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

RNA/DNA结合蛋白TDP-43的细胞质蛋白积聚在受影响的神经元中， 几乎所有肌萎缩侧索硬化症（ALS）病例和约50%的额颞叶 痴呆（FTD）。TDP-43的核清除已经在散发性脑缺血中受影响的神经元中广泛观察到。 ALS/FTD，有力支持TDP-43功能丧失是疾病关键方面的证据 ALS/FTD发病机制。我们已经鉴定了编码stathmin-2的mRNA是1）一种 轴突切断的iPSC衍生的运动神经元的轴突再生的必要因子和2）mRNA最 受TDP-43功能降低的影响，在散发性ALS和遗传性ALS中运动神经元显著丧失， 来自C9 orf 72中GGGGCC扩增的疾病。Stathmin-2是α/β-微管蛋白的丰富的直接结合伴侣 神经元胞体、轴突、生长锥和突触中的二聚体，包括神经肌肉接头（NMJ）。 使用基因组编辑，我们将确定TDP-43依赖的过早多聚腺苷酸化/隐藏的机制。 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技术 诱导多能干细胞（iPSC）衍生的人运动神经元在分隔室（compartmentedchamber）中生长（其 分离的神经元细胞体、轴突和生长锥），以确定stathmin-2如何在a）运动 神经元维持/修复，B）轴突微管稳定和/或动力学，c）神经肌肉接头 形成和/或稳定化，和d）stathmin-2的棕榈酰化如何影响其轴突功能。基因组 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 控制stathmin-2的合成或积累。最后，我们将在小鼠中确定减少 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 stathmin-2与TDP-43突变协同作用以驱动运动神经元疾病。这些努力的结果将 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是否是散发性ALS/FTD的有吸引力的治疗选择， ALS最常见的遗传原因是C9 orf 72的重复扩增。

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.