Activation of Sterol Regulatory Element Binding Factors by Fenofibrate and Gemfibrozil Stimulates Myelination in Zebrafish

Abstract

Oligodendrocytes are major myelin-producing cells and play essential roles in the function of a healthy nervous system. However, they are also one of the most vulnerable neural cell types in the central nervous system (CNS), and myelin abnormalities in the CNS are found in a wide variety of neurological disorders, including multiple sclerosis, adrenoleukodystrophy, and schizophrenia. There is an urgent need to identify small molecular weight compounds that can stimulate myelination. In this study, we performed comparative transcriptome analysis to identify pharmacodynamic effects common to miconazole and clobetasol, which have been shown to stimulate myelination by mouse oligodendrocyte progenitor cells (OPCs). Of the genes differentially expressed in both miconazole- and clobetasol-treated mouse OPCs compared with untreated cells, we identified differentially expressed genes (DEGs) common to both drug treatments. Gene ontology analysis revealed that these DEGs are significantly associated with the sterol biosynthetic pathway, and further bioinformatics analysis suggested that sterol regulatory element binding factors (SREBFs) might be key upstream regulators of the DEGs. In silico screening of a public database for chemicals associated with SREBF activation identified fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, as a drug that increases the expression of known SREBF targets, raising the possibility that fenofibrate may also stimulate myelination. To test this, we performed in vivo imaging of zebrafish expressing a fluorescent reporter protein under the control of the myelin basic protein (mbp) promoter. Treatment of zebrafish with fenofibrate significantly increased expression of the fluorescent reporter compared with untreated zebrafish. This increase was attenuated by co-treatment with fatostatin, a specific inhibitor of SREBFs, confirming that the fenofibrate effect was mediated via SREBFs. Furthermore, incubation of zebrafish with another PPARα agonist, gemfibrozil, also increased expression of the mbp promoter-driven fluorescent reporter in an SREBF-dependent manner. These results suggest that activation of SREBFs by small molecular weight compounds may be a feasible therapeutic approach to stimulate myelination.

Keywords: SREBFs; comparative transcriptomics; fenofibrate; gemfibrozil; myelination; oligodendrocytes; systems pharmacology; zebrafish.

FIGURE 1

Venn diagrams of differentially expressed genes in mEpiSC-OPCs treated with miconazole or clobetasol compared with control mEpiSC-OPCs. Transcriptome data from mEpiSC-OPCs treated with miconazole or clobetasol (GSE63804) were downloaded from a public database. Genes differentially expressed in control mEpiSC-OPCs versus mEpiSC-OPCs treated with miconazole or clobetasol for 2, 6, or 12 h were identified using a false discovery rate of 20% as the threshold. The number of DEGs in each group and the overlap between groups are shown in the Venn diagrams for 2, 6, and 12 h of treatment.

FIGURE 2

Biological pathways significantly enriched in genes regulated by both miconazole and clobetasol. DEGs common to mEpiSC-OPCs treated with miconazole or clobetasol for 6 or 12 h were independently subjected to ClueGO using Gene Ontology Biological Pathway as the database. The pathways significantly enriched in the common DEGs at 6 and 12 h treatment are shown in (A,B), respectively. Each circle represents one biological pathway. Pairs of biological pathways with similar kappa scores are connected by lines. Biological pathways clustered in the same group are shown in the same color.

FIGURE 3

Identification of SREBFs as key transcription factors for the genes regulated by both miconazole and clobetasol. (A,B) DEGs common to mEpiSC-OPCs treated with miconazole or clobetasol for 6 or 12 h were independently subjected to iRegulon to identify TFs potentially regulating the DEGs. The DEGs potentially regulated by SREBF1 and SREBF2 after 6 h (A) and 12 h (B) treatment are shown.

FIGURE 4

Effects of thyroid hormone modulators on mbp promoter-driven fluorescence in zebrafish. (A) Representative images from in vivo analysis of Tg (mbp: mCitrine, eno2: Cerulean) zebrafish incubated with or without the indicated concentrations of methimazole (MMI), propylthiouracil (PTU), or thyroxine (T4). (B) Quantification of mCitrine fluorescence intensity within the area of Cerulean fluorescence. (Top) Zebrafish were untreated (n = 40) or treated with MMI (n = 16 for 0.5 mM, n = 26 for 1 mM, n = 27 for 2 mM). (Middle) Zebrafish were untreated (n = 11) or treated with PTU (n = 5 for 1 mM, n = 10 for 2 mM, n = 3 for 3 mM). (Bottom) Zebrafish were untreated (n = 20) or treated with T4 (n = 6 for 10 nM, n = 5 for 20 nM, n = 9 for 30 nM). ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001 compared with control.

FIGURE 5

Fenofibrate and gemfibrozil increase mbp promoter-driven fluorescent reporter expression through activation of SREBFs. (A) Representative images from in vivo analysis of Tg (mbp: mCitrine, eno2: Cerulean) zebrafish incubated with or without the indicated concentrations of fenofibrate, gemfibrozil, and fatostatin. (B,C) Quantification of mCitrine fluorescence intensity within the area of Cerulean fluorescence. (B) Zebrafish were untreated (n = 28) or treated with fenofibrate (n = 27), fatostatin (n = 20), or fenofibrate and fatostatin (n = 5). ^∗p < 0.05 compared with control. (C) Zebrafish were untreated (n = 7) or treated with gemfibrozil (n = 7), fatostatin (n = 8), or gemfibrozil and fatostatin (n = 8). ^∗p < 0.05 compared with control.