2 treatment (Figures 4.3.1A and B). No significant difference in dysbindin1A protein level was observed between SH-SY5Y cells treated with PBN and those treated with vehicle (Figure 4.3.1A and B). 47 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro 4.3.2 Effect of proteasome inhibitor and the PEST sequence on dysbindin1A expression A 4.0 Dysbindin-1 Normalized to β-Actin 3.5 * 3.0 2.5 2.0 # 1.5 # 1.0 0.5 0.0 B FL -MG132 FL +MG132 WP -MG132 WP +MG132 Figure 4.3.2. Analysis of SH-SY5Y cells treated with or without MG132 after 24 h. A: Western blots showing: 1) SH-SY5Y cells overexpressing full-length dysbindin-1, treated with vehicle (Lanes 1-3), 2) SH-SY5Y cells overexpressing full-length dysbindin-1, treated with 0.5 µM MG132 (Lanes 4-6), 3) SH-SY5Y cells overexpressing dysbindin-1A without its PEST sequence treated with vehicle (Lanes 7-9), 4) SH-SY5Y cells overexpressing dysbindin-1 without its PEST sequence treated with 0.5 µM MG132 (Lanes 10-12). B: Dysbindin-1 normalized to β-actin. Cells 48 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro that stably overexpress full-length dysbindin-1 showed significantly greater protein expression after treatment with MG132, compared to vehicle controls, or cells expressing the protein without its PEST sequence, indicating an effect of the proteasome inhibitor and the PEST sequence, in dysbindin-1 protein level. The mean +SE are shown. * indicates significant difference (P < 0.05) compared to SH-SY5Y cells overpressing full-length dysbindin-1 treated with vehicle. # indicates significant difference compared to SH-SY5Y cells overpressing full-length dysbindin-1 treated with MG132. Abbreviations: FL: SH-SY5Y cells overexpressing full-length dysbindin-1A, WP: SHSY5Y cells overexpressing dysbindin-1A without its PEST sequence. To study the effects of proteasome and the PEST sequence of dysbindin1A in its degradation, SH-SY5Y cells that stably overexpress full-length dysbindin-1A or dysbindin-1A without its PEST sequence were treated with MG132. A significant 2.73-fold increase in dysbindin-1A protein expression was detected in SH-SY5Y cells that overexpress full-length dysbindin-1A after MG132 treatment, compared to vehicle controls (Figures 4.3.2A and B). In contrast, no increase was found in SH-SY5Y cells that overexpress dysbindin-1A without its PEST sequence after MG132 treatment (Figures 4.3.2A and B). 4.3.3 Effect of the PEST sequence of dysbindin-1A on protein expression Dysbindin-1 Normalized to β-Actin after oxidative stress B 3.0 2.5 * * 2.0 1.5 1.0 0.5 0.0 FL WP V 49 Dysbindin-1 Normalized to β-Actin Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro Dysbindin-1 Normalized to β-actin D F 4.0 3.5 3.0 2.5 2.0 * * 1.5 1.0 0.5 0.0 FL WP 6.0 # 5.0 * V 4.0 3.0 2.0 1.0 0.0 FL WP V Figure 4.3.3. Western blot analysis of SH-SY5Y cells transfected with dysbindin-1A, or without its PEST sequence or vector control. A, C, E: Transfected or vector control SH-SY5Y cell lines treated with 0 µm, 50 µm, and 500 µm H2O2, respectively. B, D, F: Dysbindin-1 normalized to β-actin. Cells overexpressing full-length dysbindin-1 (FL) showed significantly greater degradation after high level of oxidative stress induced by 500 µm H2O2, than cells expressing dysbindin-1 without its PEST sequence (WP). The expression level of exogenous dysbindin-1A is negligible in the control condition. The mean +SE are shown. * indicates significant difference (P < 0.05) compared to SH-SY5Y cells overexpressing vector control. # indicates significant difference compared to SH-SY5Y cells overexpressing full-length dysbindin-1. Abbreviations: FL: SH-SY5Y cells overexpressing full-length dysbindin-1, WP: SH-SY5Y cells overexpressing dysbindin-1 without its PEST sequence. V: control SH-SY5Y cells overexpressing empty vector and depict endogenous expression of dysbindin-1. Dysbindin-1A expression observed in the vector control depicts the basal level of dysbindin-1 found in SH-SY5Y cells, and any significant increase in the dysbindin-1 expression is hence mainly due to overexpressed dysbindin-1A levels. Significantly greater dysbindin-1 expression was found in SH-SY5Y cell lines that stably expressing full-length dysbindin 1A or dysbindin-1A without its 50 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro PEST sequence, compared to vector expressing controls (Figures 4.3.3A and B). Likewise, significantly greater dysbindin-1 expression was observed in cells expressing full-length dysbindin 1 or dysbindin-1 without its PEST sequence, after treatment with 50 µM H2O2, compared to vector controls (Figures 4.3.3C and D). After exposure to 500 µM of H2O2, levels of dysbindin-1 were significantly greater in SH-SY5Y cells expressing dysbindin-1 without its PEST sequence, compared to cells expressing full-length dysbindin-1, and vector controls (Figures 4.3.3E and F). 51 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro 4.4 Discussion In this chapter, the effects of oxidative stress and free radicals on dysbindin-1 protein expression were elucidated. Normal SH-SY5Y cells treated with the free radical scavenger PBN showed less degradation of dysbindin-1 protein after H2O2 induced oxidative stress than those without PBN, indicating the role of reactive oxygen species in dysbindin-1 degradation. This effect is likely at the protein level, since oxidative stress has shown to increase mRNA expression of dysbindin-1 through action at the core promoter, as observed in the previous chapter. An increase in dysbindin-1 gene expression could be a compensatory mechanism possibly through the activation of riboswitches in response to a mark reduction in dysbindin-1 protein level after oxidative stress (Cheah et al., 2007). Interestingly, dysbindin-null (sdy) mice are characterized to have reduced levels of H2O2 scavenging peroxiredoxins, a finding that is also observed in schizophrenic subjects (Gokhale et al., 2012). The above finding indicates that dysbindin-1 protein degradation is increased by oxidative stress, and the presence of an antioxidant such as PBN could modulate its degradation. It is also interesting to note that due to the spin-trapping properties of PBN, it could prevent the excessive accumulation of free radicals and thus display protective characteristics against excitotoxicity and oxidative stress (Lancelot et al., 1997). Besides, being proven as a possible therapy against disease related due to excessive free radical production (e.g. aging and stroke) (Kotake, 1999), PBN have shown to be a compound effective against extrapyramidal side effects due to long-term antipsychotic drugs dependence (Daya et al., 2011). 52 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro Since the increase in dysbindin-1 mRNA expression after oxidative stress could not account for the decrease in dysbindin-1 protein levels observed, posttranslational modifications such as ubiquitination and phosphorylation could promote the degradation of dysbindin-1A, and thus explain its reduction (Locke et al., 2009). Dysbindin-1A and -1C contain the PEST sequence in their C-terminus and we hypothesize that the presence of these sequences promotes degradation in a proteasome dependent manner. The effects of PEST sequence and proteasome inhibitor on dysbindin-1 protein expression were then determined in this chapter. A PEST sequence often marks a protein for proteasomal degradation (Rechsteiner and Rogers, 1996; Garcia-Alai et al., 2006); for example, it is critical for oxidative-stress induced phosphorylation-dependent degradation of the protein IκBα (Schoonbroodt et al., 2000). Phosphorylation at kinases sites (Talbot et al., 2009) apparent in the PEST domain of dysbindin-1 suggests a change in conformation of proteins, recognizable by the ubiquitinproteasome which may be responsible for its degradation (Rechsteiner and Rogers, 1996; Garcia-Alai et al., 2006). Moreover, the PEST sequence of dysbindin-1 contains a recognition site for TRIM32, which is an E3 ubiquitin ligase involved in the ubiquitin-proteasome degradation system (Locke et al., 2009). A possible role of proteasome in degradation of dysbindin-1 proteins was studied in vitro using a proteasome inhibitor, MG132 while a role of PEST sequence in degradation was examined, using stable cell lines that overexpress full-length dysbindin-1 or truncated dysbindin-1 isoform without its PEST sequence. Cells that overexpress dysbindin-1A showed a 2.73-fold increase in 53 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro expression after treatment with the proteasome inhibitor, MG132. This suggests that the protein turnover rate of full length dysbindin-1 is dependent on proteasome as a significant increased dysbindin-1 expression is observed after cells were treated with a proteasome inhibitor. Evidence also showed that dysbindin-1 protein is degraded in a PEST sequence dependent manner. Cells that overexpress dysbindin-1A without its PEST sequence showed no increase in expression. This finding not only ascertained that the truncated dysbindin-1A has been amplified and transfected successfully; it also suggests that dysbindin-1A devoid of its PEST sequence is less susceptible to protein degradation. It is noted that cells overexpressing the truncated protein treated with or without MG132 express reduced dysbindin-1A levels as compared to cells overexpressing full length dysbindin-1A treated with MG132. This finding could be due to lower transfection efficiency in cells that overexpress the truncated protein than cells that overexpress its full length isoform (Figure 4.3.2C). Taken together, converging findings in this study propose that dysbindin-1 is degraded in a proteasome and PEST sequence manner. The effect of oxidative stress on dysbindin-1 protein with or without its PEST sequence was further elucidated. At 0 µM and 50 µM H2O2, cells overexpressing full-length dysbindin-1A and cells overexpressing dysbindin-1A without its PEST sequence showed no difference in dysbindin-1A expression. At higher concentration of 500 µM H2O2, significantly greater dysbindin-1A protein expression was found in SH-SY5Y cells overexpressing dysbindin-1A without its PEST sequence, compared to those expressing its full-length protein. These 54 Chapter 4 Role of oxidative stress and PEST sequence on Dysbindin-1 expression in vitro results indicate that cells expressing dysbindin-1A without its PEST sequence were more resistant to H2O2 induced damage than the native protein; and also highlight the role of the PEST sequence in mediating dysbindin-1 protein degradation after oxidative stress. Similar to the truncated dysbindin-1 protein, short-lived proteins such as HCLS1-associated protein X-1 (Hax-1) (Li et al., 2012) and heat shock transcription factor 2 (HSF2) (Xing et al., 2010) with its PEST sequence deleted were more stable with slower turnover rates. Despite previous and present studies showing convincing evidence that dysbindin-1 are degraded in a proteasome and PEST sequence dependent manner, the possibility of a calpain-dependent degradation pathway should not be dismissed. The production of ROS induced by H2O2 can increase Ca2+ and activate Ca2+-dependent proteases; calpain (Hill et al., 2008; Rasbach et al., 2008; Wei et al., 2009). Proteins containing PEST sequence can also bind Ca2+, and such sites are likely targets for calpain to cleave, resulting in its degradation (Shumway et al., 1999; Rasbach et al., 2008). 55 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo CHAPTER 5 ROLE OF KAINATE EXCITOTOXICITY ON DYSBINDIN-1 EXPRESSION IN VIVO 56 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo 5.1 Introduction In previous chapters (Chapter 3 and 4), the effects of oxidative stress on dysbindin-1 gene and protein expression in vitro were studied. Present results found at the gene level upon oxidative stress, DTNBP1 is regulated by its core promoter (i.e. Sp1 binding sites) while dysbindin-1 protein degradation is modulated by its PEST sequence found in its C-terminus. In this chapter, the role of oxidative stress on dysbindin-1 expression in vivo was examined. Extensive studies on subjects with schizophrenia have found a strong association between oxidative stress and schizophrenia. Specifically, reduced antioxidant enzymes levels (e.g. catalase, glutathione peroxidase, and superoxide dismutase) (Reddy et al., 1991; Ranjekar et al., 2003; Zhang et al., 2010), lower levels of plasma and serum antioxidants (i.e. albumin, bilirubin, thioredoxin, and uric acid) (Reddy et al., 2003; Zhang et al., 2009), and elevated levels of plasma and serum lipid peroxidative products (i.e. thiobarbiturate reactive substances and malondialdehyde) (McCreadie et al., 1995; Zhang et al., 2006) were observed in schizophrenia patients. Taken together, these findings indicate an imbalance in redox regulation could play an imperative role in the onset and pathogenesis of schizophrenia. Interesting, it is noteworthy that several animal studies on oxidative stress displayed behavioral abnormalities and cognition dysfunction similar to symptoms observed in schizophrenia patients (Cabungcal et al., 2007; Steullet et al., 2010). Cabungal et al. showed through many behavioral studies, one of which; the homing hole board task that despite the aid of visual and/or olfactory cues, rats exposed to oxidative stress in 57 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo the early prenatal stage took more time to find the path (out of five possibilities) to its home cage as compared to vehicle-treated rats (Cabungcal et al., 2007). Together, oxidative stress is likely to exert an adverse effect on spatial learning and memory ability in early development. In a recent study, Steullet et al. have also found oxidative stress resulted in reduced neural oscillation in the hippocampus of young male rats, and abnormalities in response to object recognition task (Steullet et al., 2010). Hence, in this chapter, changes in dysbindin-1 expression in the hippocampus will be the main focus as there is much emphasis of an impaired cognitive function seen in schizophrenia patients and its pertinent role in learning and memory (Jarrard, 1993; Chambers et al., 1996; Bruel-Jungerman et al., 2007). Moreover, the hippocampus is known to be particularly vulnerable to oxidative stress and excitotoxicity (Jellinger, 2010). To elucidate the effect of oxidative stress and excitotoxicity on dysbindin-1 expression, the KA-mediated neurodegenerative model was used in this study. KA has shown to depict an increase in ROS and RNS which in turn lead to mitochondrion dysfunction and trigger apoptosis in neurons found in different brain regions (Wang et al., 2005a). It is also an established experimental model in inducing seizures and selective neuronal damage in susceptible limbic structures (i.e. CA3 of the hippocampus) (Schwob et al., 1980; Ben-Ari, 1985). KA could be administered systemically (i.e. subcutaneously, intravenously, and intraperitoneally), however studies showed that only a small amount of the injected KA could reach its receptors found in the brain (Berger et al., 1986). In this study, KA-mediated excitotoxicity was carried out via intracerebroventricular 58 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo injection as this route of administration induces more consistent lesioned sites in the hippocampus (Ong et al., 1999). Unilateral KA injection was carried out on the right lateral ventricle to provide a more distinct comparison between its ipsilateral and contralateral hippocampus upon neurodegeneration. Rats were injected with saline and used as a vehicle control in this study. 59 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo 5.2 Material and Methods 5.2.1 Kainate injections Wistar rats of approximately 200g each were ordered and purchased from the Centre of Animal Resources (CARE). The rats were each deeply anesthetized with 0.4 ml (0.2 ml/100g body weight) of an old rat anesthesia cocktail consisting ketamine (75 mg/kg), xylazine (10 mg/kg), and sterile water. 1.0 µl of KA (1 mg/ml dissolved in saline, Tocris, MN, USA) was stereotaxically injected into the right lateral ventricle of the rat brain as previously illustrated (Kim and Ong, 2009). After surgery, animals were assessed for the severity of their seizure based on the Racine scale (Racine, 1972) and all KA-injected rats were found to have scored 3 or above. Post-operative care was carried out to minimize animal suffering. All procedures pertaining to the animal study conducted were certified by the Institutional Animal Care and Use Committee (IACUC), National University of Singapore. 5.2.2 Immunohistochemistry A total of sixteen rats, consisting of four 1 day saline-treated controls, four 2 weeks saline-treated controls, four 1 day post-KA, and four 2 weeks post-KA injected rats were used for this study. They were deeply anesthetized intraperitoneally (please refer to Chapter 5.2.1 for more details on the type of anesthesia used) and were perfused via the left cardiac ventricle with Ringer’s solution (i.e. 8.5 g NaCl, 0.25 g KCl, 0.3 g CaCl2, 0.2 g NaHCO3 dissolved in 1 L of distilled water) to allow drainage of blood, followed by 4% paraformaldehyde in 60 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo 0.1 M phosphate buffer (pH 7.4) for fixation. 100 µm thin sections of the fixed brain were cut coronally using a vibrating microtome. To remove any traces of paraformaldehyde, sections were washed for 3 h, 5 min interval with gentle agitation in phosphate-buffered saline (PBS). The sections were then incubated overnight with the goat polyclonal antibody to dysbindin-1 (1:250 of SC-46931, Santa Cruz Biotechnology). To remove any unbound primary antibody, sections were washed 6 times, 5 min interval with gentle agitation in PBS and incubated for 1 h at room temperature in 1:2000 dilution of biotinylated horse anti-goat IgG secondary antibody (Vector, CA, USA). Sections were then incubated with an avidin-biotinylated horseradish peroxidase complex for 1 h at room temperature, and visualized with 0.05% of 3, 3-diaminobenzidine tetrahydrochloride (DAB) solution in Tris buffer containing 0.05% H2O2 treatment for 10 min. The reaction was halted with 4 washes of Tris buffer, 5 min intervals with gentle agitation. Stained sections were mounted on gelatinized glass slides and lightly counterstained with methyl green for better visualization (0.25% solution in 0.1 M acetate buffer, pH 4.8, Sigma). Control sections were incubated with antigenabsorbed antibody prepared by pre-incubating 200 µg/mL immunizing peptide with dysbindin-1 antibody overnight, instead of primary antibody. Cell counts were performed to quantify the mean number of dysbindin-1 labeled neurons in the DG, CA1 and CA3 regions of saline-treated controls, 1 day and 2 weeks post-KA-injected rats. Four sections from each region of each rat were quantified. The mean number of positively labelled neurons and its standard error were calculated and any possible significant differences between KA-injected and 61 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo saline-control groups were analyzed using the Student’s t-test. P < 0.05 was considered significant. 5.2.3 Real time RT-PCR analyses A total of eighteen rats, consisting of six saline-treated controls, six 1 day post-KA, and six 2 weeks post-KA injected rats, were used for this study. Decapitation was carried out after the rats were deeply anaesthetized (please refer to Chapter 5.2.1 for more details on the type of anesthesia used), certified by an absence of tail, cornea, and toe-pinch reflexes. The right HF ipsilateral to the injection was harvested and kept in RNAlater® (Ambion, TX, USA), immediately frozen in liquid nitrogen, and kept at -80oC for preservation until further analysis. Trizol reagent (Invitrogen) was used for total RNA extraction, according to the manufacturer’s protocol. The extracted RNA was then purified using the RNeasy® MiniKit (Qiagen). Thereafter, samples were reverse transcribed via RT-PCR and the amplified transcripts were analyzed by real time RT-PCR as mentioned above, except that probes for rat Dtnbp1 and β-actin (Rn01434739_m1, #4352340E respectively) were used. The means and standard errors were calculated and any possible significant differences between the KA-injected HF and its saline-control were determined, using the Student’s ttest (n=6 in each group). P < 0.05 was considered significant. 62 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo 5.2.4 Western blot analyses A total of sixteen rats, consisting of four 1 day saline-treated control, four 1 day post-KA, four 2 weeks saline-treated control, and four 2 weeks post-KA injected rats, were used in this study. The HF (ipsilateral to KA injection) was harvested and immediately frozen in liquid nitrogen, and kept at -80oC for preservation until further analysis. Total protein was extracted using the T-PER® Tissue Protein Extraction Reagent (Pierce) according to the manufacturer’s instructions while its protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad). The expression level of dysbindin-1 was analyzed by Western blot as mentioned above. A goat polyclonal antibody to dysbindin-1 (diluted 1:200 in Tris-buffered saline [TBS]) (#sc-46931, Santa Cruz Biotechnology) which recognizes the C-terminus of dysbindin-1 was used. The mean densities and standard errors were calculated and any possible significant differences were analyzed by 1 way ANOVA with Tukey’s multiple comparison post-hoc test (n=4 in each group). P < 0.05 was considered significant. 63 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo 5.3 Results 5.3.1 Effect of oxidative stress on dysbindin-1 localization in the hippocampal formation Figure 5.3.1.1. Dysbindin-1 immunoreactivity in the HF of rats post 1 day KA injection. A: dentate gyrus of a saline-treated rat, showing light immunoreactivity to dysbindin-1 (arrows). B: field CA3 of a saline-treated rat, showing light immunoreactivity to dysbindin-1 (arrows). C: field CA1 of a saline-treated rat, showing light immunoreactivity to dysbindin-1 (arrows). D: dentate gyrus from a 1 day post-KA injected rat, showing scattered positive cells in the subgranular zone (arrows). E: field CA3 from a 1 day post-KA injected rat, showing decreased dysbindin-1 labelling at the injection site (asterisk). F: field CA1 from a 1 day post-KA injected rat, showing light immunoreactivity to dysbindin-1 (arrows). G: dentate gyrus from a 1 day post-KA injected rat 64 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo contralateral to injection, showing scattered positive cells in the subgranular zone (arrows). H: field CA3 from a 1 day post-KA injected rat contralateral to injection, showing increased dysbindin-1 labelling in scattered neurons (arrows). I: field CA1 from a 1 day post-KA injected rat contralateral to injection, showing large numbers of immunopositive pyramidal neurons (arrows). J: dentate gyrus of a control section incubated with antigen-absorbed antibody showing only background staining. K: field CA3 of a control section, showing background staining. L: field CA1 of a control section, showing background staining. Scale = 20µm. Figure 5.3.1.2. Dysbindin-1 immunoreactivity in the HF of rats 2 weeks post KA injection. A: dentate gyrus from a 2 weeks post-saline injected rat ipsilateral to injection, showing light immunoreactivity to dysbindin-1 (arrows). B: field CA3 from a 2 weeks post-saline injected rat ipsilateral to injection, showing light immunoreactivity to dysbindin-1 (arrows). C: field CA1 from a 2 weeks post-saline injected rat ipsilateral to injection, showing light immunoreactivity to dysbindin-1 (arrows). D: dentate gyrus from a 2 weeks post-KA injected rat ipsilateral to injection, showing decreased dysbindin-1 labelling (arrows). E: field CA3 from a 2 weeks post-KA injected rat ipsilateral to injection, showing decreased dysbindin-1 labelling (arrows). F: field CA1 from a 2 weeks post-KA injected rat ipsilateral to injection, showing no visible change in dysbindin-1 labelling (arrows). Scale = 20µm. 65 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo No. of Dysbindin-1 Positive Neurons in DG 50 * 45 40 35 30 25 20 15 10 * 5 0 A 1D Saline 1D KA 2W Saline 2W KA No. of Dysbindin-1 Positive Neurons in CA3 50 45 40 35 30 25 20 15 10 * 5 * 0 1D Saline B 1D KA 2W Saline 2W KA 2W Saline 2W KA No. of Dysbindin-1 Positive Neurons in CA1 50 45 40 * 35 30 25 20 15 10 5 0 C 1D Saline 1D KA 66 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo Figure 5.3.1.3. Number of positive dysbindin-1 labelled neurons in the rat HF, 1 day and 2 weeks after KA or saline treatment ipsilateral to injection. A: dentate gyrus from 1 day postKA injected rats showed an increase in dysbindin-1 labelling, compared to 1 day post-saline injected rats, while the dentate gyrus from 2 weeks post-KA injected rats showed a decrease in dysbindin-1 labelling, compared to 2 weeks post-saline injected rats. B: CA3 from 1 day and 2 weeks post-KA injected rats showed a decrease in dysbindin-1 labelling, compared to their respectively saline controls. C: CA1 from 1 day post-KA injected rats showed an increase in dysbindin-1 labelling, compared to 1 day post-saline injected rats, while the CA1 from 2 weeks post-KA injected rats showed no significant change in dysbindin-1 labelling, compared to 2 weeks post-saline injected rats. The mean +SE are shown. * indicates significant difference (P < 0.01) as compared to its respective saline control. The distribution of dysbindin-1 positive cells after KA treatment was then analyzed by immunohistochemistry. Light staining was observed on granule neurons (Figure 5.3.1.1A) and neurons of field CA3 (Figure 5.3.1.1B) and CA1 (Figure 5.3.1.1C) in the normal rat hippocampus. At 1 day post-KA injection, increased staining was observed in subgranular zone and the adjacent polymorph layer of the dentate gyrus (DG) (Figure 5.3.1.1D) and in field CA1 (Figure 5.3.1.1F), while decreased immunoreactivity was observed in field CA3 (Figure 5.3.1.1E) relative to saline controls on the HF ipsilateral to the KA injection. Dense dysbindin-1 immunolabelled neurons were also observed in the DG (Figure 5.3.1.1G), CA3 (Figure 5.3.1.1H) and CA1 (Figure 5.3.1.1I) on the contralateral HF. Control sections were incubated with antigen-absorbed antibody and showed background staining and this confirmed the specificity of the antibody (Figures 5.3.1.1J, K and L). The immunoreactivity of dysbindin-1 was quantified. At 1 day post-KA injection, a significant 2.70- (Figure 5.3.1.3A) and 4.39-fold (Figure 5.3.1.3C) increase in the number of dysbindin-1 positive neurons relative to saline controls were found in the DG and CA1 respectively, 67 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo while a significant 0.19-fold (Figure 5.3.1.3B) decrease was observed in the CA3 in the HF ipsilateral to the KA injection. At 2 weeks post-KA injection, as compared to saline controls (Figures 5.3.1.2A-C), decreased immunoreactivity was observed in the DG and CA3 (Figures 5.3.1.2D and E), whereas no visible change in dysbindin-1 labelling in the CA1 (Figure 5.3.1.2F) ipsilateral to the KA injection. Significant decrease in the number of dysbindin-1 labelled neurons to 0.36-fold and 0.14-fold in the DG and CA3 (Figures 5.3.1.3A and B), while no significant change in the CA1 (Figure 5.3.1.3C), was found in the ipsilateral HF relative to saline controls at this time. 5.3.2 Effect of oxidative stress on dysbindin-1 mRNA and protein expression in vivo 1.4 Dysbindin-1 normalized to β-Actin Fold Change in Dysbindin-1 mRNA Level 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 A 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1D Saline 1D KA B 1D Saline 1D KA 68 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo Figure 5.3.2.1. Dysbindin-1 expression in the rat HF 1 day after KA treatment. A: Fold change in dysbindin-1 mRNA level. B: Dysbindin-1 protein normalized to β-actin after 2 weeks KA treatment. KA treatment results in no signifcant change in dysbindin-1 mRNA and protein expression at 1 day post-KA. The mean +SE are shown. C: Western blots showing: 1) Homogenates from saline-treated control hippocampus (lanes 1-4), 2) Homogenates from the hippocampus, 1 day after intracerebroventricular KA injection (lanes 5-8). No change in DTNBP1 mRNA expression was found by real time RT-PCR in the HF at 1 day after excitotoxicity induced by KA (Figure 5.3.2.1A). The affinity purified polyclonal antibody to dysbindin-1 showed a single band at approximately 33 kDa in the saline-treated and KA-treated rat HF in Western blots and this corresponds to the molecular weight of dysbindin-1C (Talbot et al., 2009). No significant difference in the dysbindin-1 protein expression was detected at 1 day after KA injury compared to saline controls (Figures 5.3.2.1B and C). 69 1.4 1.4 1.2 1.0 0.8 * 0.6 0.4 0.2 Dysbindin-1 Normalized to β-Actin Fold Change in Dysbindin-1 mRNA Level Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo 0.0 A 1.2 1.0 0.8 * 0.6 0.4 0.2 0.0 2W Saline 2W KA B 2W Saline 2W KA Figure 5.3.2.2. Dysbindin-1 expression in the rat HF 2 weeks after KA treatment. A: Fold change in dysbindin-1 mRNA level 2 weeks after KA treatment. B: Dysbindin-1 protein normalized to β-actin after 2 weeks KA treatment. KA treatment results in decreased expression at 2 weeks postinjection. The mean +SE are shown. *indicates significant difference (P < 0.05) compared to its respective saline control. C: Western blots showing: 1) Homogenates from saline-treated control hippocampus (lanes 1-4), 2) Homogenates from the hippocampus, 2 weeks after intracerebroventricular KA injection (lanes 5-8). A significant decrease in dysbindin-1 mRNA expression to 0.51-fold (Figure 5.3.2.2A) was found in the HF at 2 weeks post-KA treatment, and was accompanied by significantly reduced dysbindin-1 protein level to 0.57-fold, compared to saline-treated controls (Figures 5.3.2.2B and C). 70 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo 5.3 Discussion In vivo studies were carried out in parallel to determine possible changes in dysbindin-1 expression after KA excitotoxicity. Many studies have shown increased oxidative stress after KA induced excitotoxicity (Nakaso et al., 1999; Ong et al., 2000; Ibarretxe et al., 2006; Zheng et al., 2011), and protein and lipid oxidative markers were found to persist even 24 h after KA injury (Gluck et al., 2000). Furthermore, antioxidant enzymes such as glutathione and superoxide dismutase were reduced post-KA treatment (Wang et al., 2005a). These findings indicate that KA excitotoxicity could have some features of H2O2 induced oxidative stress. At 1 day after KA injection, immunohistochemical analyses showed increase in dysbindin-1 expression in the DG and CA1, but loss of immunoreactivity in neurons in the CA3, and real time RT-PCR and Western blot analyses showed no significant change in dysbindin-1 mRNA expression or protein expression. Increase in expression could be due to the effect of oxidative stress on the core promoter activity of dysbindin-1, as shown by our in vitro studies. An upregulation of dysbindin-1 could suggest an early compensatory response in those areas to acute oxidative stress and/or activation of synaptic NMDA receptors, which can acutely boost antioxidant defenses (Papadia et al., 2008). An increase in dysbindin-1 expression was also found in the HF contralateral to KA injection. Compared to the structures ipsilateral to the injection, neurons in the contralateral CA1 and DG showed much higher dysbindin-1 immunoreactivity 1 day after KA injection. While KA promotes death 71 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo of vulnerable neurons locally, it is possible that milder excitotoxicity exerts remotely triggers a protective mechanism and, in the case of the HF, activates commissural projections from CA3 and the DG hilus to the contralateral HF (Raisman G, 1965). In contrast to the DG and CA1, loss of dysbindin-1 immunoreactivity was observed in the CA3 ipsilateral to KA injection. This variability in KA vulnerability could be due to a higher distribution of AMPA/kainate receptors and sensitivity to Ca2+ induced cellular injury in CA3 pyramidal neurons than granule cells in the dentate gyrus (McGeer and McGeer, 1982; Wang et al., 2005b). Together, an increase in dysbindin-1 in the DG and CA1, but decrease in CA3, could account for no net change in dysbindin-1 mRNA and protein levels at 1 day post-KA injection. Unlike results observed at 1 day post-KA, decreased dysbindin-1 gene and protein expression were found at 2 weeks post-KA injection. This reduction was also evident in the postmortem HF of schizophrenia cases (Talbot et al., 2004; Talbot et al., 2011). Decreased dysbindin-1 immunolabelling was observed in DG and CA3 while no significant change was observed in the CA1. It is possible that high level of oxidative stress after KA especially in the CA3 (Ong et al., 2000) could result in increased proteasomal degradation of dysbindin-1 protein, as found by our in vitro studies. Interestingly, dysbindin-1 protein expression found in our immunohistochemical study after 2 weeks post-KA showed a trend akin to the DTNBP1 gene expression in the DG, CA1 and CA3 of schizophrenia cases reported by Weickert et al (2008). Taken together, besides 72 Chapter 5 Role of kainate excitotoxicity on dysbindin-1 expression in vivo a proteasome-dependent degradation pathway, decreased DTNBP1 transcription may also be involved in decreased dysbindin-1 protein expression reported after long term exposure to oxidative stress. Since a majority of the brain dysbindin may exist as a biogenesis lysosome-related organelles complex-1 (BLOC-1) (Ghiani et al., 2010), it may also be important to determine whether other subunits of the BLOC-1 complex are affected by oxidative stress or excitotoxicity in future studies. Intriguingly, converging evidence suggests dysbindin binding partners such as JARID2, MUTED, and ATXN1 may be associated with schizophrenia. Specifically, repeat polymorphism in JARID2 (Pedrosa et al., 2009), multiple SNPs in MUTED (Straub RE, 2005), mutation in ATXN1 (Joo et al., 1999; Duenas et al., 2006) have been associated to the added risk and/or pathogenesis of schizophrenia. Moreover, in the dysbindin-1 null model (sdy), reduced dysbindin-1A and dysbindin-1C expression was accompanied with a decrease in other subunits (e.g. MUTED and pallidin) of the BLOC-1 complex (Li et al., 2003). Hence, future studies on the interactions and effects of different BLOC-1 subunits after oxidative stress could allow a better understanding on the etiology and pathogenesis of schizophrenia. 73 Chapter 6 Conclusion CHAPTER 6 CONCLUSION 74 Chapter 6 Conclusion In conclusion, this thesis has investigated the effects of dysbindin-1 gene and protein expression in vitro and in vivo after oxidative stress. In Chapter 3, the effect of oxidative stress on the activity of dysbindin-1 core promoter was studied in vitro. Dual luciferase reporter assay results have shown an upregulation in dysbindin-1 core promoter activity after H2O2 induced oxidative stress and this induction was abolished by a Sp1 inhibitor, WP631. This suggests an increase in dysbindin-1 gene expression could be mediated by the activation of Sp1 binding sites present in its core promoter. Hence, this finding could serve as one of the possible means in restoring dysbindin-1 levels in schizophrenia patients. However, to validate Sp1 as an activator of dysbindin-1 promoter, further studies such as site-specific mutagenesis of the Sp1 promoter and siRNA knockdown experiments are needed to be carried out. In Chapter 4, the role of PEST sequence and proteasome inhibitor on dysbindin-1 protein expression was explored. Endogenous dysbindin-1 expression in SH-SY5Y cells after oxidative stress was significantly reduced but was restored after treatment with a free radical scavenger, PBN prior to oxidative stress. This result suggests the decrease in dysbindin-1 protein expression as seen in schizophrenia patients (Talbot et al., 2004; Talbot et al., 2012), could be due to the accumulation of excessive free radicals. A recent study has found PBN to be a promising agent against extrapyramidal side effects due to longterm antipsychotic drugs dependence (Daya et al., 2011). Hence, antioxidants such as PBN could present itself as a promising therapy against neurodegenerative diseases such as schizophrenia (Seybolt, 2010; Reddy, 75 Chapter 6 Conclusion 2011). The effects of PEST sequence and proteasome inhibitor on dysbindin-1 protein expression were studied using SH-SY5Y cells stably overexpressing either full length dysbindin-1 or its truncated form without its PEST sequence. Findings from this study found cells stably overexpressing 1) dysbindin-1 without its PEST sequence or 2) full length dysbindin-1A treated with a proteasome inhibitor more resistant to oxidative stress as compared to its vehicle control. These results put forth the notion that dysbindin-1 protein expression could be regulated by its PEST sequence and its protein turnover rate by a proteasomedependent manner. However, the possibility of dysbindin-1 proteins degraded by a calpain mediated system should not be dismissed as proteins containing PEST sequence can also bind Ca2+, and form complexes that are targets for calpain to cleave, resulting in its degradation (Shumway et al., 1999; Rasbach et al., 2008). Hence, further studies on the role of calpain could also be investigated to shed light on another possible pathway modulating dysbindin-1 protein degradation. In Chapter 5, possible changes in dysbindin-1 expression after KA excitotoxicity was determined. Many studies have shown increased oxidative stress after KA induced excitotoxicity (Nakaso et al., 1999; Ong et al., 2000; Ibarretxe et al., 2006; Zheng et al., 2011). Hence, KA excitotoxicity could share features of H2O2 induced oxidative stress. At 1 day after KA injection, real time RT-PCR and Western blot analyses showed no significant change in dysbindin-1 mRNA expression or protein expression, and immunohistochemical analyses which showed an increased dysbindin-1 expression in the DG and CA1, but loss of immunoreactivity in neurons in the CA3 could account for this finding. Increase 76 Chapter 6 Conclusion in dysbindin-1 expression could be due to increased dysbindin-1 core promoter activity, as shown in Chapter 3, or a possible early compensatory response in those areas to acute oxidative stress and/or activation of synaptic NMDA receptors, which can acutely boost antioxidant defenses (Papadia et al., 2008). In contrast, a loss in immunoreactivity in the CA3 ipsilateral to KA injection could be due to a higher distribution of AMPA/kainate receptors and sensitivity to Ca2+ induced cellular injury in CA3 pyramidal neurons than granule cells in the dentate gyrus (McGeer and McGeer, 1982; Wang et al., 2005b). At 2 weeks after KA injection, decreased dysbindin-1 immunolabelling was observed in DG, CA3 while no significant change was observed in CA1. It is possible that high level of oxidative stress after KA especially in CA3 (Ong et al., 2000) could result in increased proteasomal degradation of dysbindin-1 protein, as found by our in vitro studies. Since a majority of the brain dysbindin may exist as a biogenesis lysosome-related organelles complex-1 (BLOC-1) (Ghiani et al., 2010), it may also be important to determine whether other subunits of the BLOC-1 complex are affected by oxidative stress or excitotoxicity in future studies to have a better understanding on the etiology and pathogenesis of schizophrenia. Taken together, our present results indicate that oxidative stress can induce increase in dysbindin-1 transcription through action at its core promoter, and also facilitate protein degradation in a proteasome and PEST sequence dependent manner. 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Behav Brain Funct 3:19. 100 [...]... oxidative stress SH-SY5Y human neuroblastoma cells that stably overexpress dysbindin- 1A or dysbindin- 1A without its PEST sequence were also used to determine the effects of oxidative stress, the proteasome inhibitor and the PEST sequence of dysbindin- 1 on protein expression The effect of the potent glutamate analog, kainic acid (KA), on hippocampal dysbindin- 1 expression was also elucidated KA induces... Hence, dysbindin- 1 will be the focus of this thesis 1. 2 .1. 2 Dysbindin- 1 and its functions Figure 1. 2 .1. 2 Schematic diagram of dysbindin- 1 isoforms in human Dysbindin- 1 isoforms are characterized by 3 main regions: 1) C- terminus region (CTR), 2) Coiled coil domain (CCD), and 3) N -terminus region (NTR) Dysbindin- 1A is the full length dysbindin isoform, while dysbindin- 1B and dysbindin- 1C are exactly like its... medication and substance abuse are of great importance as they affect the onset of schizophrenia Though medication may alleviate the severity of symptoms, substance abuse may increase the chance of relapses Additionally, psychosocial factors such as stress and social support also play a pertinent role in the course of schizophrenia (Mueser and McGurk, 2004) Similarly, epidemiological studies also found... promoter activity after oxidative stress CHAPTER 3 CHANGES IN DYSBINDIN- 1 CORE PROMOTER ACTIVITY AFTER OXIDATIVE STRESS 23 Chapter 3 Changes in Dysbindin- 1 core promoter activity after oxidative stress 3 .1 Introduction Several studies have found significant correlations between schizophrenia and haplotypes of SNPs in specific genes following the discovery of DTNBP1 as a schizophrenia-susceptibility gene (Cloninger,... induces excitotoxicity in hippocampal neurons and since many converging evidences have shown that this is associated with oxidative stress and lipid peroxidation (Ong et al., 2000; Wang et al., 2005b; Sanganahalli et al., 2006), analyses of dysbindin- 1 expression after KA might provide insights into effects of oxidative stress on dysbindin- 1 expression in vivo 22 Chapter 3 Changes in Dysbindin- 1 core promoter... levels or indices of oxidative stress are increased, while antioxidant defenses are decreased in the serum and plasma of schizophrenia cases (Gysin et al., 2007; Zhang et al., 2 010 ; Ciobica et al., 2 011 ; Li et al., 2 011 ; Yao and Keshavan, 2 011 ) The same imbalance of oxidants and antioxidant defenses has also been reported in the cerebrospinal fluid and brain tissue (Do et al., 2009; Ciobica et al., 2 011 ;... hydrophobic index and, greater probability of the sequence acting as a proteolytic signal Phosphorylation of predicted kinases sites in the PEST sequence may elicit a change in conformation which is recognizable by proteasome, causing the rapid degradation of its protein (Rechsteiner and Rogers, 19 96; Garcia-Alai et al., 2006) For example, oxidative stress- induced degradation of a protein, nuclear factor... this thesis, the effects of oxidative stress on dysbindin- 1 expression were studied in vitro and in vivo by treating human neuroblastoma cells with hydrogen peroxide (H2O2), a reactive oxygen species, and using kainic acid (KA) in rats, respectively 1. 4 .1 Kainic acid-mediated excitotoxicity Figure 1. 4 .1 Schematic diagram of KA-mediated neuronal cell death pathway (1) Binding of KA to Ca2+ AMPA/KA receptors... Dysbindin- 1A is known to be the full length isoform, a 3 51 amino acid protein expressed in 9 Chapter 1 Introduction humans and 352 amino acid protein expressed in rats Dysbindin- 1B is similar to its full length isoform except for a truncated CTR and is a 303 amino acid protein found in humans but not expressed in rats Dysbindin- 1C on the other hand is an isoform that lacks a NTR, and is a 270 amino acid... RT-PCR Reverse transcription polymerase chain reaction SCF Stem cell growth factor Sdy Dysbindin- null siRNA Small interfering ribonucleic acid SNPs Single nucleotide polymorphisms SOD Superoxide dismutase Sp1 Specificity protein 1 TBARS Thiobarbituric reactive substances TBS Tris-buffered saline TRIM32 Tripartite motif-containing protein 32 XI Chapter 1 Introduction SECTION I INTRODUCTION 1 Chapter 1 Introduction ... HindIII sites including the predicted core promoter of dysbindin- 1A was isolated using a forward CAGTCTCGAGAGGACTGGGGATGTCACTCA-3’) primer and reverse (5’primer (5’- GTACAAGCTTAACCCAGCCTTCTCCAAG-3’)... PEST sequence on Dysbindin- 1 expression in vitro PEST sequence under oxidative stress was investigated using SH-SY5Y human neuroblastoma cells that stably overexpress dysbindin- 1A or dysbindin- 1A. .. that Sp1 is involved in modulating dysbindin- 1 expression upon oxidative stress To validate and propose Sp1 as an activator of dysbindin1 , further studies such as (1) mutagenesis of Sp1 binding