Padhi Bhaja K, Rosales Marianela, Pelletier Guillaume
Hazard Identification Division, HECSB, Health Canada, Tunney's Pasture, Ottawa, Ontario K1A 0L2, Canada.
Hazard Identification Division, HECSB, Health Canada, Tunney's Pasture, Ottawa, Ontario K1A 0L2, Canada.
Neurotoxicology. 2015 May;48:223-30. doi: 10.1016/j.neuro.2015.04.006. Epub 2015 Apr 29.
Early life exposure to environmental chemicals can interfere with myelin formation in the developing brain, leading to neurological disorders. The Proteolipid Protein 1 (Plp1), Myelin Basic Protein (Mbp) and 2',3'-Cyclic Nucleotide 3'Phosphodiesterase (Cnp) genes expressed in oligodendrocytes and involved in myelination processes can be useful biomarkers of potential developmental neurotoxicity. In an earlier study, we concluded that the reduction in the expression levels of Mbp splice variants in juvenile rat cerebellum following perinatal methylmercury (MeHg) exposure were compatible with an overall reduction of mature oligodendrocytes population. This observation prompted us to analyze the expression of Plp1 and Cnp in developing rat cerebellum to further confirm and investigate the toxic effects of MeHg on vulnerable oligodendrocytes. Splice variants of Plp1 in human and of Cnp in mouse are curated in NCBI RefSeq database, but not for rat. Lack of annotation of splice variants can pose significant challenge for the reliable quantification of gene expression levels in toxicological studies. Therefore, we applied a "comparative sequence analysis" approach, relying on annotated splice variants in human/mouse and on evolutionary conservation of intron-exon structures, to identify additional splice variants of Plp1 and Cnp in rat. Then, we confirmed their identity by nucleotide sequencing and characterized their temporal expression patterns during brain development by RT-PCR. The measurement of total transcripts and individual splice variants of Plp1 and Cnp in the cerebellum of MeHg-exposed rat pups revealed a relatively similar level of reduction in their expression levels. This study further confirms that perinatal exposure to MeHg can impact oligodendrocytes in pups. Based on these observations, we conclude that monitoring the expression of these oligodendrocyte-enriched genes can be useful to identify toxic chemicals affecting myelination.
生命早期暴露于环境化学物质会干扰发育中大脑的髓鞘形成，导致神经紊乱。在少突胶质细胞中表达且参与髓鞘形成过程的蛋白脂质蛋白1（Plp1）、髓鞘碱性蛋白（Mbp）和2',3'-环核苷酸3'-磷酸二酯酶（Cnp）基因，可能是潜在发育神经毒性的有用生物标志物。在早期研究中，我们得出结论，围产期甲基汞（MeHg）暴露后幼鼠小脑Mbp剪接变体的表达水平降低，与成熟少突胶质细胞群体的整体减少相一致。这一观察结果促使我们分析发育中大鼠小脑Plp1和Cnp的表达，以进一步确认并研究MeHg对易损少突胶质细胞的毒性作用。人类Plp1和小鼠Cnp的剪接变体在NCBI RefSeq数据库中有整理，但大鼠没有。剪接变体注释的缺失可能对毒理学研究中基因表达水平的可靠定量构成重大挑战。因此，我们应用了一种“比较序列分析”方法，依靠人类/小鼠中注释的剪接变体以及内含子-外显子结构的进化保守性，来识别大鼠中Plp1和Cnp的其他剪接变体。然后，我们通过核苷酸测序确认了它们的身份，并通过逆转录-聚合酶链反应（RT-PCR）表征了它们在大脑发育过程中的时间表达模式。对暴露于MeHg的幼鼠小脑Plp1和Cnp的总转录本和单个剪接变体的测量显示，它们的表达水平降低程度相对相似。这项研究进一步证实，围产期暴露于MeHg会影响幼鼠的少突胶质细胞。基于这些观察结果，我们得出结论，监测这些富含少突胶质细胞的基因的表达，有助于识别影响髓鞘形成的有毒化学物质。
