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

项目摘要 脑干和脊髓受累的白质脑病和乳酸升高（LBSL）是一种罕见的， 由DARS 2基因突变引起的常染色体隐性遗传神经系统疾病，DARS 2基因编码 线粒体氨酰-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移动辅助， 严重的话会导致死亡DARS 2在线粒体蛋白质翻译中具有明确的作用，然而， LBSL患者的典型功能没有改变。此外，突变家族内的 线粒体tRNA合成酶（mt-ARS）各自产生一种独特的功能障碍模式，表明其独特的 独立于翻译。DARS 2的普遍表达和选择性CNS功能障碍 在LBSL患者中的发现强调了在相关细胞类型中研究疾病机制的必要性。诱导 多能干细胞（iPSC）使我们能够将患者的外周细胞分化为成熟的功能性运动细胞。 神经元（Aim 1）。在这个提议中，我们计划培养LBSL患者和同基因控制运动神经元（Aim 1.1)在LBSL患者系中建立表型（目标1.2）。初步数据显示LBSL神经元的缺陷 也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体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 建议通过探测DARS 2相互作用伙伴（Aim 2）将我们的研究扩展到疾病机制。 据报道，胞质tRNA合成酶参与蛋白质信号传导，甚至表现为RNA结合 影响基因表达、翻译和自我调节的蛋白质，因此我们怀疑DARS 2作为一种 线粒体tRNA合成酶可能具有类似的非翻译功能。RNA和蛋白质的评价 通过交联免疫沉淀（CLIP-seq; Aim 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DARS 2相关的RNA和蛋白质伴侣 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物， 理解患者突变可能影响这些实验的技术方面。新功能数据 仅从健康患者样本中收集的数据就足以改变我们对细胞中mt-ARS的理解， 决定了mt-ARS疾病的治疗发展。由于LBSL病理学与其他合成酶不同 疾病，可能与疾病机制有关的替代功能，并确定这些功能 为干预提供了目标。患者的iPSC和iPSC衍生的运动神经元对于 了解内源性DARS 2行为和LBSL病理生理学。我们的总体目标是使用患者iPSC- 衍生神经元以了解LBSL疾病机制并利用这些信息指导治疗 发展

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.