Determining stathmin-2 function and potential as a therapeutic target in ALS/FTD

确定 Stathmin-2 的功能和作为 ALS/FTD 治疗靶点的潜力

• 批准号: 10370327
• 负责人: Don W Cleveland
• 金额: $ 79.73万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10370327
• 关键词: Affect; Amyotrophic Lateral Sclerosis; Antisense Oligonucleotides; Axon; Axotomy; Binding; Binding Sites; C9ORF72; CRISPR screen; Cells; Cognition Disorders; Custom; Cytoplasmic Protein; DNA-Binding Proteins; Dementia; Denervation; Disease; Flow Cytometry; Frontotemporal Dementia; Genes; Genetic; Genetic Screening; Growth Cones; Guide RNA; Hereditary Disease; High-Throughput RNA Sequencing; Human; Induced pluripotent stem cell derived neurons; Inherited; Injections; Introns; Libraries; Link; Maintenance; Messenger RNA; Microtubule Stabilization; Microtubules; Motor Neuron Disease; Motor Neurons; Mus; Muscle; Mutation; Natural regeneration; Neurobiology; Neuromuscular Junction; Neurons; Nuclear; Optical Methods; Optics; Outcome; Paralysed; Pathogenesis; Pathology; Polyadenylation; Post-Translational Protein Processing; Proteins; RNA; RNA Sequences; RNA Splicing; Role; Site; Synapses; Therapeutic; Tubulin; Variant; axon growth; axon regeneration; base; beta Tubulin; dimer; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genome editing; genome-wide; induced pluripotent stem cell; insight; loss of function; mRNA Precursor; motor neuron function; nerve supply; neuron regeneration; neuronal cell body; palmitoylation; premature; prevent; protein TDP-43; repaired; restoration; small hairpin RNA; sporadic amyotrophic lateral sclerosis; stathmin; therapeutic target

Cytoplasmic protein accumulations of the RNA/DNA binding protein TDP-43 are found in affected neurons in almost all instances of amyotrophic lateral sclerosis (ALS) and approximately 50% of frontotemporal dementia (FTD). Nuclear clearance of TDP-43 has been widely observed in affected neurons in sporadic ALS/FTD, evidence strongly supporting the proposal that TDP-43 loss of function is a key aspect of disease mechanism underlying ALS/FTD pathogenesis. We have identified that the mRNA encoding stathmin-2 is 1) an essential factor for axonal regeneration of axotomized iPSC-derived motor neurons and 2) the mRNA most affected by reduction in TDP-43 function, with a striking loss from motor neurons in sporadic ALS and inherited disease from GGGGCC expansion in C9orf72. Stathmin-2 is an abundant, direct binding partner of α/β-tubulin dimers in neuronal perikarya, axons, growth cones, and synapses, including neuromuscular junctions (NMJs). Using genome editing, we will identify the mechanism of TDP-43-dependent premature polyadenylation/cryptic splicing that suppresses stathmin-2 synthesis when TDP-43 levels are lowered. We will use genome editing of induced pluripotent stem cells (iPSCs) derived human motor neurons grown in compartmented chambers (that separate neuronal cell bodies, axons and growth cones) to determine how stathmin-2 functions in a) motor neuron maintenance/repair, b) axonal microtubule stabilization and/or dynamics, c) neuromuscular junction formation and/or stabilization, and d) how palmitoylation of stathmin-2 affects its axonal function(s). Genome wide CRISPR/Cas9 screens using flow cytometry and optical methods will be undertaken to identify factors that control stathmin-2 synthesis or accumulation. Finally, we will determine the consequences in mice of reduction or loss of stathmin-2 on motor neuron function and muscle innervation/denervation and whether reduction in stathmin-2 synergizes with TDP-43 mutation to drive motor neuron disease. Outcomes of these efforts will provide key insights for understanding basic aspects of axonal and synaptic neurobiology and for evaluating whether maintaining or restoring stathmin-2 is an attractive therapeutic option in sporadic ALS/FTD and ALS from its most frequent genetic cause, repeat expansion in C9orf72.

在受影响的神经元中发现 RNA/DNA 结合蛋白 TDP-43 的细胞质蛋白积累 几乎所有肌萎缩侧索硬化症 (ALS) 病例和大约 50% 的额颞叶硬化症病例 痴呆 (FTD) 中 TDP-43 的核清除已在散发的受影响神经元中广泛观察到。 ALS/FTD，强有力的证据支持 TDP-43 功能丧失是疾病的一个关键方面的提议 ALS/FTD 发病机制的潜在机制 我们已确定编码 stathmin-2 的 mRNA 是 1) 轴突 iPSC 衍生运动神经元轴突再生的重要因素，2) mRNA 最重要 受TDP-43功能减少的影响，散发性ALS和遗传性ALS中运动神经元显着丧失 C9orf72 中 GGGGCC 扩增引起的疾病，Stathmin-2 是 α/β-微管蛋白的丰富的直接结合伴侣。 神经元核周、轴突、生长锥和突触（包括神经肌肉接头 (NMJ)）中的二聚体。 使用基因组编辑，我们将确定 TDP-43 依赖性过早多聚腺苷酸化/神秘的机制 当 TDP-43 水平降低时，我们将使用基因组编辑来抑制 Stathmin-2 合成。 诱导多能干细胞 (iPSC) 衍生的人类运动神经元在隔室中生长（即 分离神经元细胞体、轴突和生长锥）以确定 stathmin-2 在 a) 运动中的功能 神经元维护/修复，b) 轴突微管稳定和/或动力学，c) 神经肌肉接头 形成和/或稳定，以及 d) stathmin-2 的棕榈酰化如何影响其轴突功能。 将使用流式细胞术和光学方法进行广泛的 CRISPR/Cas9 筛选，以确定影响 控制stathmin-2的合成或积累最后，我们将确定减少对小鼠的影响。 或 stathmin-2 的缺失对运动神经元功能和肌肉神经支配/去神经支配的影响，以及是否减少 stathmin-2 与 TDP-43 突变协同作用，驱动运动神经元疾病。 为理解轴突和突触神经生物学的基本方面以及评估提供关键见解 维持或恢复 Stathmin-2 是否是散发性 ALS/FTD 中有吸引力的治疗选择 ALS 的最常见遗传原因是 C9orf72 中的重复扩增。