BACKGROUND 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. OBJECTIVES AND METHODS 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. RESULTS 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. CONCLUSIONS 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.

中文翻译：

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SIRT1激活可挽救RNA结合蛋白的错误定位和遗传性钴胺素疾病引起的认知缺陷。

背景技术维生素B12或钴胺素代谢的先天性错误的分子后果尚不清楚。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系统结局。我们先前的发现表明，SIRT1，细胞应激与RNA结合蛋白（RBP）定位错误之间的联系是由维生素B12代谢受损引起的病理机制。目的和方法本研究的目的是研究使用SRT1720激活SIRT1的药理作用对甲硫氨酸合酶活性降低引起的分子机制的影响。实验是利用cblG和cblC遗传了维生素B12代谢缺陷的患者的成纤维细胞在体外进行的，并使用了特定于神经元细胞的甲硫氨酸合酶缺乏症的原始转基因小鼠模型进行了体内实验。通过免疫染色和共聚焦显微镜检查患者成纤维细胞中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试验研究了RBP的甲基化和磷酸化。用水生迷宫测量用SRT1720处理的转基因小鼠的认知能力。结果患有维生素B12代谢的cblC和cblG缺陷的患者成纤维细胞表现为内质网应激，HuR，HnRNPA1和RBM10的甲基化，磷酸化和亚细胞定位改变，全局mRNA定位错误和IRF3外显子的HnRNPA1依赖性跳跃增加。将成纤维细胞与钴胺素，S-腺苷甲硫氨酸和冈田酸一起孵育可拯救RBP和mRNA的定位。SIRT1激活化合物SRT1720抑制内质网应激，并挽救了RBP和mRNA的错误定位和IRF3剪接。用这种SIRT1激动剂进行的治疗可以预防患者成纤维细胞中的所有这些特征，但它也可以改善蛋氨酸合酶条件基因敲除小鼠的海马依赖性学习能力不足。结论通过阐明由cbl代谢的先天性错误引起的与ER应激，RBP定位错误和mRNA交易相关的分子机制，我们的研究为治疗维生素B12代谢性先天性错误开辟了新的治疗前景。