Analysis of DARS2 in Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation (LBSL) Patient Neurons

DARS2 在脑干和脊髓受累及乳酸升高 (LBSL) 患者神经元白质脑病中的分析

• 批准号: 10524935
• 负责人: S. Ali Fatemi
• 金额: $ 44.57万
• 依托单位: HUGO W. MOSER RES INST KENNEDY KRIEGER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10524935
• 关键词: Affect; Amino Acyl-tRNA Synthetases; Aminoacylation; Antibodies; Aspartate-tRNA Ligase; Beds; Behavior; Biological Assay; Brain Stem; Cell Line; Cell physiology; Cells; Cessation of life; Chemicals; Co-Immunoprecipitations; Communities; Complex; Data; Defect; Demyelinations; Differentiation and Growth; Diffuse; Disease; Disease Progression; Electrophysiology (science); Embryo; Enzymes; Evaluation; Family; Family member; Fostering; Functional disorder; Future; Gene Expression; Genes; Glean; Goals; Growth; Heterozygote; Immunoprecipitation; Individual; Induced pluripotent stem cell derived neurons; Informal Social Control; Intervention; Knock-out; Knockout Mice; Leukoencephalopathy; Ligase; Magnetic Resonance Imaging; Mass Spectrum Analysis; Microelectrodes; Mitochondria; Mitochondrial Proteins; Modeling; Motor Neurons; Mutation; Nerve Degeneration; Neuraxis; Neurons; Pathogenesis; Pathology; Patients; Pattern; Peripheral; Phenotype; Play; Positioning Attribute; Proteins; Proteome; RNA; RNA-Binding Proteins; RNA-Protein Interaction; Reporting; Role; Sampling; Series; Signaling Protein; Spinal Cord; Suggestion; Teenagers; Testing; Therapeutic; Transfer RNA; Translations; Variant; Western Blotting; Work; cell type; crosslink; crosslinking and immunoprecipitation sequencing; disease mechanisms study; dorsal column; early childhood; embryonic stem cell; enzyme activity; experience; experimental study; induced pluripotent stem cell; insight; live cell imaging; motor control; mutant; neglect; nerve stem cell; nervous system disorder; neurite growth; novel; novel therapeutics; patient population; peripheral blood; stem; therapeutic development; therapeutic target; transcription factor; transcriptome sequencing; ultraviolet; white matter

Project Summary Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare, autosomal recessive neurological disorder caused by mutations in the gene DARS2, which encodes the mitochondrial aspartyl-tRNA synthetase. LBSL patients are compound heterozygote, and experience slow and progressive dorsal column dysfunction, resulting in the need for mobility assistance by the early teenage years, or in severe cases, death. DARS2 has a well-defined role in mitochondrial protein translation, however, this canonical function appears unaltered in patients with LBSL. Furthermore, mutations within the family of mitochondrial tRNA synthetases (mt-ARS) each produces a unique pattern of dysfunction, suggesting unique mechanisms independent of translation. Ubiquitous expression of DARS2 and selective CNS dysfunction within LBSL patients underscores the need to study disease mechanism in relevant cell types. Induced pluripotent stem cells (iPSCs) allow us to differentiate patient peripheral cells into mature and functional motor neurons (Aim 1). Within this proposal we plan to culture LBSL patient and isogenic control motor neurons (Aim 1.1) to establish phenotypes in LBSL patient lines (Aim 1.2). Preliminary data reveals deficits in LBSL neurons and also shows feasibility of cell phenotyping experiments. Characterization of CNS cell types in LBSL is novel and will inform the field on how patient variant combinations affect cell function. Preliminary evaluation of the mitochondrial proteome in LBSL revealed no errors in translation, and only diffuse effects overall, thus we propose to expand our search into disease mechanism by probing DARS2 interaction partners (Aim 2). Cytosolic tRNA synthetases are reported to participate in protein signaling and even to behave as RNA-binding proteins influencing gene expression, translation, and self-regulation, we therefore suspect that DARS2 as a mitochondrial tRNA synthetase may have similar non-translational functions. Evaluation of RNA and protein interaction through cross-linking immunoprecipitation (CLIP-seq; Aim 2.1) and cross-linking co- immunoprecipitation (XL-MS; Aim 2.2) will reveal RNA and proteins partners pertinent to normal DARS2 activity. We plan to assess interactors in both control and LBSL patient iPSC-derived motor neurons, with the understanding that patient mutations may affect technical aspects of these experiments. Novel functional data collected from healthy patient samples alone stand to change our understanding of mt-ARS in the cell and may dictate therapeutic development for mt-ARS disorders. As LBSL pathology is unique from other synthetase disorders, it is possible that alternative functions relate to disease mechanism, and identifying these functions would provide targets for intervention. Patient iPSCs and iPSC-derived motor neurons are essential to understand endogenous DARS2 behavior and LBSL pathophysiology. Our overall goal is to use patient iPSC- derived neurons to understand LBSL disease mechanism and to use this information to guide therapeutic development.

项目概要 累及脑干和脊髓并伴有乳酸升高的白质脑病 (LBSL) 是一种罕见的、 由 DARS2 基因突变引起的常染色体隐性神经系统疾病，该基因编码 线粒体天冬氨酰-tRNA 合成酶。 LBSL 患者是复合杂合子，并且经历缓慢和 进行性背柱功能障碍，导致青少年早期需要行动辅助， 或严重时死亡。 DARS2 在线粒体蛋白质翻译中具有明确的作用，然而，这 LBSL 患者的典型功能似乎没有改变。此外，家族内的突变 线粒体 tRNA 合成酶 (mt-ARS) 各自产生独特的功能障碍模式，表明其独特的功能障碍 独立于翻译的机制。 DARS2 的普遍表达和选择性 CNS 功能障碍 LBSL 患者的研究强调了研究相关细胞类型疾病机制的必要性。诱发 多能干细胞 (iPSC) 使我们能够将患者外周细胞分化为成熟且有功能的运动细胞 神经元（目标 1）。在该提案中，我们计划培养 LBSL 患者和等基因控制运动神经元（目标 1.1) 建立 LBSL 患者系的表型（目标 1.2）。初步数据显示 LBSL 神经元存在缺陷 并显示了细胞表型实验的可行性。 LBSL 中 CNS 细胞类型的表征是新颖的 并将告知该领域患者变异组合如何影响细胞功能。初步评估 LBSL 中的线粒体蛋白质组显示没有翻译错误，总体上只有扩散效应，因此我们 建议通过探索 DARS2 相互作用伙伴来扩大我们对疾病机制的研究（目标 2）。 据报道，胞质 tRNA 合成酶参与蛋白质信号转导，甚至起到 RNA 结合的作用 影响基因表达、翻译和自我调节的蛋白质，因此我们怀疑 DARS2 作为一个 线粒体 tRNA 合成酶可能具有类似的非翻译功能。 RNA和蛋白质的评估 通过交联免疫沉淀（CLIP-seq；目标 2.1）和交联共沉淀相互作用 免疫沉淀（XL-MS；目标 2.2）将揭示与正常 DARS2 相关的 RNA 和蛋白质伙伴 活动。我们计划评估对照和 LBSL 患者 iPSC 衍生的运动神经元中的相互作用因子， 了解患者突变可能会影响这些实验的技术方面。新颖的功能数据 仅从健康患者样本中收集的样本就足以改变我们对细胞中 mt-ARS 的理解，并可能 决定了 mt-ARS 疾病的治疗开发。由于 LBSL 病理学不同于其他合成酶 疾病，替代功能可能与疾病机制有关，并识别这些功能 将为干预提供目标。患者 iPSC 和 iPSC 衍生的运动神经元对于 了解内源性 DARS2 行为和 LBSL 病理生理学。我们的总体目标是使用患者 iPSC- 派生神经元来了解 LBSL 疾病机制并利用这些信息来指导治疗 发展。