Université de Lorraine, Inserm, UMRS 1256, NGERE - Nutrition, Genetics, and Environmental Risk Exposure, F-54000 Nancy, France.
Université de Lorraine, Inserm, UMRS 1256, NGERE - Nutrition, Genetics, and Environmental Risk Exposure, F-54000 Nancy, France.
Metabolism. 2019 Dec;101:153992. doi: 10.1016/j.metabol.2019.153992. Epub 2019 Oct 28.
The molecular consequences of inborn errors of vitamin B12 or cobalamin metabolism are far from being understood. Moreover, innovative therapeutic strategies are needed for the treatment of neurological outcomes that are usually resistant to conventional treatments. Our previous findings suggest a link between SIRT1, cellular stress and RNA binding proteins (RBP) mislocalization in the pathological mechanisms triggered by impaired vitamin B12 metabolism.
The goal of this study was to investigate the effects of the pharmacological activation of SIRT1 using SRT1720 on the molecular mechanisms triggered by impaired methionine synthase activity. Experiments were performed in vitro with fibroblasts from patients with the cblG and cblC inherited defects of vitamin B12 metabolism and in vivo with an original transgenic mouse model of methionine synthase deficiency specific to neuronal cells. Subcellular localization of the RBPs HuR, HnRNPA1, RBM10, SRSF1 and Y14 was investigated by immunostaining and confocal microscopy in patient fibroblasts. RBPs methylation and phosphorylation were studied by co-immunoprecipitation and proximity ligation assay. Cognitive performance of the transgenic mice treated with SRT1720 was measured with an aquatic maze.
Patient fibroblasts with cblC and cblG defects of vitamin B12 metabolism presented with endoplasmic reticulum stress, altered methylation, phosphorylation and subcellular localization of HuR, HnRNPA1 and RBM10, global mRNA mislocalization and increased HnRNPA1-dependent skipping of IRF3 exons. Incubation of fibroblasts with cobalamin, S-adenosyl methionine and okadaic acid rescued the localization of the RBPs and mRNA. The SIRT1 activating compound SRT1720 inhibited ER stress and rescued RBP and mRNA mislocalization and IRF3 splicing. Treatment with this SIRT1 agonist prevented all these hallmarks in patient fibroblasts but it also improved the deficient hippocampo-dependent learning ability of methionine synthase conditional knock-out mice.
By unraveling the molecular mechanisms triggered by inborn errors of cbl metabolism associating ER stress, RBP mislocalization and mRNA trafficking, our study opens novel therapeutic perspectives for the treatment of inborn errors of vitamin B12 metabolism.
先天性维生素 B12 或钴胺素代谢错误的分子后果远未被理解。此外，需要创新的治疗策略来治疗通常对常规治疗有抵抗力的神经学结果。我们之前的研究结果表明，SIRT1、细胞应激和 RNA 结合蛋白 (RBP) 定位错误之间存在联系，这些都与维生素 B12 代谢受损引发的病理机制有关。
本研究的目的是研究使用 SRT1720 对蛋氨酸合酶活性受损引发的分子机制的药理学激活 SIRT1 的影响。在患有先天性维生素 B12 代谢 cblG 和 cblC 缺陷的成纤维细胞中进行体外实验，并在特定于神经元细胞的蛋氨酸合酶缺陷的原始转基因小鼠模型中进行体内实验。通过免疫染色和共聚焦显微镜研究了患者成纤维细胞中 RBPs HuR、HnRNPA1、RBM10、SRSF1 和 Y14 的亚细胞定位。通过共免疫沉淀和邻近连接分析研究了 RBPs 的甲基化和磷酸化。使用水迷宫测量用 SRT1720 治疗的转基因小鼠的认知表现。
患有先天性维生素 B12 代谢 cblC 和 cblG 缺陷的患者成纤维细胞表现出内质网应激、HuR、HnRNPA1 和 RBM10 的甲基化、磷酸化和亚细胞定位改变、全局 mRNA 定位错误以及 IRF3 外显子的 HnRNPA1 依赖性跳过增加。用钴胺素、S-腺苷甲硫氨酸和 okadaic 酸孵育成纤维细胞可挽救 RBPs 和 mRNA 的定位。SIRT1 激活化合物 SRT1720 抑制内质网应激并挽救 RBP 和 mRNA 定位错误和 IRF3 剪接。该 SIRT1 激动剂的治疗不仅可以防止患者成纤维细胞出现所有这些特征，还可以改善蛋氨酸合酶条件敲除小鼠的海马依赖性学习能力缺陷。
通过阐明与内质网应激、RBP 定位错误和 mRNA 运输相关的 cbl 代谢先天错误引发的分子机制，我们的研究为治疗先天性维生素 B12 代谢错误开辟了新的治疗前景。
