Exp Neurobiol 2016; 25(6): 296-306
Published online December 31, 2016
© The Korean Society for Brain and Neural Sciences
Yoonhee Kim1†, Yinhua Zhang1†, Kaifang Pang2,3†, Hyojin Kang4, Heejoo Park5, Yeunkum Lee1, Bokyoung Lee1, Heon-Jeong Lee6, Won-Ki Kim1, Dongho Geum5 and Kihoon Han1*
1Department of Neuroscience and Division of Brain Korea 21 Biomedical Science, Korea University College of Medicine, Seoul 02841, Korea, 2Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, 3Department of Pediatrics, Computational and Integrative Biomedical Research Center, Baylor College of Medicine, Houston 77030, USA, 4HPC-enabled Convergence Technology Research Division, Korea Institute of Science and Technology Information, Daejeon 34141, 5Department of Biomedical Sciences, Korea University College of Medicine, 6Department of Psychiatry, Korea University College of Medicine, Seoul 02841, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-2286-1390, FAX: 82-2-953-6095
†These authors contributed equally to this work.
Bipolar disorder (BD), characterized by recurrent mood swings between depression and mania, is a highly heritable and devastating mental illness with poorly defined pathophysiology. Recent genome-wide molecular genetic studies have identified several protein-coding genes and microRNAs (miRNAs) significantly associated with BD. Notably, some of the proteins expressed from BD-associated genes function in neuronal synapses, suggesting that abnormalities in synaptic function could be one of the key pathogenic mechanisms of BD. In contrast, however, the role of BD-associated miRNAs in disease pathogenesis remains largely unknown, mainly because of a lack of understanding about their target mRNAs and pathways in neurons. To address this problem, in this study, we focused on a recently identified BD-associated but uncharacterized miRNA, miR-1908-5p. We identified and validated its novel target genes including
Keywords: Bipolar disorder, microRNA, miR-1908-5p, glutamatergic synapse
Bipolar disorder (BD) is a highly heritable, chronic, and devastating mental illness characterized by recurrent mood swings between depression and mania with intervening euthymic states. With a lifetime prevalence of 1~2%, BD is recognized as the sixth leading cause of disability worldwide and also incurs huge social and economic costs . Although the underlying pathogenic mechanisms are poorly defined, recent genomewide molecular genetic studies have identified many single-nucleotide polymorphisms (SNPs) associated with BD in multiple chromosomal regions [2,3,4]. These loci include several protein-coding genes (e.g.
MicroRNAs (miRNAs) are 21~25-nucleotide small non-coding RNAs that regulate the expression of target mRNAs by directly interacting with the 'seed' complementary sequences in 3' untranslated regions (3'UTRs) . miRNA binding downregulates the expression of target mRNAs either by decreasing their stability or by inhibiting translation. Notably, the expression levels of many miRNAs are altered in postmortem brains and blood samples of BD patients, suggesting a potential role for miRNAs in BD pathophysiology [12,13,14]. In many cases, however, it is unclear whether such changes in miRNA expression have any causative role in BD or simply reflect disease progression or patient medication. Independently, molecular genetic studies have also identified several miRNAs associated with BD, such as miR-499, miR-708, miR-1908, and miR-2113 [4,15]. Although these miRNAs could be potentially involved in BD pathogenesis, the key target mRNAs that could mediate the pathogenic process remain largely unknown. Importantly, it has been shown that a single miRNA can regulate a specific biological function by modulating the expression of a group of proteins interacting with each other or participating in the same pathway . Therefore, we reasoned that identifying the target genes of BD-associated miRNAs and understanding their enriched functions could provide important insights into the key pathogenic mechanisms of BD.
In this study, we tested this hypothesis by focusing on a recently identified BD-associated, primate-specific but uncharacterized miRNA, miR-1908-5p. We identified and validated its novel target genes including
The 3'UTR regions of human
The control and bipolar patient iPS cell lines were generated by transfection of integration-free episomal expression vectors containing p53shRNA, Oct3/4, Sox2, Klf4, L-Myc and Lin28 as described in Okita et al. . The iPSC lines were maintained in mTeSR1 medium (Stemcell Technologies). For differentiation of iPSC to NPC, we followed Cho et al. with minor modifications . In brief, iPSC colonies were detached with dispase and cultured with STEMdiff Neural Induction Medium (Stemcell Technologies) in a bacterial dish for 5 days to form EBs. EBs then were plated onto matrigel-coated dishes in STEMdiff Neural Induction Medium for 7 days to form neural rosettes. The neural rosettes were mechanically isolated and cultured in DMEM/F12 plus N2 and bFGF, in order to form Spherical Neural Masses (SNMs). For differentiation of SNMs to NPCs, SNMs were chopped using a stainless steel blade (Dorco) and plated onto matrigel-coated dishes in DMEM/F12 plus N2 and B27. NPCs were chronically exposed to 1 mM lithium chloride (Sigma-Aldrich) or valproic acid sodium salt (Sigma-Aldrich) for a week.
Total RNA was extracted from human NPCs using a miRNeasy minikit (Qiagen) according to the manufacturer's instruction. From 50 ng of total RNA, cDNAs for
The target genes of miRNAs were predicted by using the TargetScan database (Release 7.0, http://www.targetscan.org) . The predicted target genes were sorted by context++ scores of the binding sites, and low scored target genes were discarded to remove less likely target genes. To further restrict the target genes, low-expressed genes whose expression levels were less than a 50th quantile of the average expression in mouse brain were also discarded. The expression values of mouse brain were obtained from 84 GDS datasets downloaded from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO). The gene ontology (GO) analysis for the remaining target genes was carried out using DAVID software (v6.7, https://david.ncifcrf.gov) . GO terms with an adjusted p-value (Benjamini) less than 0.05 were considered significant. Barplots were generated using the R (v.3.2.3) package ggplot2 (v.2.0.0).
The miRNA sequencing data of the developing human brain were downloaded from the BrainSpan database (http://www.brainspan.org). This dataset contains 216 samples spatially covering 16 brain regions and temporally spanning developmental periods from 4 months to 23 years of age. Reads were normalized to reads per million mapped reads (RPM) using the formula:
where C is the number of reads mapped to one miRNA, N is the total number of mapped reads in the sample, and L is the length of the miRNA. The gene expression data of the human brain were downloaded from the NCBI GEO (GEO accession number GSE25219) . This dataset contains 1,340 samples spatially covering multiple brain regions and temporally spanning periods from 4 post-conceptional weeks to 82 years of age. The expression levels of genes were assayed using the Affymetrix GeneChip Human Exon 1.0 ST Array platform, and represented as normalized log2-transformed signal intensity values. For brain regions with both left and right hemispheres of the same donor profiled, gene expression values were averaged. The samples in the miRNA expression data and those in the gene expression data were matched using the sample mapping table downloaded from the BrainSpan database, and 213 matched samples were used for analyses. To explore the regional expression pattern of miR-1908-5p and its target genes, both the miRNA and gene expression datasets were divided into 16 brain regions, with the samples from the same brain region being grouped together. To investigate correlations between the expression level of miR-1908-5p and those of its target genes, Spearman's correlation coefficient was calculated among the matched samples for each brain region.
miR-1908 is an intronic miRNA of the fatty acid desaturase 1 (
To solve these problems and to better understand the genes and biological pathways regulated by miR-1908-3p and miR-1908-5p, we decided to use the newest version of TargetScan (Release 7.0) . We found that TargetScan (Release 7.0) predicted a total of 481 and 2,500 target genes of miR-1908-3p and miR-1908-5p, respectively. In TargetScan (Release 7.0), targets are ranked based on the cumulative weighted context++ score, which combines the contribution of 14 features irrespective of binding site conservation . To focus on the high-ranked and more likely targets, we set thresholds for context++ scores and narrowed down the gene list to 303 and 358 genes for miR-1908-3p and miR-1908-5p, respectively (Additional file 1: Fig. S3). We further restricted the targets by selecting brain-expressed genes (184 and 225 genes, respectively) and performed Gene Ontology (GO) analysis (Fig. 1a and b). After applying adjusted p-value (Benjamini, <0.05), we found some significant terms, including 'regulation of cell development' and 'regulation of axonogenesis' in the Biological Process category for miR-1908-3p targets (Fig. 1a and Additional file 1: Fig. S4a). For miR-1908-5p, 'synapse' and 'synaptic vesicle' related terms were significantly enriched in the Cellular Component category (Fig. 1b and Additional file 1: Fig. S4b).
For the validation of putative miR-1908 targets, we decided to focus on the 'synapse' related targets of miR-1908-5p, which was the most significant term in our entire GO analysis (Additional file 1: Fig. S4). Moreover, both human genetic and animal model studies have proposed that abnormalities in neuronal synapses could be one of the major pathogenic mechanisms of BD [8,23,24]. We searched the literature and found that most of the putative synaptic targets of miR-1908-5p are located in excitatory glutamatergic synapses, either pre- or post-synaptic side (Fig. 1c). We selected the top 6 synaptic genes based on the target rank from TargetScan (Release 7.0) (Fig. 1c) and cloned their 3'UTRs to generate luciferase constructs. Each 3'UTR of the 6 genes had at least two putative miR-1908-5p binding sites (Fig. 1d). In HEK293T cells, miR-1908-5p overexpression significantly decreased the luciferase activities of constructs with
Among those synaptic targets of miR-1908-5p,
The human and primate-specific interactions of miR-1908-5p and its synaptic target genes prompted us to investigate their expression profiles and correlations in the human brain. We first investigated the regional expression profiles of
Next, we investigated correlations between the expression level of miR-1908-5p and those of its target genes. We reasoned that if miR-1908-5p functions as an important regulator of
Lithium and valproate are the two most common medications for BD . Previously, it was shown that treatment with lithium or valproate could change the expression levels of several miRNAs in the rat hippocampus and human lymphoblastoid cell lines [33,34]. Therefore, we decided to investigate whether miR-1908-5p expression could also be affected by these drugs in human neural progenitor cells (NPCs). First, we established two human NPC lines derived from dermal fibroblasts of either a control or a BD subject. We then treated the cultured NPCs with vehicle or 1 mM lithium or valproate for a week (Fig. 3a). During this period, no overt difference in growth and morphology of the NPCs was observed among the different treatment conditions (data not shown). After the chronic drug treatment, total RNAs were purified and processed for quantitative real-time reverse transcription PCR (qRT-PCR) to measure the levels of mature miR-1908-5p. We found that valproate, but not lithium, increased miR-1908-5p expression by about 70% in control NPCs (Fig. 3b). In contrast, valproate, but not lithium, decreased miR-1908-5p expression by about 60% in BD NPCs (Fig. 3b). Together, we found that miR-1908-5p expression might be possibly affected in the opposite direction in control and BD NPCs after chronic treatment of valproate. Further experiments with more control and BD NPC lines are necessary to confirm this intriguing but preliminary result.
BD is a highly heritable (at least 80%) and polygenic disease, meaning that there are many risk alleles with small effects [6,35]. Therefore, understanding the common functional pathways of BD-associated genes, rather than focusing on each, might provide better insight into the key pathophysiology of BD. Indeed, abnormalities in calcium signaling have been considered as a potential pathogenic mechanism for BD based on the identification of both common and rare variants in genes encoding calcium channels [6,36]. In a similar manner, 'synaptic pathology' has been proposed to be involved in the pathogenesis of BD. For example, BD-associated
Notably, none of the synaptic target genes of miR-1908-5p identified in this study were listed in the original study by Forstner et al.  where they used the previous version of TargetScan (Release 6.2) that considered the conservation of miRNA binding sites in 3'UTRs. Their approach could have missed meaningful targets of miR-1908-5p, especially considering that miR-1908-5p expression itself is limited to humans and other primates. Indeed, we found that the validated miR-1908-5p binding sites in the
Among the miR-1908-5p synaptic targets,
As a preliminary study, we compared the expression levels of miR-1908-5p in human NPCs derived from dermal fibroblasts of control and BD subjects under vehicle- and drug-treated conditions. Interestingly, we found that chronic treatment with valproate, but not lithium, increased the miR-1908-5p expression in control NPCs. In contrast to the control NPCs, valproate treatment decreased miR-1908-5p expression in BD NPCs. Although it is known that lithium and valproate exert their therapeutic efficacy by targeting both common and specific biological pathways [42,43,44], the detailed mechanisms that explain how miR-1908-5p expression was selectively affected by valproate, and how NPCs from control and BD subjects showed the opposite changes are not clear at this moment. Recently, Kuang et al. showed that miR-1908 expression is not correlated with its host gene,
In conclusion, we focused on a recently identified BD-associated miRNA, miR-1908-5p, and identified and validated its novel target genes functioning in neuronal glutamatergic synapses. Further studies, such as functional experiments using human induced pluripotent stem cell (iPSC)-derived neurons, will help us better understand the role of miR-1908-5p in synaptic function and its potential implications for BD. We also propose that similar approaches could be applied to other BD-associated miRNAs, such as miR-499, miR-708, and miR-2113 [4,15] since none of their target mRNAs has been directly validated yet. When combined, these studies will provide more insights into the molecular basis of BD pathophysiology and potentially into better diagnostic and therapeutic approaches for this devastating mental illness.
Conservation of the miR-1908-3p and miR-1908-5p sequences among 100 aminal genomes. The red and blue boxes indicate seed and full sequences of the mature miRNAs, respectively.en-25-296-s001.pdf
Conservation of the miR-34a-3p and miR-34a-5p sequences among 100 aminal genomes. The red and blue boxes indicate seed and full sequences of the mature miRNAs, respectively.en-25-296-s002.pdf
The thresholds of context++ scores for miR-1908-3p and miR-1908-5p putative target genes predicted by TargetScan. The thresholds of −0.2 and −0.5 were selected for miR-1908-3p and miR-1908-5p to narrow down the target gene list to 303 and 358, respectively.en-25-296-s003.pdf
GO analysis of miR-1908-3p and miR-1908-5p target genes. There was no significant term in the Molecular Function and Cellular Component categories from miR-1908-3p targets (a). There was no significant term in the Molecular Function and Biological Process categories from miR-1908-5p targets (b).en-25-296-s004.pdf
Conservation of the first (770-776) and second (803-809) miR-1908-5p binding sites in the
Human brain expression of
Summary of statistical analyses for the experiments.en-25-296-s007.pdf