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

基本信息

批准号：
10835733
负责人：
Don W Cleveland
金额：
$ 85.38万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
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/10835733
关键词：
Affect Amyotrophic Lateral Sclerosis Antisense Oligonucleotides Axon Axotomy Binding Binding Sites C9ORF72 CRISPR screen Cells Coculture Techniques 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 Poly A Polyadenylation Post-Translational Protein Processing Proteins RNA RNA Sequences RNA Splicing Role Site Synapses Therapeutic Tubulin Variant axon regeneration beta Tubulin dimer frontotemporal lobar dementia amyotrophic lateral sclerosis functional restoration 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 synergism therapeutic target

项目摘要

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.

RNA/DNA结合蛋白TDP-43的细胞质蛋白积累在受影响的神经元中发现几乎所有肌萎缩性侧索硬化症（ALS）的实例和额颞的大约50％痴呆（FTD）。 TDP-43的核清除率在偶发的神经元中广泛观察到 ALS/FTD，有证据支持以下提议，即TDP-43功能丧失是疾病的关键方面 ALS/FTD发病机理的基础机制。我们已经确定编码Stathmin-2的mRNA为1）轴突化IPSC衍生运动神经元的轴突再生的重要因素，2）mRNA最大受TDP-43功能的降低影响，零星ALS中的运动神经元的打击损失并继承 C9orf72中GGGGCC扩张的疾病。 Stathmin-2是α/β-微管蛋白的丰富，直接结合的伴侣神经元周围，轴突，生长锥和突触的二聚体，包括神经肌肉连接（NMJS）。使用基因组编辑，我们将确定TDP-43依赖性早产/隐性的机制当TDP-43水平降低时，可以抑制Stathmin-2合成的剪接。我们将使用基因组编辑诱导的多能干细胞（IPSC）衍生出在隔室中生长的人类运动神经元（该神经元（该神经元）单独的神经元细胞体，轴突和生长锥），以确定Stathmin-2在A）运动中的功能神经元维护/维修，b）轴突微管稳定和/或动力学，c）神经肌肉连接形成和/或稳定，以及d）Stathmin-2的棕榈酰化如何影响其轴突功能。基因组将采用流式细胞仪和光学方法进行广泛的CRISPR/CAS9筛选，以确定因素控制Stathmin-2合成或积累。最后，我们将确定减少小鼠的后果或在运动神经元功能和肌肉神经神经支配/神经支配上失去Stathmin-2，以及是否减少 Stathmin-2与TDP-43突变协同驱动运动神经元疾病。这些努力的结果将提供关键见解，以理解轴突和突触神经生物学的基本方面并评估在零星ALS/FTD中，维护或恢复Stathmin-2是有吸引力的治疗选择 ALS来自最常见的遗传原因，在C9orf72中重复扩张。