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

在受影响的神经元中发现RNA/DNA结合蛋白TDP-43的胞浆蛋白积聚 几乎所有的肌萎缩侧索硬化症(ALS)和大约50%的额颞部 痴呆症(FTD)。TDP-43的核清除在散发性神经元中被广泛观察到。 ALS/FTD，强有力的证据支持TDP-43功能丧失是疾病的关键方面的提议 ALS/FTD发病机制。我们已经鉴定出编码stathmin-2的mRNA1)和 轴突切断后IPSC来源运动神经元轴突再生的必备因子和2)最重要的基因 受TDP-43功能降低的影响，散发性ALS和遗传性ALS的运动神经元显著丧失 C9orf72的GGGGGCC扩张性疾病。Stathmin-2是α/β-微管蛋白丰富的直接结合伙伴 神经细胞膜、轴突、生长锥体和突触中的二聚体，包括神经肌肉接头(NMJ)。 利用基因组编辑，我们将确定依赖TDP-43的过早多聚腺苷/隐含的机制 当TDP-43水平降低时，抑制stathmin-2合成的剪接。我们将使用基因组编辑 诱导多能干细胞(IPSCs)来源的人类运动神经元生长在隔室(即 分离神经元胞体、轴突和生长锥)，以确定stathmin-2在)运动中如何发挥作用 神经元维持/修复，b)轴突微管稳定和/或动力学，c)神经肌肉连接 以及d)stathmin-2的棕榈酰化如何影响其轴突功能(S)。基因组 将使用流式细胞术和光学方法进行CRISPR/CAS9宽屏筛查，以确定 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。 或者Stathmin-2对运动神经元功能和肌肉神经支配/去神经的影响 Stathmin-2与TDP-43突变协同作用，驱动运动神经元疾病。这些努力的结果将是 为理解轴突和突触神经生物学的基本方面以及评估 维持或恢复stathmin-2是治疗散发性ALS/FTD的有吸引力的选择 ALS来自其最常见的遗传原因，在C9orf72中重复扩张。

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.