Author’s Accepted Manuscript Cellular models to study bipolar disorder: A systematic review Biju Viswanath, Sam P. Jose, Alessio Squassina, Jagadisha Thirthalli, Meera Purushottam, Odity Mukherjee, Vladimir Vladimirov, Georg P Patrinos, Maria Del Zompo, Sanjeev Jain www.elsevier.com/locate/jad

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S0165-0327(15)00344-4 http://dx.doi.org/10.1016/j.jad.2015.05.037 JAD7473

To appear in: Journal of Affective Disorders Received date: 20 January 2015 Revised date: 20 May 2015 Accepted date: 20 May 2015 Cite this article as: Biju Viswanath, Sam P. Jose, Alessio Squassina, Jagadisha Thirthalli, Meera Purushottam, Odity Mukherjee, Vladimir Vladimirov, Georg P Patrinos, Maria Del Zompo and Sanjeev Jain, Cellular models to study bipolar disorder: A systematic review, Journal of Affective Disorders, http://dx.doi.org/10.1016/j.jad.2015.05.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cellular models to study bipolar disorder: A systematic review

Biju Viswanath1, 5, 8 Sam P. Jose2 Alessio Squassina3 Jagadisha Thirthalli1 Meera Purushottam1 Odity Mukherjee5 Vladimir Vladimirov6 Georg P Patrinos7 Maria Del Zompo3, 4 Sanjeev Jain1, 5

1 - Department of Psychiatry, National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2 - Department of Psychiatry, Elite Mission Hospital, Koorkanchery, Thrissur, Kerala, India. 3 - Laboratory of Pharmacogenomics, Section of Neuroscience and Clinical Pharmacology, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy. 4 - Unit of Clinical Pharmacology, Teaching Hospital, Cagliari, Italy. 5 - National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India. 6 - Virginia Institute for Psychiatric and Behavioral Genetics, Virginia Commonwealth University, Richmond, VA, USA.

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7 - University of Patras, School of Health Sciences, Department of Pharmacy, Patras, Greece. 8 – Centre for Brain Development and Repair, Institute of Stem Cell Biology and Regenerative Medicine, Bangalore, India.

Corresponding Author Prof. Sanjeev Jain Professor of Psychiatry, Molecular Genetics Laboratory, National Institute of Mental Health and Neuro Sciences, Bangalore, India. Phone: +918026995263 Email: [email protected]

Running head: Cell models in bipolar disorder Word count (abstract): 249 Word count (main text): 5639 Number of tables: 3 Number of figures: 1

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Abstract Background There is an emerging interest in the use of cellular models to study psychiatric disorders. We have systematically reviewed the application of cellular models to understand the biological basis of bipolar disorder (BD). Method Published scientific literature in MEDLINE, PsychINFO and SCOPUS databases were identified with the following search strategy: [(Lymphoblastoid OR Lymphoblast OR Fibroblast OR Pluripotent OR Olfactory epithelium OR Olfactory mucosa) AND (Bipolar disorder OR Lithium OR Valproate OR Mania)]. Studies were included if they had used cell cultures derived from BD patients. Results There were 65 articles on lymphoblastoid cell lines, 14 articles on fibroblasts, 4 articles on olfactory neuronal epithelium (ONE) and 2 articles on neurons reprogrammed from induced

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pluripotent stem cell lines (IPSC). Several parameters have been studied, and the most replicated findings are abnormalities in calcium signaling, endoplasmic reticulum (ER) stress response, mitochondrial oxidative pathway, membrane ion channels, circadian system and apoptosis related genes. These, although present in basal state, seem to be accentuated in the presence of cellular stressors (e.g.: oxidative stress – rotenone; ER stress – thapsigargin), and are often reversed with in-vitro lithium. Conclusion Cellular modeling has proven useful in BD, and potential pathways, especially in cellular resilience related mechanisms have been identified. These findings show consistency with other study designs (genome-wide association, brain-imaging, post-mortem brain expression). ONE cells and IPSC reprogrammed neurons represent the next generation of cell models in BD. Future studies should focus on family-based study designs and combine cell models with deep sequencing and genetic manipulations. Keywords: Bipolar disorder; Cell model; Lymphoblast; Pluripotent; Olfactory; Fibroblast

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Introduction Bipolar disorder (BD) is a chronic, severe psychiatric illness affecting 0.7-6% of the general population and uses a substantial portion of mental health resources worldwide (McCarthy et al., 2012).Clinical research in susceptibility to BD has as yet, yielded inadequate insight into its biology. Heritable influences are well established, with heritability estimates ranging between 80-90% (Shih et al., 2004), and these influences are likely to be polygenic in nature (Gibson, 2010). Further research is inhibited by the difficulty of generating large enough data sets to separate true biological signal from background noise (Seifuddin et al., 2012). Understanding ex vivo cellular networks from patients with BD may allow us to understand the interface between genetic variations, and both susceptibility and treatment response in BD. Although brain cells are ideal for studying biological determinants of BD, live sampling of such tissues is impossible. In addition, post-mortem brain is generally not suitable for culture or in vitro experiments. Many genes that have been identified as risk factors for neuro-psychiatric disorders are not uniquely expressed in brain, but also in many other tissues, including whole blood. Hence, the use of cell cultures as an appropriate surrogate model for disease in general, and for neuropsychiatric disease in particular, has become prominent currently (Brennand et al., 2012). Various cell culture systems have been used for such studies including non-neuronal [lymphoblastoid cell lines (LCL), fibroblasts] and neuronal [olfactory neuronal epithelium (ONE), neurons reprogrammed from induced pluripotent stem cells (IPSC)] cell lines. The IPSC

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derived neuron system is emerging as the most exciting area for such research (O'Shea and McInnis, 2015). The field seems to hold a lot of promise. However, many questions have been raised about the validity of cell models (Choy et al., 2008; Matigian et al., 2008; Min et al., 2010). So are these cell models useful and valid in BD? We attempt to address this question by systematically reviewing current literature on the application of the above cellular models to understand the biology of BD. Method (Figure 1) We searched published scientific literature in MEDLINE, PsychINFO and SCOPUS databases with the following search strategy between April 7th to May 7th, 2013: [(Lymphoblastoid OR Lymphoblast OR Fibroblast OR Pluripotent OR Olfactory epithelium OR Olfactory mucosa) AND (Bipolar disorder OR Lithium OR Valproate OR Mania)]. Studies were included if they had used cell cultures (lymphoblastoid cell lines, fibroblasts, olfactory neuronal epithelium, induced pluripotent stem cell lines and reprogrammed neurons) derived from BD patients. Only articles in English language were selected. A total of 709 citations were identified. Articles were selected based on title and abstract and, when needed, on inspection of the full text to assess relevance. Records retrieved through references of these articles were screened for additional studies not previously identified. The authors were contacted if additional information was required. The first and second author of this study independently carried out the search. Any inconsistency was discussed and resolved. A total of 76 articles fulfilled the inclusion criteria and were included in this review. Additional publications till January 2015 (n=7) and those identified by reviewers (n=2) were also included, thus the number increased to 85 articles. The

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literature search details are given in figure 1. The first and second authors extracted information from the full text reports/ supplemental material independently. Authors were contacted if the method/results of the study were unclear. Meta-analysis and funnel-plot (for publication bias) could not be performed due to the wide variations in methodology and multiple publications (with overlap of clinical sample) from the few laboratories which are performing such experiments. However, selection bias was examined by reviewing the study methodologies. The review is compliant with PRISMA guidelines (Moher et al., 2009). Results and discussion Non-neuronal models: Lymphoblastoid cell lines and Fibroblasts Studies using non-neuronal cell models in BD have used hypothesis-driven and hypothesis-free (genome-wide) approaches. Hypothesis-driven approach (Table 1) Calcium (Ca2+) signaling Given the importance of Ca in regulating many cellular processes including neuroplasticity, Ca2+signaling has been the most investigated aspect in cell models of BD. Ca2+signaling studies have focused on various key components of the system [e.g.: mitochondrial channels, sarco(endo)plasmic reticulum ATPase (SERCA)and transient receptor potential (TRP) ion channels] to understand the mechanisms of aberrant signaling. The most replicated finding across Ca2+signaling studies is the higher cytoplasmic level of Ca2+ in LCLs derived from BD patients compared to controls (Corson et al., 2001; Emamghoreishi et al., 2000; Emamghoreishi et al., 1997; Kato et al., 2003; Machado-Vieira et al., 2011; Perova et al., 2010; Perova et al., 2008; Uemura et al., 2011). In addition, cytosolic Ca2+ response to Thapsigargin (TG) (SERCA inhibitor – first depletes ER Ca2+, later activates Ca2+ entry at membrane)(Kato et al., 2003) and

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Lysophosphatidic acid (LPA) (Ca2+ entry through a diacylgycerol dependent TRP like channel)(Perova et al., 2008) have been found to be greater in BD LCLs than in control LCLs. The mood stabilizers lithium (Andreopoulos et al., 2004; Perova et al., 2010; Wasserman et al., 2004) and valproate (Perova et al., 2010) have been found to attenuate agonist-induced Ca2+ responses in these cell models. Among the Ca2+signaling related gene expression studies in BD LCLs, the only replicated finding has been that of the anti-apoptotic gene, Bcl-2. Lower Bcl-2 gene expression and protein levels have been found in LCLs with higher Ca2+, suggesting that the Bcl-2 gene has a protective role in BD (Uemura et al., 2011). Lithium treatment in vitro has been found to increase Bcl-2 expression, leading to restoration of Ca2+ levels (Machado-Vieira et al., 2011). A SNP located in the Bcl-2 gene (rs956572) has been found to modulate the above changes in LCLs (MachadoVieira et al., 2011; Uemura et al., 2011). Other findings include reduced TRPC7 expression (Yoon et al., 2001b) and greater frequency of the TRPM2-intron 19 SNP T/T genotypein BD LCLs with higher Ca2+(Xu et al., 2006).TRPM2- and TRPC3-mediated Ca2+fluxes after rotenone (oxidative stress) are also impaired in BD LCLs (Roedding et al., 2012). G protein system Differences in cyclic AMP (cAMP) production between BD LCLs having high and low Ca 2+ levels have been noted (Emamghoreishi et al., 2000). The G-protein receptor kinase (GRK)-3 protein levels have been found to be higher in BD LCLs than in control LCLs (Niculescu et al., 2000). The gene which encodes GRK3, namely ADRBK2 at the locus 22q13, was examined in another study (McCarthy et al., 2010). They studied chromatin immunoprecipitation of acetylated histone H3 at individual ADRBK2 regulatory alleles (for promotor activity) and measured allele expression imbalance among transcribed, synonymous SNPs in the ADRBK2

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transcript, and did not find any difference. Stimulatory G protein (Gsα) protein levels were also examined in LCLs from BD patients with good lithium response versus controls, with no significant differences (Alda et al., 2001). The cAMP – Protein-Kinase A (PKA) signaling pathway and downstream Brain Derived Neurotrophic Factor (BDNF) signaling has also been examined (Karege et al., 2004a; Karege et al., 2004b). Basal PKA activity, catalytic PKA subunit and p-CREB protein levels were increased(Karege et al., 2004a), whereas 3H–cAMP binding to PKA regulatory subunits was reduced in BD (Karege et al., 2004b). The induction of PKA activity with an activator/inhibitor was found to increase/decrease BDNF expression respectively, especially in BD LCLs (Karege et al., 2004a; Karege et al., 2004b). A recent examination of reporter based variations in lithium induced cAMP-CREB signaling revealed greater magnitude in fibroblasts derived from BD(Gaspar et al., 2014). Older studies on Isoprenaline induced cAMP response in fibroblasts (Berrettini et al., 1986; Berrettini et al., 1987a) did not find differences between BD and control lines. Inositol signaling system The inositol signaling system has been considered important in BD because of the proposed link between this system and Ca signaling, as well as in the mechanism of action of lithium.The expression of the inositol monophosphatase gene has been found to be lower in BD LCLs with higher basal Ca levels when compared to BD LCLs with low Ca levels (only in men) (Yoon et al., 2001a). Lower activity of the inositol monophosphatase (IMPase) enzyme has been found in LCLs from patients with BD (Shaltiel et al., 2001; Shamir et al., 1998). Lithium treatment of LCLs in-vitro was associated with significant increase in the IMPase mRNA levels; however, effect on enzyme activity was not studied (Shamir et al., 1998). Inositol incorporation (Banks et

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al., 1990) and inositol content (Belmaker et al., 2002) has been found to be lower in BD LCLs, in comparison with control LCLs. Endoplasmic Reticulum (ER) Many genes of the ER stress pathway have been studied in BD LCLs. The gene HSPF1 has been found to be up-regulated in post-mortem brain from BD patients (Iwamoto et al., 2004b) as well as in two independent samples of BD LCLs, when compared with control LCLs (Iwamoto et al., 2004a; Iwamoto et al., 2004b). Transcriptional activation of another ER stress gene XBP1gene is attenuated in BD LCLs, when exposed to an ER stress inducing agent (TG/Tunicamycin) (Hayashi et al., 2009; So et al., 2007). Similar findings have also been found for GRP94 (Hayashi et al., 2009) and CHOP (So et al., 2007).Other genes studied in BD LCLs with negative results include ATF4 (Kakiuchi et al., 2007b), ATF5(Kakiuchi et al., 2007b), HSP90B1 (Kakiuchi et al., 2007a), GRP78(Hayashi et al., 2009; So et al., 2007) and calreticulin (Hayashi et al., 2009). Mitochondria LCLs from BD patients have been found to display abnormalities in mitochondrial morphology and distribution (Cataldo et al., 2010). The mitochondria were distributed more in the perinuclear regions than in distal processes. Atypically shaped mitochondria were also found in fibroblasts and post-mortem brains derived from patients with BD. LCLs from BD patients (in Japan) have been found to have reduced expression of the nuclear encoded mitochondrial electron transport chain complex 1 (NDUFV2) gene, in comparison to the LCLs from healthy controls (Washizuka et al., 2009; Washizuka et al., 2003). However, this difference was not found in European patients (Washizuka et al., 2009; Xu et al., 2008). Nominal associations have also been found with expression of NDUFA1, NDUFA6, NDUFS7, NDFUV6

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and COX6C genes (Washizuka et al., 2005). The expression of NDUFV2 directly correlates with the expression of these genes (Washizuka et al., 2003; Washizuka et al., 2005). Membrane Sodium (Na)-Potassium (K) pump Greater Na-K pump site number (Banks et al., 1989) and lower pump activity (Cherry and Swann, 1994) has been found in BD LCLs, when compared to control LCLs. No difference has been found in terms of transmembrane potential (Buss et al., 1996). Studies (Huff et al., 2010; Li and El-Mallakh, 2004) have also examined the effect of oxidative stress using ethacrynic acid in BD and control LCLs. When exposed to stress, the control cells could upregulate Na-K pump numbers, activity, mRNA and protein levels. This allowed them to maintain normal Na levels within the cell. However, the BD cells were unable to do so, leading to increased Na levels in these LCLs. Neuroplasticity related genes Synapsin II is a phosphoprotein involved in synaptic plasticity, transmission and synaptogenesis. The expression of the Synapsin II gene (SYN2A and SYN2B variants) has been examined in LCLs derived from lithium responders (Li-Rs) and lithium non-responders (Li-NRs) in BD (Cruceanu et al., 2012). A broader distribution of lithium induced SYN2A and SYN2B expression was found in Li-Rs. BDNF has also been studied in BD LCLs. Basal and lithium induced BDNF protein levels have been found to lower in BD LCLs than control LCLs. (Tseng et al., 2008). However, another study was negative (Gao et al., 2012). Circadian rhythm Circadian clock systems have been suspected in BD in view of disruptions in circadian rhythms such as sleep and daily activity that often normalize after lithium treatment(McCarthy et al., 2011a). When the circadian clock genes were examined with respect to Lithium response in BD

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using LCLs, it was found that lithium response was best predicted by the A allele of NR1D1 rs2071427and C allele of GSK3B rs6438552. The NR1D1 A/A genotype was also found to be associated with a lower amount of full length NR1D1 transcript and a greater amount of truncated NR1D1 transcript, when exposed to lithium in-vitro (McCarthy et al., 2011a).The 12 hour amplitude of the gene BMAL1, overall expression of DBP and phosphorylated GSK-3β protein levels have been found to be lower in fibroblasts from patients with BD and those from healthy controls (Yang et al., 2009a). However, when fibroblasts from BD/controls were incorporated with promoter sequences for BMAL1 to drive the expression of the firefly luciferase gene, no differences were found between the groups in terms of period length or phase response curves (Bamne et al., 2013). Similar experiment with PER2 showed that baseline period was longer in BD, and that lithium resynchronised the circadian clock (McCarthy et al., 2013). An enzyme related to this system, Acetyl Serotonin N-Methyl Transferase (ASMT) of the melatonin biosynthesis pathway, has also been investigated (Etain et al., 2012). The ASMT mRNA and enzyme levels were significantly lower in BD LCLs than in control LCLs. Neurotransmitter & neuroendocrine systems Neurotransmitter systems examined in BD cell models include adrenergic (Berrettini et al., 1987b; Kay et al., 1993; Kay et al., 1994; Wright et al., 1984), serotonergic (Pinto et al., 2011) and cholinergic(Lin and Richelson, 1986; Nadi et al., 1984); most studies have had negative results. The binding of 125I-iodohydroxybenzylpindolol to β-adrenoceptors in BD LCLs has been examined (Berrettini et al., 1987b; Kay et al., 1993; Kay et al., 1994; Wright et al., 1984), but only one study (Wright et al., 1984) found lower binding in BD. Incubation with β-adrenoceptor agonist Isoprenaline leads to reduced downregulation of the adrenergic system in BD LCLs,

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which is normalized by lithium (Kay et al., 1993; Kay et al., 1994). Monoamine oxidase A enzyme level has been studied in BD fibroblasts with negative results (Breakefield et al., 1980). There was also preliminary evidence of greater number of muscarinic receptor binding sites (Nadi et al., 1984) in LCLs derived from patients with BD, but this could not be replicated(Lin and Richelson, 1986). HPA axis dysregulation has also been investigated in LCLs derived from BD (Henning et al., 2005). Basal glucocorticoid receptor numbers and receptor downregulation with hydrocortisone treatment were not different between cells derived from BD and healthy controls. MicroRNA Basal expression of 13 miRNAs and effect of in vitro lithium were examined in LCLs derived from BD patients and their unaffected siblings (Chen et al., 2009). Although no differences were found between groups, lithium upregulated four miRNAs (miR-34a, miR-152, and miR-155, and miR-221). Predicted mRNA targets for these microRNAs were significantly enriched on three gene ontology defined biological processes - macromolecular complex assembly, protein complex assembly, and cellular component assembly. Amino acids and polyamines The maximal transport capacity for Tyrosine has been found to be lower in fibroblasts derived from patients with BD (Persson et al., 2009). In a recent report, baseline production of kynurenic acid and 3-hydroxykynurenine in BD fibroblasts was higher than in controls and case-control interaction analyses revealed that cells from patients with BD respond differently to proinflammatory cytokines in terms of L-kynurenine metabolism (Johansson et al., 2013). SpermineN1-acetyltransferase (SAT1), the key regulator of cellular polyamine content, has been examined in BD LCLs. Lithium in-vitro was found to have differential effects on SAT1

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expression and protein levels, in LCLs derived from BD patients with and without suicidality (Squassina et al., 2013b). Polyglutamine (CAG, which is translated into glutamine) expansions have also been explored in the proteome of BD LCLs, with negative results (Jones et al., 1997; Zander et al., 1998). Others A recent study (Kato et al., 2011) compared the expression of 17 genes(selected from previous gene expression studies, animal studies, convergent functional genomics and those related to mitochondria) between LCLs derived from BD and healthy controls. Significant findings were up-regulation of ANK3 and down-regulation of RASGRP1 and POLG1. ANK3 codes for Ankyrin G, an adapter protein linking ion channels to spectrin in membrane; RASGRP1 codes for a guanyl nucleotide exchange factor that activates ras, and has binding domains for Ca2+ and diacylglycerol; POLG1 codes for the catalytic subunit of mitochondrial DNA polymerase. Lithium incorporation (Breslow et al., 1985)and plasma membrane fatty acid composition (Mahadik et al., 1996)have been compared in fibroblasts derived from BD and controls, with negative results. Hypothesis-free approach (Table 2) The hypothesis-free approaches use high-throughput technologies to investigate whole genome (DNA and RNA) or proteome, generating new hypotheses to be tested in further studies. Most studies have focused on monozygotic twins. Findings span various pathways and await replication in independent samples. One finding (Kakiuchi et al., 2003)which has been replicated in an independent sample is the impaired feedback regulation of ER stress genes (Hayashi et al., 2009; So et al., 2007). Upregulation of the apoptotic pathway (Matigian et al., 2007)and proteins

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related to cell death (Kazuno et al., 2013)has additional support from hypothesis-driven studies on Bcl-2 expression(Machado-Vieira et al., 2011; Uemura et al., 2011). Critical overview of non-neuronal models: Are they useful in BD? Studies in non-neuronal models have found biological signals in various pathways and processes in BD. Most of the findings from hypothesis-free studies await replication. Among the 54 hypothesis-driven studies, 40 have had positive findings, but there have been only some attempts at replication. Attempts to replicate findings in BD LCLs are given in table 3. Thus, the most replicated findings from the studies on non-neuronal cell models include abnormalities in Ca2+signaling, ER stress response, mitochondrial oxidative pathway, membrane ion channels, circadian system and apoptosis related genes (Bcl-2).These processes are critical to cellular homeostasis, plasticity, signaling and cell viability. These pathways are interlinked in many different ways - Ca2+ is a widespread second-messenger, and plays a major role in cellular ionic homeostasis, plasticity, and survival by directly affecting mitochondria and ER; the antiapoptotic protein bcl-2 has also been found to regulate Ca2+signaling by interacting with ER and mitochondria(Machado-Vieira et al., 2013); circadian clock has been found to be regulated by membrane potential and intracellular Ca2+(Noguchi et al., 2012); oxidative stress is commonly caused by mitochondrial Ca2+ overload, but oxidative stress has also been found to induce dysregulation of Ca2+ homeostasis and apoptosis(Andreazza et al., 2008). These abnormalities, although present in basal state, seem to be accentuated in the presence of cellular stressors (e.g.: oxidative stress – rotenone; ionic stress – ethacrynic acid; ER stress – thapsigargin), highlighting reduced cellular resilience in BD cells. The impaired cellular resilience in BD has been hypothesized to contribute to the increased vulnerability of BD patients on exposure to stressful

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environmental situations (Fries et al., 2012). Many of these cellular perturbations are reversed with in-vitro application of mood stabilizers, especially lithium. So are these models useful and valid in the study of BD? It is clear from the reviewed literature that these models do provide biological signals by deep interrogation of relatively fewer numbers of samples. The major findings from these studies also show consistency with findings using other study designs. Ca2+ channel related genes, especially CACNA1C have been some of the top hits in the recently completed psychiatric genomic consortium genome wide association studies, both in the BD sample (PGC, 2011)and the cross-disorder meta-analytic sample(Smoller et al., 2013). Recent studies in post-mortem brains have identified impairment of mitochondrial complex I activity in BD, which is associated with increased oxidation and nitration (Andreazza et al., 2010; Andreazza et al., 2013). The Bcl-2rs956572 genotype, which has been associated with high Ca2+ levels in LCLs, has also been found to be associated with high anterior cingulate glutamate in a recent magnetic resonance spectroscopy study(Soeiro-de-Souza et al., 2012). When put together, available evidence does indicate that signals derived from cellular models are valid; similar findings have been derived from post-mortem brain studies, brain imaging studies and genome-wide association studies. Limitations of available literature need to be discussed. As previously mentioned, many of the findings await replication. One reason for negative studies/ non-replication could be inadequate power of the study. None of the hypothesis-driven studies, including the negative studies have mentioned a power calculation. We calculated desired sample size with R Software 3.0.1 (2013) using literature on the most investigated aspect of BD namely Ca signaling in LCLs. To detect a mean difference of 7.4nM between groups, with standard deviation 8.8nM (Emamghoreishi et al., 1997), 80% power and significance level of 0.05 using two-sided independent-Samples T

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test; the expected sample size is 46 LCLs (23 LCLs per group). The median number of LCLs used per study was 34.5 (range 12-456).The numbers are far fewer in hypothesis-free studies (median = 4, range 4-46).False positive results are a constant source of concern in such studies, and it is crucial to have adequate numbers. Further, the most important limitation of these models is that it is non-neuronal. Although LCLs/fibroblasts express metabolic pathways specific for an individual, to detect specific cellular perturbations occurring in neuropsychiatric disease, a neuronal cell model is still the best. Many genes could be expressed differentially in neurons and non-neuronal cells to have variable effects. It may still be possible to differentiate a patient and healthy control/ treatment responder and non-responder with non-neuronal models, but one will have to fall back to neurons to determine the exact pathophysiology of BD. Neuronal models: Olfactory neuronal epithelium Biopsy derived ONE cells from BD patients and healthy controls have been investigated for Ca2+signaling (Hahn et al., 2005). Two odorant mixtures A and B were used to stimulate cAMP and inositol triphosphate second messenger systems respectively. Cells derived from BD patients responded only with mixture B, whereas control cells responded to both. The response frequency was also lower in cells from medicated BD patients. Cellular proliferation, cell death and GWGE profiles have also been studied in biopsy derived ONE cells from patients with BD, schizophrenia and healthy controls (McCurdy et al., 2006). In comparison with controls, ONE cells from patients with schizophrenia had more mitosis, whereas cells from patients with BD had greater cell death. GWGE profiling identified inositol metabolism, apoptosis, catecholamine metabolism and protein biosynthesis related genes to be differentially expressed in cells from BD patients.

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A novel non-invasive method to isolate ONE cells by exfoliation of the anterior region of the medial lateral turbinate using a brush has been reported (Benitez-King et al., 2011). Functionality of the olfactory sensory neuron was demonstrated by measuring voltage operated Ca2+current in the cell lines using patch clamp recording. In the most recent study by the same group (SolisChagoyan et al., 2013), microtubule organization and quantification of assembled tubulin was performed in ONE cells derived from patients with BD and schizophrenia. BD neurons demonstrated short microtubules and a reduction in the amount of tubulin in total homogenates and 40% reduction in the globular fraction. Neuronal models: Induced pluripotent stem cell line derived neurons Studies using IPSC neurons in psychiatry have primarily focused on schizophrenia (Brennand et al., 2012), and there are only two reports of such cell lines derived from patients with BD (Chen et al., 2014; Wang et al., 2014). In the first study (Chen et al., 2014), fibroblasts derived from 3 BD patients and 3 healthy controls were reprogrammed to IPSC neurons. Microarray analysis revealed a significant increase in the expression of transcripts for membrane bound receptors and ion channels, especially those related to Ca2+signaling. In addition, there was differential expression of transcription factors which determine telencephalic fate – genes involved in the differentiation of ventral regions were expressed in BD neurons while those involved in differentiation of dorsal regions were expressed in control neurons. Ca2+ signaling experiments were also carried out. Lithium pre-treatment of BD neurons significantly reduced their Ca2+ transient and wave amplitude, in comparison to control neurons. In the second study (Wang et al., 2014), skin fibroblasts from 12 BD patients and 6 controls were directly converted to induced neuronal like cells using lentivirus vectors. Label free advanced

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optical imaging (BIND imaging) was performed using a nanostructured photonic crystal biosensor. Cells derived from Li-NR BD patients were found to have reduced adhesion to the biosensor used, in comparison to Li-Rs and controls. Lithium treatment in vitro did not lead to any significant changes. Critical overview of neuronal models in BD It is obvious that the next generation of research in the field will focus on neuronal cell models – the ONE cells and IPSC neurons. Cortical glutamatergic and GABAergic neuronal populations, midbrain dopaminergic neuronal populations and hippocampal neurons can now be efficiently differentiated in vitro from IPSCs (Chambers et al., 2009; Yu et al., 2014a). Technology to differentiate specific type of neurons from ONE cells is in progress. Neuronal cell models have been used extensively in many neuropsychiatric disorders like Huntington’s disease (Mu et al., 2014) Alzheimer’s disease (Machairaki et al., 2014) Parkinson’s disease (Zhao et al., 2014) Autism (Kim et al., 2014) and Schizophrenia (Brennand et al., 2014). These studies have revealed many technical issues, which have been detailed in other eminent methodology focused reviews (Brennand et al., 2013). Some of the major limitations are the following: 1. Neural populations generated through IPSC reprogramming have mixed temporal and spatial identities. Cell surface markers to determine the temporal and regional identities are unavailable at this point. In a given culture, one cannot make out whether a neuron is glutamatergic, dopaminergic or GABAergic (Brennand et al., 2013). 2. IPSC neurons require many months to mature, lack myelination and resemble fetal neural tissue (Brennand et al., 2013).This is one reason why the sample sizes of some of the reported studies are very small. In this regard, ONE can be quickly generated in larger

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numbers; although bearing in mind that acquiring these cells needs an invasive and relatively uncomfortable procedure (Mackay-Sim et al., 2011). 3. Genetic and epigenetic mutations can occur during the process of reprogramming. This is a potential disadvantage as the effect of these mutations on disease-associated genes is unknown. Also, this could cause variations between clones generated from each patient. One suggestion to overcome this problem is to use multiple clones derived from the same patient for validation(Brennand et al., 2013). ONE cells however, are not genetically modified (Mackay-Sim et al., 2011).

In spite of the above said limitations, there is no doubt that proper use of neuronal cell models is likely to lead to gene and pathway discoveries in BD. As previously stated, many lines of studies suggest the role of calcium in BD. Studies using non-neuronal cells have showed the role of mitochondria and endoplasmic reticulum, both of which are intracellular reservoirs of calcium. The role of membrane calcium channels has been suggested by GWAS, and it should be tested by neuronal cells in the future. Using neuronal cells, functional consequence of altered calcium signaling such as neural plasticity could be tested. Future directions Peripheral versus neuronal models: The majority of studies reviewed have used nonneuronal/peripheral cell models. With these cell lines (LCL, fibroblast) which are convenient to derive, the primary question is whether we can assume that mechanisms associated with the risk of major psychiatric disorders cause systemic effects (Matigian et al., 2008). Even if they do, genes could be expressed differently in neurons and LCLs/fibroblasts. In addition, differences between the transcriptomes of LCLs and fibroblasts(Matigian et al., 2008), as well as LCLs and blood (Min et al., 2010)has been a cause of major concern. With respect to neuronal models, 20 Viswanath et al.

ONE cells are easy to culture and are not genetically modified, but the process to obtain these cells is relatively invasive. IPSC neurons, although more readily accessible as they can be derived from fibroblasts, keratinocytes (Petit et al., 2012), LCLs or urine (Mackay-Sim et al., 2011; Zhou et al., ; Zulfiqar et al., 2013), are difficult to grow in culture and are often genetically modified (Brennand et al., 2013). However, common sense indicates that fundamental mechanisms of neuropsychiatric disease are likely to be revealed better with these cell lines. Study designs: A case-control approach (cells derived from patients versus controls) has been used by most studies on non-neuronal cell lines, and this design is likely to be used further with neuronal cell lines. However, other powerful study designs also need to be considered: 1. Using familial cell lines to identify heritable cellular processes in BD. Cellular models derived from dense families of BD have been used in few studies (Berrettini et al., 1986; Houlihan et al., 2009; Kakiuchi et al., 2003; Kazuno et al., 2013; Kuratomi et al., 2008; Matigian et al., 2007; Nadi et al., 1984; Sugawara et al., 2011; Yang et al., 2009a; Yang et al., 2009b). Some of them were studies on monozygotic twins (Kakiuchi et al., 2003; Kazuno et al., 2013; Kuratomi et al., 2008; Matigian et al., 2007; Sugawara et al., 2011). Considering that BD is a disorder with high heritability and that even Lithium response in BD is familial(Grof et al., 2002), there is a need for studies to focus on the heritability of biological processes itself in BD. LCLs/fibroblasts from many large families affected with BD are already available. Biological studies (Ca2+ signaling, cell survival, apoptosis, and expression studies) in cortical neurons derived from these cells would be a logical step forward. Having multiple affected and unaffected individuals from the same family/ group of families would also reduce the genetic heterogeneity within the study sample resulting in less false positive signals.

21 Viswanath et al.

2. Integrating psychiatric genomics consortium data with cell biology data to find the genetic basis of biological processes in BD. With the advent of next generation deep sequencing technologies and the psychiatric genomics consortium (PGC, 2011; Smoller et al., 2013), it will be possible to identify the genes/ genetic variations are responsible for the biological alterations in BD. This may be achieved by using a combined cell model- deep sequencing approach in a relatively smaller number of samples (especially family-based samples). Sequencing of the genome, transcriptome and methylome as has been achieved via the consortium, should be combined with cell biology approaches.The optimism regarding this approach is tempered by the fact that genomewide association study (GWAS) peaks have not shown strong SNPs having large effect sizes. One hopes that such SNPs/ combination of SNPs having very small effect size on bipolar disorder could have a larger effect on cellular phenotype. 3. Experiments using genetic manipulations in cell models Many genetic variations of ‘probable’ impact on the BD phenotype have been reported in recent GWAS samples (e.g.: CACNA1C). The biological actions/effects of these variations are largely unknown. One interesting approach would be to use homogeneous samples of patients with such known mutations and examine the effect of gene editing. Advances in genome engineering techniques based on the CRISPR-associated RNA-guided endonuclease Cas9 has enabled targeted interrogation of specific gene functions (Hsu et al., 2014). DNA sequences within the human genome and their functional outputs can be easily edited. This will allow each cell line to be its own control, and permit a study of the effect of particular genotypes since rest of the genomic background is unchanged. 4. Pharmacogenomics based experiments.

22 Viswanath et al.

Cell models, such as ONE, are useful tools for the investigation of molecular predictors in pharmacogenomics (Severino et al., 2013). Also, they can be used for testing drug effects in vitro and for the identification of new drug targets (Morag et al., 2010). Although Lithium has been used in vitro in many of the reviewed reports, only few studies (Cruceanu et al., 2012; McCarthy et al., 2011b; McEachin et al., 2010; Shamir et al., 1998; Squassina et al., 2008; Squassina et al., 2013a; Sun et al., 2004; Tseng et al., 2008)have used cell lines (LCLs) to model clinical response to lithium in BD.The approach has been to use LCLs derived from BD patients with extremes of lithium response (responders versus non-responders) to maximize phenotypic differences. Similar approach has been recently attempted with induced neuron like cells reprogrammed from fibroblasts (Wang et al., 2014). ONE cells, with the potential of longitudinal monitoring, could intuitively prove more useful. Ideal approach would be to combine clinical drug response phenotype with in vitro application of lithium to maximize the probability of identifying its mechanisms of action. 5. Modeling specific aspects of BD One critical aspect missing in most of the reviewed studies is genotype-phenotype correlation. Except for a handful of studies which have examined suicidality in BD, other clinical aspects of BD have been rarely correlated with cell related parameters. Some specific clinical aspects (e.g.: predominant polarity of episodes, presence of psychotic symptoms/catatonia, course and outcome) and clinical endophenotypes (e.g. neuropsychological/imaging) could be used in an attempt to model subtypes of BD rather than the full spectrum of this heterogeneous disorder. Synthesis of leads emerging from different methods of enquiry (from cell biology to imaging and neuropsychology) will require the creation of model systems that approximate the clinical

23 Viswanath et al.

condition, albeit with varying degrees of fidelity. Patient derived neuronal cell lines may prove to be a critical component of this effort. Glial cell models Diffusion tensor imaging studies have consistently found white matter differences to be present in first onset BD patients (Bauer et al., 2015) and even in subjects at-risk for BD (Roybal et al., 2015). These changes also tend to progress during the course of illness (Nortje et al., 2013). Considering that glial cells play an important role in trophic support to neurons, early onset of glial abnormalities in BD may indicate a primary role for glial abnormalities in the pathogenesis of BD. Genetic studies have also shown that myelin related genes and pathways are important in BD (Yu et al., 2014b). It is currently possible to directly study oligodendrocytes/ astrocytes derived from reprogrammed IPSCs (Stacpoole et al., 2013). Hence, such an approach may deserve merit in BD. Conclusions To conclude, cellular modeling has proven useful in BD, and potential pathways, especially in cellular resilience related mechanisms have been identified. ONE cells and IPSC derived neurons promise to represent the next generation of cell models in BD. Further refined study designs (family-based,

pharmacogenomics)

and

combination

of

cellular

models

with

deep

sequencing/genetic manipulation will hopefully provide us with a deeper understanding of the biology of this intriguing and complex disorder.

Acknowledgements: We thank Prof. Sumantra Chattarji and Dr.Vijayalaxmi Nalavadi from the Centre for Brain Development and Repair, Institute of Stem Cell Biology and Regenerative

24 Viswanath et al.

Medicine, Bangalore, India, and Prof. Siddharthan Chandran from the Centre of Clinical Brain Sciences, University of Edinburgh, UK, for their expert comments and inputs on the manuscript.

Contributors: BV and SPJ performed the literature search with inputs from JT. BV and AS wrote the first draft of the manuscript. OM, MP and MDZ contributed significantly to the nonneuronal section of the manuscript. VV, GPP and SJ contributed significantly to the neuronal section of the manuscript. All authors approved the final version of the manuscript.

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Funding Sources: Research on cellular models of neuropsychiatric disorders at NIMHANS is funded by a Centre of Excellence grant of the Department of Biotechnology, Government of India. Dr. BV is funded by the INSPIRE faculty fellowship of the Department of Science and Technology, Government of India. Dr. AS is a post-doctoral research fellow funded with a grant from the Sardinia Regional Government (POR Sardegna FSE Operational Program of the Autonomous Region of Sardinia, European Social Fund 2007–2013 – Axis IV Human Resources, Objective l.3, Line of Activity l.3.1.). GPP was a visiting scientist, during his sabbatical leave, at the Laboratory of Pharmacogenomics, Department of Biomedical Sciences, University of Cagliari between 1-10-2014 and 31-3-2015.

Financial Disclosures: Nil

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Table 1: Non-neuronal cell models in bipolar disorder: Hypothesis-based studies Author and year (location of laboratory)

Sample Cells Methodology Significant results details (method of sample selection where specified) Calcium signaling (Also So et al. 2007, Iwamoto et al. 2004 & 2004a in the section ‘Endoplasmic reticulum’ and Xu et al. 2008 in the section ‘Mitochondria’ Emamghores 28BD1 , LCLs Ca2+ levels by Basal Ca2+ level higher in hi et al.1997 11BD2, ratiometricfluorimetry BD (Toronto) 14MDD 14 Nonmood psychiatric disorder, 44 Viswanath et al.

20C (Sequentia l sampling) Emamghores 22BD1, LCLs hi et al.2000 11C (Toronto) (Sequentia l sampling)

Corson et al. 72BD1, 2001 36BD2, (Toronto) 21MDD, 37C (Sequentia l sampling) Yoon et al. 40BD1, 2001(Toront 9BD2, o) 8MDD, 20C (Sequentia l sampling) Yoon et al. 24BD1, 2001a 12C (Toronto) (Sequentia l sampling) Kato et al. 12BD, 2003 11C (Tokyo) (Euthymic patients)

LCLs

LCLs

LCLs

LCLs

Andrepoulos 15BD1, LCLs et al. 2004 15C (Toronto) (Sequentia l sampling)

Wasserman 26BD1, et al. 2004 17C (Toronto) (Sequentia 45 Viswanath et al.

LCLs

Ca2+ levels by ratiometricfluorimetry cAMP protein levels with and without Isoproterenol (beta adrenergic stimulant) in LCLs cAMP protein levels with and without sodium fluoride in LCL membrane Ca2+ levels by ratiometricfluorimetry Genotyping - SNP C825T in exon 10 of G protein beta 3 subunit gene (GNB3) Ca2+ levels by ratiometricfluorimetry TRPC7 &ATP2A2 expression using RTPCR

Basal Ca2+ level higher in BD Reduced Isoproterenol stimulated cAMP formation in LCLs with high Ca level Elevated effector-dependent cAMP production in LCL membranes with high Ca levels.

Ca2+ levels by ratiometricfluorimetry IMPA2 expression using RT-PCR Ca2+ levels by ratiometricfluorimetry Mitochondrial DNA 5178/10398 genotyping Agonists – PAF, CCCP, TG TRPC1 and TRPC3 expression by RT-PCR TRPC1 and TRPC3 protein levels by immunolabeling Li treatment of LCLs – chronic (7days) Ca2+ levels by ratiometricfluorimetry Agonists – LPA, TG

High Ca2+ group (men only) had lower IMPA2 transcript level

No association of genotype with Ca2+ levels

High Ca2+ group had lower TRPC7 transcript level than others, negative with ATP2A2

TG induced Ca2+ response greater in BD Mitochondrial DNA genotype was related to CCCP induced Ca2+ response Li reduced TRPC1 protein levels in all LCLs except male controls

Chronic Li treatment reduced TG and LPA induced Ca2+ response in all LCLs

l sampling)

Xu et al. 124BD1, LCLs 2006 64BD2, (Toronto) 268C (Sequentia l sampling) Squassina et 15 Li-R LCLs al. 2008 BD, 15 Li(Sardinia) NR BD (Random selection of Li-Rs and matched NRs) Perova et al. 52BD1, LCLs 2008 30C (Toronto) (Sequentia l sampling) Perova et al. 24BD1, LCLs 2010 16C (Toronto) (Sequentia l sampling)

Uemura et al. 150BD1, LCLs 2011 65BD2, (Toronto) 30MDD, 70C (Sequentia l sampling)

MachadoVieira et al. 2011 (New Jersey)

18BD1 LCLs (Selected on the basis of Bcl-2 genotype)

46 Viswanath et al.

Li treatment of LCLs – acute (24hours), chronic (7days) Ca2+ levels by ratiometricfluorimetry Genotyping 7 SNPs in TRPM2 gene

Basal Ca2+ level higher in BD Ca2+ levels higher in BD1 with the TRPM2-intron 19 SNP T/T genotype compared to other genotypes LIM expression using No differences RT-PCR

Ca2+ levels by LPA induced Ca2+ response ratiometricfluorimetry greater in BD Agonists – LPA, TG Ca2+ levels by ratiometricfluorimetry Agonists – LPA, TG Li/valproate/lamotrigin e treatment of LCLs – acute (24hours), chronic (7days) Ca2+ levels by ratiometricfluorimetry Agonists – LPA, TG Bcl-2 rs956576 genotyping Bcl-2 expression using RT-PCR Bcl-2 protein levels by western blot Ca2+ levels by ratiometricfluorimetry Agonists –TG, Adenophostin A, BH3-I Bcl-2 rs956576 genotyping Bcl-2 expression using RT-PCR

Chronic valproate attenuated TG/LPA induced Ca2+ responses Chronic Li attenuated LPA induced Ca responses Lamotrigine – no effect Basal Ca2+ level higher in BD Basal and LPA induced Ca2+ levels associated with genotype in BD (GG>AG>AA) Gene expression and protein inversely correlated to Ca2+ level Basal Ca2+ levels associated with genotype (AA>AG>GG) Gene expression and protein inversely correlated to Ca2+ level Adenophostin led to further reduction of ER Ca2+ in AA

Bcl-2 protein levels by western blot Li treatment of LCLs – chronic (7days) Roeddig et 6BD1, 5C LCLs al.2012 (Sequentia (Toronto) l sampling)

G protein system Berrettini et 13BD and al. 1986 15UR (Bethesda) from 3 families, 13C (High density families) Berrettini et 14BD, 9C al. 1987 (High (Bethesda) density families) Niculescu et 6BD, 6C al. 2000 (San (Families Diego) with high LOD score at a specific locus in chromoso me 22) Alda et al. 23BD, 2001 23C (Montreal) (Patients 47 Viswanath et al.

BH3-I in GG caused similar changes as in AA Lithium increased Bcl-2 expression and reversed changes Rotenone treatment - Chronic rotenone reduced acute (24 hours) cell viability, TRPM2 and chronic (4days) TRPC3 protein levels and TRPM2- and TRPC3- functionality (Ca flux) mediated Ca2+fluxesin presence of their respective activators: H2O2 /1-oleoyl-2acetyl-sn-glycerol TRPM2&TRPC3expres sion by RT-PCR TRPM2 & TRPC3 protein levels by western blot Cell viability estimated using propidium iodide

Fibroblas ts

Radioimmunoassay for Negative when controlled for cAMP activity; cell cell density density measured using protein content

Fibroblas ts

Radioimmunoassay for Negative when controlled for cAMP activity; cell cell density density measured using protein content GRK3 protein level Higher in BD using western blot

LCLs

LCLs

Gsα – stimulatory G No difference protein levels

stabilized on lithium selected from a larger cohort) Karege et al. 10BD, LCLs 2004 10C (Geneva) (Euthymic patients)

Karege et al. 12BD, 2004a 12C (Geneva) (Euthymic patients)

LCLs

McCarthy et 11BD, 6C LCLs al. 2010 (Selected (San Diego) on the basis of ADRBK2 genotype)

Gaspar et al. 10BD, 2014 10C (Zurich) (Bipolar depression 48 Viswanath et al.

Fibroblas ts

Basal and cAMP activated PKA activity 3H–cAMP binding to PKA regulatory subunits (fluorimetry) BDNF expression using RT-PCR Basal and cAMP activated PKA activity 3H–cAMP binding to PKA regulatory subunits (fluorimetry) Catalytic PKA subunit and p-CREB levels using western blot BDNF expression using RT-PCR Two powerful cAMPanalogs, Rp-8Br-cAMPS (inhibitor) and Sp-8-Br-cAMPS (activator) used to manipulate PKA activity Chromatin immunoprecipitation (ChIP) of acetylated histone H3 at individual ADRBK2 regulatory alleles Allele expression imbalance among transcribed, synonymous SNPs in the ADRBK2 transcript with RT-PCR Lentiviral reporter system to profile transcriptional activation of major

PKA activity higher in BD 3H–cAMP binding lower in BD No difference in BDNF expression

PKA activity higher in BD 3H–cAMP binding lower in BD Catalytic PKA subunit and p-CREB levels greater in BD No difference in baseline BDNF expression, but increased expression with activator and vice versa, especially in BD

No difference

Magnitude of cAMP response element - binding protein (CREB) signaling higher in BD

)

signal transduction pathways Inositol signaling system (Also Yoon et al.2001 & 2001a in the section ‘Calcium signaling’) Banks et al. 6BD, 6C LCLs Incubated with 3H- Reduced in BD by 50-60% 1990 (St. (High Inositol Andrews) density Incorporation into families) phosphoinositides measured using liquid chromatography Shamir et al. 77BD (56 LCLs Inositol Li-R BD < Li-NR/C 1998 (Beer Li-R, 21 monophosphatase Lithium treatment increased Sheva) Li-NR), enzyme activity using mRNA levels 29C ELISA (Selection Cells incubated with on basis of lithium for 5 days – li response) mRNA level measured Shaltiel et 12BD, LCLs Inositol Enzyme activity in BD lower al. 2001 51MDD, monophosphatase than other groups (Beer 28SZ, 39C enzyme activity using Li-R BD > Li-NR/C Sheva) (Selection ELISA on basis of Cells incubated with Li lithium for 5 days response) Belmaker et 37BD, 40C LCLs Inositol content Inositol content less in BD al. 2002 (Selection measured by gas LCLs (Beer on basis of chromatography Sheva) Li Incubated with 3Hresponse) Inositol Incorporation into phosphoinositides measured using liquid chromatography Houlihan et 4BD, LCLs PI4K2B expression Negative al. 2009 4MDD, using RT-PCR and (Edinburgh) 4UR, protein levels by 4Founders Western Blot (All from one family) Endoplasmic reticulum (ER) Iwamoto et 11BD, LCLs HSPF1&LIMexpression HSPF1 up-regulation &LIM al. 2004 11MDD, 13 using RT-PCR down-regulation in BD (Tokyo) SZ, 15 controls (Euthymic patients) Iwamoto et Japanese: LCLs HSPF1&LIMexpression HSPF1 up-regulation in BD 49 Viswanath et al.

al. 2004a 26BD, 33C (Tokyo) Caucasian: 10BD1, 14BD2 12SZ, 13C (Euthymic patients) So et al. 20BD, 10C 2007 (Sequential (Toronto) sampling)

LCLs

Kakiuchi et 23BD, 19C al. 2007 (Euthymic (Tokyo) patients)

LCLs

Kakiuchi et al. 2007a (Tokyo) Hayashi et al. 2009 (Tokyo)

LCLs

18BD, 24C (Euthymic patients) 59BD, 59C (Euthymic patients)

Mitochondria Washizuka 13BD1, et al. 2003 8BD2, 11C (Tokyo) (Euthymic patients) Washizuka 13BD1, et al. 2005 8BD2, 11C 50 Viswanath et al.

LCLs

using RT-PCR

No difference LIM expression BD and controls

Expression of XBP1, GRP78, CHOP using RT-PCR Induction of ER stress using TG/ Tunicamycin in LCLs Basal Ca level in LCLs HSP90B1 expression using RT-PCR Southern blot for insertion/deletion polymorphisms; sequencing for upstream polymorphisms ATF4 and ATF5 expression using RTPCR Expression of XBP1, GRP78 (HSPA5), GRP94(HSP90B1), CHOP (DDIT3), and calreticulin using RT-PCR Genotyping of XBP1 (rs2269577), GRP94 (rs391957, rs17840761 and rs16927997)and GRP78 (HSPA5) Induction of ER stress using TG/Tunicamycin in LCLs

XBP1 and CHOP: No differences I baseline expression; induction with TG/ Tunicamycin attenuated

C allele of rs14034977 associated with lower expression; no difference BD and controls

Negative study

XBP1 and GRP94 levels higher in BD; Induction with TG or Tunicamycin is attenuated C haplotype of GRP94 normalizes the induction Interaction between XBP1 rs2269577and induction with Tunicamycin

LCLs

NDUFV2 expression Expression reduced in BD using RT-PCR

LCLs

Expression NDUFA1,

of Nominally NDUFA6, expression

of

reduced NDUFA1,

(Tokyo)

(Euthymic patients)

Xu et al. 178BD, 2008 120C (Toronto) (Sequential sampling) Washizuka Group 1 et al. 2009 (Japanese) (Tokyo) 25BD1, 10BD2, 33C Group 2 (Caucasian) 14BD1, 12BD2, 13SZ, 10C (Euthymic patients) Cataldo et 8BD, 8C al. 2010 (Fibroblasts (Boston) ) 6BD, 6C (LCLs) (High density families) Sodium-Potassium pump Banks et al. 11BD, 11C 1989 (St. (High Andrews) density families) Cherry et al. 9BD, 7UR, 1994 8C (Texas) (All from Old Order Amish pedigrees) Buss et al. 14BD1, 1986 9UR, 8C (Kentucky) (All from Old Order Amish pedigrees) Li et al. 15BD, 15C 51 Viswanath et al.

LCLs

LCLs

NDUFS2, NDUFS7, NDUFA6, NDUFS7, NDUFV6, COX5A, NDFUV6 and COX6C COX6C, COX7C, NRF1 and NRF2 NDUFV2 expression Negative using RT-PCR Basal Ca level in LCLs NDUFV2 expression Expression reduced in BD using RT-PCR from group1 No difference in group 2

LCLs & Electron microscopy to Mitochondria in BD cells Fibroblas examine mitochondrial concentrated in perinuclear ts morphology and region than in distal regions distribution More ring and cusp shaped Effects of lithium mitochondria in BD treatment for 5 days No effect of lithium

LCLs

3

H-ouabain binding (Na pump site number) 86 Rb flux (Na pump activity) 3 H-ouabain binding (Na pump site number) 86 Rb flux (Na pump activity), cell volume

Greater Na number in BD

LCLs

Transmembrane potential

No difference

LCLs

Ethacrynic

LCLs

pump

site

Lower 86Rb flux per pump site, no other differences

acid Ethacrynic

acid

induced

2004 (Kentucky)

(All from Old Order Amish pedigrees)

application in-vitro for upregulation of Na pump 72 hours number, protein, mRNA and 3 H-ouabain binding (Na activity leading to normal Na pump site number) levels in control LCLs. 86 Rb flux (Na pump BD LCLs – no upregulation activity), Na pump leading to increased Na protein by western blot, Na pump mRNA by RT-PCR, intracellular Na/K measurements Ethacrynic acid application in-vitro for 72 hours Huff et al. 16BD, LCLs Ethacrynic acid Ethacrynic acid induced 2010 9siblings, application in-vitro for upregulation of Na pump (Kentucky) 11C 72 hours protein in control LCLs, but 3 (All from H-ouabain binding (Na not in BD LCLs Old Order pump site number) 86 Amish Rb flux (Na pump pedigrees) activity), Na pump protein by western blot, intracellular Na/K measurements Ethacrynic acid application in-vitro for 72 hours Neuroplasticity related genes (Also Karege et al. 2004 and 2004a in the section ‘G protein system’) Tseng et al. 12 Li-R LCLs BDNF protein using Reduced protein levels in 2008 BD, 14UR, ELISA BD (Montreal) 13C lithium exposure for 7 Li reduced levels in all (Selection days in-vitro groups; differences still on basis of significant Li response) Gao et al. 30BD LCLs BDNF rs12273363 No association between 2012 (Selected genotyping and genotype and expression (Kentucky) on the basis expression using RT- levels of BDNF PCR genotype) Serum deprivation and monesin treatment for induction of apoptosis and BDNF production Cruceanu et 11 Li-R LCLs Expression of Broader distribution of al. 2012 BD, 12 LiSYN2A&SYN2B using expression in Li-R BD LCLs (Montreal) NR BD, RT-PCR 52 Viswanath et al.

13C (Selection on basis of Li response) Circadian system Yang et al. Group1 - Fibroblas 2009 12BD, ts (Philadelphi 5siblings, a) 12C Group 2 6BD/23C (Older Order Amish cohort)

Lithium exposure for 7 days in-vitro

McCarthy et 13 Li-R LCLs al. 2011 BD, 18 Li(San Diego) NR BD (Selection on basis of Li response) Etain et al. 41BD, 18C LCLs 2012 (Euthymic (Creteil) patients)

Expression of circadian gene NR1D1 and GSK3B with & without Li exposure NR1D1rs2071427and GSK3B rs6438552 genotyping ASMT gene expression and enzyme levels ASMT SNP rs4446909 genotyping

Bamne et al. 13BD1, 2013 12C (Pittsburgh)

Fibroblas ts

McCarthy et 19BD1,

Fibroblas

53 Viswanath et al.

Expression(RT-PCR) of core clock genes at various time intervals till 72h in group1 RT-PCR for various kinases and Western blot for GSK3b and phosphorylated-GSK3b in group 1+2

12 hour amplitude of expression of BMAL1 lower in BD Overall expression of DBP lower in BD Phosphorylated-GSK3b level lower in BD

Li-treated samples with the NR1D1A/A genotype expressed lower ratio of full length/ truncated NR1D1 transcript compared with untreated samples G/G genotype had lower mRNA expression and enzyme levels Enzyme levels also lower between BD and controls Cells infected ex vivo No differences with a lentiviral reporter incorporating the promoter sequences for Bmal1, a circadian gene to drive expression of the fireflyluciferase gene Following synchronization, bioluminescence used to estimate period length. Phase response curves generated following forskolin challenge Cells transduced with Baseline period longer in BD

al. 2013 19C (San Diego) (Inpatients)

ts

Per2::luc, a rhythmically expressed, bioluminescent circadian clock reporter gene Rhythms measured for 5 consecutive days with and without Li Genotyping of clock gene variants

Neurotransmitter and Neuroendocrine systems Breakefiled 18 BD, 18C Fibroblas Monoamine oxidase-A et al. 1980 (Euthymic ts enzyme activity (Connecticu patients) t) Nadi et al. 12BD1, Fibroblas Muscarinic binding 1984 5BD2, ts using radioligand (3H) (Bethesda) 1MDD, 17 assay relatives with affective illness, 5 UR, 12C (High density families) 125 Wright et al. 6BD, 7UR, LCLs Binding of I1984 11C iodohydroxybenzylpind (Edinburgh) (High olol to β-Adrenoceptors density families) Lin et al. 3BD, Fibroblas Muscarinic binding 1986 1SZAFF, ts using radioligand (3H) (Rochester) 1MDD, 2C assay Berrettini et 17BD, 14C al. 1987 (High (Bethesda) density families) Kay et al. 12BD, 12C 1993 (Bipolar (Oxford) depression) 54 Viswanath et al.

Li 1mM increased amplitude in controls only PER3 and RORA genotype predicted period lengthening by Li GSK3b genotype predicted rhythm effects of Li Cells from past suicide attempters more likely to show period lengthening with Li Li enhanced the resynchronization of damped rhythms Negative

Binding increased in all affected individuals than in unaffected

Reduced in BD

Negative

LCLs

125 Binding of I- No difference iodohydroxybenzylpind olol to β-Adrenoceptors

LCLs

125 Binding of I- No difference in baseline iodohydroxybenzylpind Downregulation with olol to β-Adrenoceptors Isoprenaline reduced in BD

Kay et al. 12BD,12C 1994 (Bipolar (Oxford) depression)

LCLs

Henning et al. 2005 (Duesseldor f)

4BD1, LCLs 2BD2, 8MDD, 13C (High density families) Pinto et al. 21 BD LCLs 2011 (13suicide (Toronto) attempters and 8 BD nonattempters) MicroRNA Chen et al. 10BD1, LCLs 2009 10siblings (Michigan) (Ten families having patient and sibing) Amino acids and polyamines Jones et al. 18BD, LCLs 1997 18SZ (Cardiff) (Selected of the basis of CAG/CTG repeats) Zander et al. 119BD, LCLs 1998 88C (Paris) (Consecutiv e admissions)

Persson

et 10BD1,

55 Viswanath et al.

Fibroblas

cAMP protein levels Incubation with agonist Isoprenaline 125 Binding of Iiodohydroxybenzylpind olol to β-Adrenoceptors Incubation with agonist Isoprenaline Incubation with lithium for 7days before assay Glucocorticoid receptor quantification by radioligand binding assay

No difference in baseline Downregulation with Isoprenaline reduced in BD, but normalized with lithium treatment in-vitro

Number of receptors low in MDD Downregulation with hydrocortisone lower in affective disorders as a group

Allele specific No difference expression of the SNP expression G2651T of the 5HTT gene

Micro RNA expression

in

gene

No difference BD and siblings Upregulation of miR-34a, miR-152, and miR-155, and miR-221 with Lithium

Western blot to detect Negative study expanded polyglutamine sequences in proteome

Repeat expansion No differences detection analysis in genomic DNA Western blot to detect expanded polyglutamine sequences in proteome Amino acid transport Maximal transport capacity

al. 2009 10C (Stockholm)

ts

Squassina et 9 BD LCLs al. 2013 suicide (Sardinia) completers, 19BD HR, 17BD LR, 21C (Selected on basis of suicidal risk) Johansson et 10BD1, Fibroblas al. 2013 11SZ, 12C ts (Lublin)

Others Breslow et 10 Li-R, al. 1985 10C (New York) (Sequential sampling) Mahadik et 6 BD, 12 al. 1996 SZ, 10C (Georgia) (Consecutiv e admissions) Kato et al. 31BD1, 2011 58C (Tokyo) (Euthymic patients)

56 Viswanath et al.

was measured using the for Tyrosine reduced in BD cluster tray method than controls 14 with C labeled 3 Tyrosine and H labeled Tryptophan Expression of SAT1 Lithium increased SAT1 using RT-PCR expression in the HR, LR Lithium exposure for 7 and control groups, but had days in-vitro no effect in suicide completers

Production of kynurenic acid, 3hydroxykynurenine (3HK) Expression of genes coding for kynurenine pathway enzymes using RT-PCR Basal values and following treatment with interferon-g, tumor necrosis factor-a, interleukin-1b/6

Production of kynurenic acid, 3-hydroxykynurenine higher in BD and SZ Treatment with cytokines further increased the production of 3hydroxykynurenine in BD and SZ

Fibroblas ts

Lithium ratio

Negative

Fibroblas ts

Phospholipid fatty acid No differences BD and composition using gas controls; Docosahexanoic chromatography acid levels lower in SZ than BD/C

LCLs

Gene expression using ANK3upregulated RT-PCR of 17 genes POLG1,RASGRP1downregul related to BD ated

BD- Bipolar disorder, MDD-Major depressive disorder, C-Control, UR-Unaffected relative, SZSchizophrenia, SZAFF-Schizoaffective disorder, LCL-Lymphoblastoid cell line, Ca-Calcium, cAMP-cyclic adenosine monophosphate, RT-PCR-Real time quantitative polymerase chain reaction, PAF-Platelet activating factor, CCCP-Carbonyl cyanide m-chlorophenylhydrazone, TG-Thapsigargin, Li-Lithium, LPA-Lysophosphatidic acid, Li-R-Lithium responder, Li-NRLithium non-responder, HR- High risk for suicide, LR – Low risk for suicide, ER-Endoplasmic reticulum, TRPM2-Transient receptor potential melastatin2, TRPC3-Transient receptor potential canonical, GRK3-G protein receptor kinase 3, PKA-Protein kinase A, CREB-cAMP response element binding protein, BDNF-Brain derived neurotrophic factor, ELISA-Enzyme linked immunosorbent assay, miR-MicroRNA, GSK3b-Glycogen synthase kinase 3 beta.

Table 2: Hypothesis-free studies using lymphoblastoid cell lines in bipolar disorder Author and year

57 Viswanath et al.

Sample

Methods

Significant findings

Yang et al. 2009 Old Order Amish pedigree, 16BD, 3MDD, 27UR

Effects of CNV on expression

11 CNVs associated with expression changes and enriched in affected individuals

Kakiuchi et al. 2003

2 pair MZ

GWGE

Impaired feedback regulation of ER stress pathway genes

Matigian et al. 2007

2 pair MZ

GWGE

Upregulation of WNT signaling pathway and apoptosis

Kuratomi et al. 2008

2 pair MZ

Genomewide DNA methylation

Aberrant DNA methylation upstream of Spermine synthase &PeptidylprolylIsomerase E-like genes

Sugawara et al. 2011

2 pair MZ

Genomewide wide DNA methylation

SLC6A4 hypermethylation

Kazuno et al. 2013

2 pair MZ

Proteomics

Upregulation of proteins involved in glycolysis and cell death. Phosphoglycerate mutase 1 (glycolysis related protein) was identified as a candidate biomarker.

Sun et al. 2004

12 Li-R and 8C

GWGE

Downregulation of alpha I βadrenoceptor (αIB-AR), acetylcholine receptor protein alpha chain precursor (ACHR) and cAMP dependent 3’, 5’cyclic phosphodiesterase 4D (PDE4D) by in-vitro lithium

McEachin et al. 2010

6 Li-R and 4 drug-naive

GWGE

CRIP1 (cysteine-rich protein 1), FOS (FBJ murine osteosarcoma viral oncogene homolog), NR4A2 (nuclear receptor subfamily 4, group A, member 2), STC2 (stanniocalcin 2) and RGC32 (regulator of cell cycle) identified as potential candidate biomarkers.

Squassina et al. 2013

10 Li-R and 10 Li-NR

GWGE

Insulin-like growth factor-1 (IGF-1) overexpression in Li-Rs.

58 Viswanath et al.

BD- Bipolar disorder, MDD-Major depressive disorder, C-Control, UR-Unaffected relative, MZ –Monozygotic twins discordant for BD, CNV – Copy number variation, GWGE – Genomewide gene expression, Li-R – Lithium responder, Li-NR – Lithium non-responder.

Table 3: Attempts to replicate findings in cell models of bipolar disorder Findings Higher cytoplasmic calcium in BD LCLs Lithium attenuates LPA-induced calcium response in BD LCLs Bcl-2 gene is association with calcium signaling abnormalities in BD LCLs TG-induced calcium response is higher in BD LCLs Lithium attenuates TG-induced calcium response in BD LCLs

Positive studies Emamghoreishi et al., 1997; Emamghoreishi et al., 2000; Uemura et al., 2011; Xu et al., 2006 Perova et al., 2010; Wasserman et al., 2004 Machado-Vieira et al., 2011; Uemura et al., 2011 Kato et al., 2003 Wasserman et al., 2004

Calcium signaling related gene LIM is downregulated in BD LCLs

Iwamoto et al., 2004b

ER stress genes are upregulated in BD LCLs

Hayashi et al., 2009;

59 Viswanath et al.

Negative studies

Perova et al., 2008 Perova et al., 2010 Iwamoto et al., 2004a: Squassina et al., 2008 So et al., 2007

Kakiuchi et al., 2003; Iwamoto et al., 2004a; Iwamoto et al., 2004b Transcriptional activation of ER stress genes in the presence of stressor (TG, Tunicamycin) is attenuated in BD LCLs Mitochondrial complex I gene NDUFV2 downregulated in BD LCLs

Hayashi et al., 2009; So et al., 2007 Washizuka et al., 2009; Washizuka et al., 2003

Na pump site number greater in BD LCLs

Banks et al., 1989

Na pump site activity reduced in BD LCLs Ionic stressor (Ethacrynic acid) upregulates Na pump activity in LCLs from normal volunteers, but not in BD LCLs

Cherry and Swann., 1994

Binding of 125I-iodohydroxybenzylpindolol to β-Adrenoceptors is reduced in BD LCLs

Xu et al., 2008 Cherry and Swann., 1994 Banks et al., 1989

Huff et al., 2010; Li and ElMallakh et al., 2004

Wright et al., 1984

Berrettini et al., 1987b; Kay et al., 1993; Kay et al., 1994; Xu et al., 2008

BD – Bipolar disorder, LPA-Lysophosphatidic acid, LCL-Lymphoblastoid cell lines, TGThapsigargin, ER-Endoplasmic reticulum, Na-Sodium

60 Viswanath et al.

Literature search (April-May 2013) Pubmed (525) PsychINFO (68) SCOPUS (116) Total 709 Limit : English only Abstract reading and removal of duplicates n=88

Cross-references n =12 Total n=100 Full length articles assessed for eligibility , n=100 Remaining n=76 Pubmed search in January 2015 (n=7) + references suggested by reviewers (n=2) Total n = 85

LCLs n=65

Fibroblasts n=14

ONE n=4

Records excluded n=621

Records excluded (n = 24) Cells from normals only (n=10) Cells from schizophrenia/dep ression (n= 5) Animal cells only (n=3) Leukocytes (n=4) Post-mortem brain only(n=2)

IPSC n=2

LCL – Lymphoblastoid cell line, ONE-Olfactory Neuronal Epithelium, IPSC-Induced Pluripotent Stem Cell Figure 1 – Flowchart of literature search

61 Viswanath et al.