Childhood-onset hypomyelinating leukodystrophy and the multi-tRNA synthetase complex

儿童期发病的低髓鞘性脑白质营养不良和多 tRNA 合成酶复合物

• 批准号: 10582441
• 负责人: Ranjan Dutta
• 金额: $ 62.66万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582441
• 关键词: Affect; Amino Acids; Amino Acyl-tRNA Synthetases; Anticodon; Ataxia; Automobile Driving; Behavioral; Binding; CRISPR/Cas technology; Cell Nucleus; Cells; Central Nervous System; Central Nervous System Diseases; Cerebellar Diseases; Cerebrum; Child; Childhood; Clinical; Code; Cognitive; Complex; Cytoplasm; Defect; Developmental Delay Disorders; Disparate; Distant; Elements; Etiology; Exhibits; Functional disorder; Gait abnormality; Genes; Genetic Code; Glutamates; Growth; Heterozygote; Human Inbreeding; Impaired cognition; Intellectual functioning disability; Knock-in Mouse; Lamin Type A; Letters; Life Expectancy; Ligase; Ligation; Mediating; Messenger RNA; Methylation; Microcephaly; Molecular; Motor; Mus; Mutant Strains Mice; Mutate; Mutation; Myelin; Neural Conduction; Neurodegenerative Disorders; Neurologic; Neurologic Deficit; Nomenclature; Nuclear; Nuclear Export; Oligodendroglia; Parents; Pathologic; Pathologic Nystagmus; Pathology; Pathway interactions; Peripheral Nerves; Phenotype; Point Mutation; Poly(ADP-ribose) Polymerases; Positioning Attribute; Post-Transcriptional Regulation; Proline; Protein Biosynthesis; Proteins; Recombinants; Repression; Role; Siblings; Specificity; System; Testing; Therapeutic; Therapeutic Intervention; Trans-Activators; Transcript; Transfer RNA; Transfer RNA Aminoacylation; Translations; Variant; adverse outcome; brain magnetic resonance imaging; cell type; curative treatments; early adolescence; exome sequencing; experimental study; glutamic acid-tRNA; glutamyl-prolyl-tRNA synthetase; in vivo; in vivo Model; knock-down; leukodystrophy; lymphoblastoid cell line; motor impairment; mouse model; mutant; myelination; novel; oligodendrocyte precursor; preclinical evaluation; precursor cell; prenatal; programs; proline-tRNA; protein expression; single nucleus RNA-sequencing; small hairpin RNA; spasticity; therapeutic RNA; transcriptomics; virtual

Project Summary/Abstract Child-onset hypomyelinating leukodystrophy (HLD) is a genetically heterogeneous group of neurodegenerative diseases characterized by reduced cerebral myelin formation. Clinical features include cognitive as well as motor impairment appearing in childhood. There are no curative treatments. Our collaborator, Dr. Grace Yoon, evaluated two siblings presenting with severe neurological deficit and a shared phenotype consisting of global developmental delay and intellectual disability, prenatal onset undergrowth and microcephaly, rotatory nystagmus, ataxia and progressive gait disturbance/spasticity, and hypomyelination on brain MRI. Whole exome sequencing revealed homozygosity of a c.4444C>A mutation in the EPRS1 gene. The mutation is in the coding sequence (cds) of the gene causing a Pro-to-Thr point mutation at aa position 1482. The EPRS1 gene encodes the bifunctional, glutamyl-prolyl tRNA synthetase (EPRS1) that resides in the cytoplasmic multi- tRNA synthetase complex (MSC). The P1482T mutation is located near the C-terminus of the protein in a region of the Pro synthetase outside the catalytic or anti-codon recognition domains. The specific activity of recombinant P1482T mutant EPRS1 is indistinguishable from wild-type. In contrast, EPRS1 protein expression in immortalized lymphoblastoid cell lines (LCL) from affected siblings is about 20% of that in unaffected controls. EPRS1 mRNA levels are identical in siblings and controls indicating post-transcriptional regulation. Our preliminary studies show a dual mechanism determining diminished EPRS1 level in affected LCLs, namely, decreased nuclear export of mutant mRNA, followed by decreased cytoplasmic translation. We show that decreased EPRS1 expression causes release of other MSC constituents. Remarkably, suppression of several other MSC components causally implicated in HLD also induce MSC constituent release, suggesting a common etiology of an entire class of HLDs. We hypothesize that inefficient nuclear export and translation of c.4444C>A EPRS1 mRNA reduces MSC-bound EPRS1, causing release of MSC constituents and driving the HLD phenotype. Likewise, defects in genes encoding other MSC constituents share a common pathway and etiology of HLD. We will test this hypothesis by pursuing these Specific Aims: In Aim 1 we will elucidate molecular mechanisms underlying reduced expression of EPRS1P1482T variant, focusing on the role of m6A methylation. Aim 2 determines the HLD-related phenotype of our newly generated, genetically-modified Eprs1P1482T mice. In Aim 3 we will investigate effect of deficiency of MSC constituents on MSC integrity and adverse consequences in myelinating oligodendrocytes. A CNS-specific transcriptomic analysis will be done by single-nucleus RNA-sequencing. Completion of these studies will elucidate a unique mechanism of gene dysregulation that induces HLD, and will provide a unique mouse model of HLD that will permit detailed in vivo analysis of the cellular defects in HLD. Importantly, these results suggest the possibility of specific, RNA-based therapeutic intervention to rescue translation of the variant EPRS1 mRNA and rescue the neurologic defect.

项目摘要/摘要 儿童起病的低髓素性白质营养不良症(HLD)是一组遗传异质性的神经退行性疾病 以脑髓鞘形成减少为特征的疾病。临床特征包括认知能力和 运动障碍出现在儿童时期。目前还没有根治的方法。我们的合作者，Grace Yoon博士， 评估了两个兄弟姐妹，他们都有严重的神经功能缺陷，并有共同的表型，包括 发育迟缓和智力残疾，产前发病发育不足和小头畸形，旋转 眼球震颤，共济失调和进行性步态障碍/痉挛，以及脑部MRI上的髓鞘减少。整体 外显子组测序显示EPRS1基因中的C.4444C&GT；A突变是纯合的。突变是在 导致AA1482位Pro-to-Thr点突变的基因的编码序列(CDS)。《环境政策与政策》第1期 该基因编码存在于细胞质中的双功能谷氨酰脯氨基tRNA合成酶(EPRS1). TRNA合成酶复合体(MSC)。P1482T突变位于蛋白的C末端附近 Pro合成酶催化或反密码子识别结构域外的区域。的特定活动 重组P1482T突变体EPRS1与野生型难以区分。相反，EPRS1蛋白的表达 在来自患病同胞的永生化淋巴母细胞系(LCL)中，约为未患病患者的20% 控制。EPRS1的mRNA水平在兄弟姐妹和对照中是相同的，表明转录后调控。 我们的初步研究表明，在受影响的LCLS中，EPRS1水平下降是一种双重机制， 也就是说，突变mRNA的核输出减少，随后细胞质翻译减少。我们展示了 EPRS1表达的降低会导致其他MSC成分的释放。值得注意的是，对 与HLD有因果关系的其他几种MSC成分也可以诱导MSC成分的释放，这表明 一整类HLDS的常见病因。我们假设低效的核出口和翻译 C.44C&GT；A EPRS1 mRNA减少与MSC结合的EPRS1，导致MSC成分释放，并推动 HLD表型。同样，编码其他间充质干细胞成分的基因缺陷共享共同的途径和 HLD的病因学。我们将通过追求以下具体目标来检验这一假设：在目标1中，我们将阐明 EPRS1P1482T变异体表达降低的分子机制，重点是M6A的作用 甲基化。目标2决定了我们新产生的、转基因的HLD相关表型 Eprs1P1482T小鼠。在目标3中，我们将调查MSC成分缺乏对MSC完整性和 少突胶质细胞髓鞘形成的不良后果。一项针对中枢神经系统的转录分析将由 单核RNA测序。这些研究的完成将阐明一种独特的基因机制 导致肝豆状核变性的调节失调，并将提供一种独特的肝豆状核变性小鼠模型，将允许在体内进行详细的 肝豆状核变性的细胞缺陷分析。重要的是，这些结果表明，特定的、基于RNA的 治疗干预，挽救EPRS1变异基因的翻译，挽救神经功能缺陷。