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Downloaded Micro- Ing in MS Ding et al. J Transl Med (2020) 18:109 https://doi.org/10.1186/s12967-020-02276-1 Journal of Translational Medicine RESEARCH Open Access Integrative systems and functional analyses reveal a role of dopaminergic signaling in myelin pathogenesis Sujun Ding1,2†, Yun Gu3†, Yunyun Cai4†, Meijuan Cai5, Tuo Yang6, Shuangxi Bao3, Weixing Shen7, Xuejun Ni2*, Gang Chen1,3,8* and Lingyan Xing3* Abstract Background: Myelin sheaths surrounding axons are critical for electrical signal transmission in the central nervous system (CNS). Diseases with myelin defects such as multiple sclerosis (MS) are devastating neurological conditions for which few efective treatments are available. Dysfunction of the dopaminergic system has been observed in multiple neurological disorders. Its role in myelin pathogenesis, however, is unclear. Methods: This work used a combination of literature curation, bioinformatics, pharmacological and genetic manipu- lation, as well as confocal imaging techniques. Literature search was used to establish a complete set of genes which is associated with MS in humans. Bioinformatics analyses include pathway enrichment and crosstalk analyses with human genetic association studies as well as gene set enrichment and causal relationship analyses with transcrip- tome data. Pharmacological and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9) genetic manipulation were applied to inhibit the dopaminergic signaling in zebrafsh. Imaging techniques were used to visualize myelin formation in vivo. Results: Systematic analysis of human genetic association studies revealed that the dopaminergic synapse signaling pathway is enriched in candidate gene sets. Transcriptome analysis confrmed that expression of multiple dopaminer- gic gene sets was signifcantly altered in patients with MS. Pathway crosstalk analysis and gene set causal relationship analysis reveal that the dopaminergic synapse signaling pathway interacts with or is associated with other critical pathways involved in MS. We also found that disruption of the dopaminergic system leads to myelin defciency in zebrafsh. Conclusions: Dopaminergic signaling may be involved in myelin pathogenesis. This study may ofer a novel molecu- lar mechanism of demyelination in the nervous system. Keywords: Dopaminergic signaling, myelin pathogenesis, genetics, transcriptome, zebrafsh, in vivo analysis Background *Correspondence: [email protected]; [email protected]; [email protected] Myelin sheaths surrounding axons ensure rapid action †Sujun Ding, Yun Gu and Yunyun Cai contributed equally to this work potential conduction. Myelin pathogenesis derived from 1 School of Medicine, Nantong University, Nantong, China developmental defcits or demyelination is deleterious 2 Department of Ultrasound, Afliated Hospital of Nantong University, Nantong, China in the central nervous system (CNS), leading to irrevers- 3 Key Laboratory of Neuroregeneration of Jiangsu and the Ministry ible and progressive neurological decline. Myelin defcits of Education, Co-innovation Center of Neuroregeneration, Nantong have been found in multiple neurological disorders, such University, Nantong, China Full list of author information is available at the end of the article as Parkinson’s disease (PD), schizophrenia, and multiple © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ding et al. J Transl Med (2020) 18:109 Page 2 of 12 sclerosis (MS) [1, 2], many of which have disturbed dopa- signifcance level from the genome-wide association minergic system. However, the relationships between study (GWAS) were also included. dopaminergic signaling and myelin defects are not well understood. Enriched pathway and aggregated category analysis PD and schizophrenia are neurological disorders at Enriched Kyoto Encyclopedia of Genes and Genomes least partially attributed to disrupted dopaminergic sign- (KEGG) pathways of MS gene sets were generated based aling [3, 4]. In patients with PD, abnormal CNS connec- on the latest KEGG database 90.1 (https ://www.genom tivity was observed in several brain regions [5, 6]. White e.jp/kegg/pathw ay.html) [19]. Hypergeometric distribu- matter defects indicate that myelin abnormalities under- tion was used to determine enrichment of the specifed lie some aspects of PD. Te potential roles of dopamin- gene sets in pathways. P values were corrected with the ergic signaling in white matter and myelin integrity were Benjamini-Hochberg procedure. Only those pathways further elucidated in patients with schizophrenia. Te with >=10 target genes were examined further. antipsychotic drug clozapine has been shown to improve white matter integrity in schizophrenia by way of block- Gene interaction analysis ing serotonin and dopamine receptors [7]; Another imag- Gene interactions between genes in the dopaminergic ing study specifcally links dopamine D2/D3 receptor synapse pathway (DS) and other literature-curated (oLC) density to myelin indices in normal and schizophrenic genes in MS were analyzed with datasets from Pathway white matter [8]. Tese indicate a potential role of dopa- Commons (http://www.pathw aycom mons.org/) [20, 21] minergic signaling in myelin defcits. and high-confdence human interactome [22]. In Path- MS is a neurological disorder characterized by demy- way Commons (http://www.pathw aycom mons.org/) [21], elination and axon loss. Interestingly, concomitance of only four common types of interactions were considered: MS and PD as well as MS and schizophrenia has been controls-state-change-of, controls-expression-of, con- observed [9, 10]. Altered dopamine receptor levels were trols-transport-of, and controls-phosphorylation-of. In found in the blood of patients with MS [11, 12]. Moreo- Pathway Commons (http://www.pathw aycom mons.org/) ver, modulation of dopamine receptors can promote or [21], directed interactions are provided; that is, interac- prevent experimental autoimmune encephalomyelitis tions between genes of DS and oLC were analyzed with (EAE) in mouse, a rodent model for human MS [13, 14]. DS either as upstream or downstream genes. In high- Based on these, we hypothesize that dopaminergic sign- confdence human interactome, undirected interactions aling may be important for demyelination in MS. are provided. In this study, we described a systems analysis which integrate multi-source-based data from human genetic Transcriptome analysis association studies and transcriptome data in patients To study the transcriptional profle of brain lesions in with MS to investigate the role of dopaminergic signal- patients with multiple sclerosis, we downloaded micro- ing in MS. Tis unbiased approach combined with in vivo array data in the GEO database (https ://www.ncbi.nlm. functional analysis will provide us with insight into a role nih.gov/geo/) [23] from GSE26927 for grey matter lesions of dopaminergic signaling in MS. and from GSE38010 for white matter lesions. GSE26927 is the microarray data on the Illumina human Ref-8 v2.0 expression beadchip [24, 25]. Data was normalized by Materials and methods Quantile algorithm. 15373 probes were detected after Identifcation of MS‑related genes fltering low expression with a detection p value <0.01. MS-related genes were obtained by a systematic analy- GSE38010 is data from the Afymetrix Human Genome sis of the human genetic association studies deposited in U133 Plus 2.0 Array [26]. Data was normalized by the Pubmed (https ://www.ncbi.nlm.nih.gov/pubme d) [15]. Rosetta error models. Only probes with log2 transformed Similar to references [16–18], we queried for publications normalized signal >=6 in at least one sample were con- about MS with the terms (Multiple sclerosis [MeSH]) sidered, of which 31275 probes were detected. and (polymorphism [MeSH] or genotype [MeSH] or alleles [MeSH]) not (neoplasms [MeSH]). 2428 publica- Gene set enrichment analysis tions in total were found by June 8th, 2018. We selected Diferentially expressed gene sets in GSE26927 and only those genes reported to be associated with MS by GSE38010 were identifed with the ROAST test (ROAST: manually reviewing abstracts and the full reports if the rotation gene set tests for complex microarray experi- abstract was not clear. When multiple genes reported to ments) [27]. We used the ‘mixed’ test, in which the act together were associated with MS, all of these genes directionality of changes in expression was not consid- were included. In addition, genes with a genome-wide ered. Multiple gene sets were selected based on
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