Center for Molecular Medicine, Department of Clinical Neuroscience, Neuroimmunology Unit, Karolinska Institutet, Stockholm, Sweden.
PLoS One. 2010 Sep 15;5(9):e12716. doi: 10.1371/journal.pone.0012716.
To elucidate mechanisms involved in multiple sclerosis (MS), we studied genetic regulation of experimental autoimmune encephalomyelitis (EAE) in rats, assuming a conservation of pathogenic pathways. In this study, we focused on Eae23, originally identified to regulate EAE in a (LEW.1AV1xPVG.1AV1)F2 cross. Our aim was to determine whether one or more genes within the 67 Mb region regulate EAE and to define candidate risk genes.
METHODOLOGY/PRINCIPAL FINDINGS: We used high resolution quantitative trait loci (QTL) analysis in the 10th generation (G10) of an advanced intercross line (AIL) to resolve Eae23 into two QTLs that independently regulate EAE, namely Eae23a and Eae23b. We established a congenic strain to validate the effect of this region on disease. PVG alleles in Eae23 resulted in significant protection from EAE and attenuated CNS inflammation/demyelination. Disease amelioration was accompanied with increased levels of Foxp3(+) cells in the CNS of the congenic strain compared to DA. We then focused on candidate gene investigation in Eae23b, a 9 Mb region linked to all clinical phenotypes. Affymetrix exon arrays were used to study expression of the genes in Eae23b in the parental strains, where none showed differential expression. However, we found lower expression of exon 4 of ZEB1, which is specific for splice-variant Zfhep1. ZEB1 is an interleukin 2 (IL2) repressor involved in T cell development. The splice-specific variance prompted us to next analyze the expression of ZEB1 and its two splice variants, Zfhep1 and Zfhep2, in both lymph node and spleen. We demonstrated that ZEB1 splice-variants are differentially expressed; severity of EAE and higher IL2 levels were associated with down-regulation of Zfhep1 and up-regulation of Zfhep2.
CONCLUSIONS/SIGNIFICANCE: We speculate that the balance between splice-variants of ZEB1 could influence the regulation of EAE. Further functional studies of ZEB1 and the splice-variants may unravel novel pathways contributing to MS pathogenesis and inflammation in general.
为了阐明多发性硬化症（MS）的发病机制，我们研究了实验性自身免疫性脑脊髓炎（EAE）在大鼠中的遗传调控，假设致病途径是保守的。在这项研究中，我们专注于 Eae23，最初是在（LEW.1AV1xPVG.1AV1）F2 杂交中被鉴定为调节 EAE 的基因。我们的目的是确定 67Mb 区域内的一个或多个基因是否调节 EAE，并定义候选风险基因。
方法/主要发现：我们使用高级互交系（AIL）第 10 代（G10）的高分辨率数量性状基因座（QTL）分析将 Eae23 分解为两个独立调节 EAE 的 QTL，即 Eae23a 和 Eae23b。我们建立了一个同系交配株来验证该区域对疾病的影响。Eae23 中的 PVG 等位基因导致 EAE 显著保护，并减轻中枢神经系统炎症/脱髓鞘。与 DA 相比，同系交配株的中枢神经系统中 Foxp3(+)细胞水平升高伴随着疾病的改善。然后，我们专注于与所有临床表型相关的 9Mb 区域 Eae23b 中的候选基因研究。我们使用 Affymetrix 外显子芯片研究了亲本株中外显子 4 的表达情况，结果显示均无差异表达。然而，我们发现 ZEB1 外显子 4 的表达降低，ZEB1 是一种白细胞介素 2（IL2）抑制剂，参与 T 细胞发育。剪接特异性变异促使我们接下来分析 ZEB1 及其两个剪接变体 Zfhep1 和 Zfhep2 在淋巴结和脾脏中的表达。我们证明 ZEB1 剪接变体存在差异表达；EAE 的严重程度和更高的 IL2 水平与 Zfhep1 的下调和 Zfhep2 的上调相关。
结论/意义：我们推测 ZEB1 剪接变体的平衡可能会影响 EAE 的调节。进一步研究 ZEB1 和剪接变体的功能可能会揭示导致 MS 发病机制和炎症的新途径。
