Using patient-derived stem cells and drug screening to unravel the mechanisms of astrocyte-induced DNA damage as a pathway for motor neurone death in

背景：肌萎缩侧索硬化症的特征是运动神经元变性，导致致命的肌肉无力和萎缩。几项体外和体内研究已经证明，星形胶质细胞积极促进ALS中的运动神经元损失（https：//www.ncbi.nlm.nih.gov/pubmed/21908873）。许多研究表明，这种非细胞自主机制涉及星形胶质细胞分泌的可溶性因子，因为暴露于来自ALS星形胶质细胞的生长培养基足以触发运动神经元死亡（https：//www.ncbi.nlm.nih.gov/pubmed/21832997）。虽然许多研究集中在揭示星形胶质细胞毒性的身份，一个中心问题仍然存在：运动神经元中发生的生物学机制导致其死亡？最近的前沿研究表明，DNA损伤在ALS的运动神经元死亡中起关键作用（https：//www.ncbi.nlm.nih.gov/pubmed/28714954）。因此，我们的假设是ALS星形胶质细胞可以通过触发DNA损伤诱导神经退行性变。我们的目标是：1 -系统地确定来自携带各种疾病相关基因突变以及散发性的ALS患者的人星形胶质细胞是否可以诱导健康和ALS人运动神经元中的DNA损伤（每个患者组和健康个体n=3）。学生将学习尖端技术，如生长和分化干细胞以及运行蛋白质印迹，免疫染色和定量PCR，以确定分化细胞中DNA损伤的存在。2 -通过使用基因治疗工具操纵星形胶质细胞或运动神经元来确定参与该机制的途径。我们将使用病毒载体抑制或过表达特定的候选物，以评估它们在两种细胞类型中的DNA损伤中的作用。3 -研究来自我们的工业合作伙伴阿斯利康的工具化合物，这些化合物可以调节目标2中确定的DNA损伤途径。学生将有机会在剑桥的阿斯利康学习3个月，学习如何设计，建立和分析药物筛选试验。翻译含义：考虑到ALS星形胶质细胞通过几种机制促进MN死亡（https：//www.ncbi.nlm.nih.gov/pubmed/25233402），理解DNA损伤是否是该过程中的中心途径至关重要。翻译的影响是直接的，因为几个批准的化合物靶向这一途径是可用的。证明增强MN中的DNA损伤反应是一种主要的防御机制对于未来的治疗方法至关重要。

Background: ALS is characterised by degeneration of motor neurons causing fatal muscle weakness and atrophy. Several in vitro and in vivo studies have demonstrated that astrocytes actively contribute to motor neuron loss in ALS (https://www.ncbi.nlm.nih.gov/pubmed/21908873). A number of studies have shown that this non-cell-autonomous mechanism involves soluble factors secreted by astrocytes as the exposure to growth medium from ALS astrocytes is sufficient to trigger motor neuron death (https://www.ncbi.nlm.nih.gov/pubmed/21832997). Although much research focuses on revealing the identity of astrocyte toxicity, a central question remains: What are the biological mechanisms taking place in motor neurons leading to their death? Recent cutting-edge research has shown that DNA damage plays a key role in motor neuron death in ALS (https://www.ncbi.nlm.nih.gov/pubmed/28714954). Therefore our hypothesis is that ALS astrocytes can induce neurodegeneration by triggering DNA damage. We aim to: 1 - Systematically determine whether human astrocytes from ALS patients carrying various disease-linked genetic mutations as well as sporadic can induce DNA damage in healthy and ALS human motor neurons (n=3 per patient group and healthy individuals). The student will learn cutting edge techniques such growing and differentiating stem cells as well as running Western blots, immunostaining and quantitative PCR to determine the presence of DNA damage in differentiated cells. 2 - Identify the pathways involved in this mechanism by manipulating the astrocytes or the motor neurones using gene therapy tools. We will suppress or overexpress specific candidates to assess their role in DNA damage in both cells types using viral vectors. 3 - Study a selection of tool compounds from AstraZeneca, our industrial partner, which can modulate the pathways involved in DNA damage identified in Aim 2. The student will have the opportunity to spend 3 months at AstraZeneca in Cambridge to learn how to design, set up and analyse drug screening assays. Translational implications: Considering that ALS astrocytes contribute to MN death through several mechanisms (https://www.ncbi.nlm.nih.gov/pubmed/25233402), understanding whether DNA damage is a central pathway in this process is of key importance. The translational implications are immediate, as several approved compounds targeting this pathway are available. Proving that enhancing DNA damage response in MNs is a primary defence mechanism is crucial for future therapeutic approaches.