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.

项目概要/摘要 儿童发病的低髓鞘性脑白质营养不良（HLD）是一组遗传异质性神经退行性疾病 以脑髓磷脂形成减少为特征的疾病。临床特征包括认知以及 儿童时期出现的运动障碍。没有治愈方法。我们的合作者 Grace Yoon 博士， 评估了两个患有严重神经缺陷和共同表型的兄弟姐妹 发育迟缓和智力障碍、产前生长发育不良和小头畸形、旋转 眼球震颤、共济失调和进行性步态障碍/痉挛，以及脑 MRI 上的髓鞘形成不足。所有的 外显子组测序揭示了 EPRS1 基因中 c.4444C>A 突变的纯合性。突变是在 导致第 1482 位氨基酸发生 Pro-to-Thr 点突变的基因的编码序列 (cds)。EPRS1 该基因编码双功能谷氨酰脯氨酰 tRNA 合成酶 (EPRS1)，该酶位于细胞质多 tRNA 合成酶复合物 (MSC)。 P1482T 突变位于蛋白质 C 末端附近 Pro 合成酶的催化域或反密码子识别域之外的区域。具体活动为 重组 P1482T 突变体 EPRS1 与野生型没有区别。相反，EPRS1蛋白表达 来自受影响兄弟姐妹的永生化​​淋巴母细胞系 (LCL) 约为未受影响的 20% 控制。兄弟姐妹和对照中的 EPRS1 mRNA 水平相同，表明转录后调节。 我们的初步研究表明，双重机制决定了受影响 LCL 中 EPRS1 水平的降低， 也就是说，突变体 mRNA 的核输出减少，随后细胞质翻译减少。我们展示 EPRS1 表达减少会导致其他 MSC 成分的释放。值得注意的是，抑制 与 HLD 有因果关系的其他几种 MSC 成分也会诱导 MSC 成分释放，这表明 整个 HLD 类别的共同病因。我们假设低效的核输出和翻译 c.4444C>A EPRS1 mRNA 减少 MSC 结合的 EPRS1，导致 MSC 成分释放并驱动 HLD表型。同样，编码其他 MSC 成分的基因缺陷具有共同的途径，并且 HLD 的病因学。我们将通过追求这些具体目标来检验这一假设：在目标 1 中，我们将阐明 EPRS1P1482T 变体表达减少的分子机制，重点关注 m6A 的作用 甲基化。目标 2 确定我们新生成的转基因细胞的 HLD 相关表型 Eprs1P1482T 小鼠。在目标 3 中，我们将研究 MSC 成分缺乏对 MSC 完整性的影响和 髓鞘少突胶质细胞的不良后果。 CNS 特异性转录组分析将由 单核RNA测序。这些研究的完成将阐明基因的独特机制 诱导 HLD 的失调，并将提供独特的 HLD 小鼠模型，该模型将允许详细的体内研究 HLD 中的细胞缺陷分析。重要的是，这些结果表明基于 RNA 的特异性研究的可能性 治疗干预以挽救变异 EPRS1 mRNA 的翻译并挽救神经系统缺陷。