Investigating the role of MAP2 in chemotherapy-induced peripheral neurotoxicity

项目摘要/摘要 化疗所致周围神经病(CIPN)是一种引起发病率的严重副作用 并限制了治疗癌症所允许的化疗剂量。在大多数类别的神经毒性 化疗后，CIPN表现为背根神经节感觉神经元损伤。这个 神经元损伤被认为是由于微管功能障碍的不明组合所致， 轴突运输和线粒体，最终导致程序性轴突变性 烟酰胺单核苷酸腺苷转移酶2(NMNA2)与无菌α和TIR基序 包含1(Sarm1)途径。然而，这些过程之间的联系仍然是完全的 未知。 用人诱导的多能干细胞来源的背根节感觉神经元 培养系统(ISN)，我们已经确定微管相关蛋白2(MAP2)是关键 CIPN的决定因素。当暴露于临床相关剂量的硼替佐米、紫杉醇或 长春新碱，MAP2水平下降，在ISN中存在亚细胞MAP2错误定位，发生在 轴突变性。关键是，map2的过度表达对硼替佐米诱导的损伤具有保护作用。 ISN的神经毒性。 在这项拨款申请中，我们建议进一步剖析MAP2在CIPN中的角色，利用一个 创新CRISPR-ErCas12a系统开发基因工程IPSC系 荧光标记MAP2或NMNA2。这些新颖的IPSC系列将允许详细分析 改变的亚细胞定位和轴突运输的时间进程，以及这些过程是如何联系的 与Sarm1激活和轴突变性有关。此外，我们将调查重叠 波特佐米、紫杉醇和长春新碱在CIPN中的作用机制 蛋白质组学分析方法，现在将采用最先进的定量蛋白质组学和 磷酸蛋白质组学技术。拟议的具体目标建立在对 CIPN的发病机制，同时创新到未知的新领域，这些领域是潜在的目标 预防性治疗的发展。

PROJECT SUMMARY/ABSTRACT Chemotherapy-induced peripheral neuropathy (CIPN) is a serious side effect that causes morbidity and limits the dose of chemotherapy allowed to treat cancers. In most classes of neurotoxic chemotherapeutics, CIPN manifests as damage to dorsal root ganglia sensory neurons. The neuronal damage is thought to be due to an unelucidated combination of dysfunctional microtubules, axonal transport and mitochondria that ultimately leads to programmed axonal degeneration via a nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) and Sterile Alpha and TIR Motif Containing 1 (SARM1) pathway. The link between these processes, however, remains entirely unknown. Using a human induced pluripotent stem cell (iPSC)-derived dorsal root ganglia sensory neuronal culture system (iSN), we have identified microtubule-associated protein 2 (MAP2) as a key determinant of CIPN. When exposed to clinically-relevant doses of bortezomib, paclitaxel or vincristine, MAP2 levels drop and there is subcellular MAP2 mislocalization in iSN that occurs prior to axonal degeneration. Critically, overexpression of MAP2 is protective for bortezomib-induced neurotoxicity in iSN. In this grant application, we propose to further dissect the role of MAP2 in CIPN by utilizing an innovative CRISPR-ErCas12a system to develop genetically-engineered iPSC lines that fluorescently-tag either MAP2 or NMNAT2. These novel iPSC lines will allow for detailed analyses of the time course of altered subcellular localization and axonal transport, and how these processes link to SARM1 activation and axonal degeneration. Furthermore, we will investigate the overlapping mechanisms in CIPN due to bortezomib, paclitaxel, and vincristine using our previously successful proteomics analysis approach, which now will employ a state-of-the-art quantitative proteomic and phosphoproteomic technology. The proposed Specific Aims build on existing understanding of the pathomechanisms of CIPN while innovating into unexplored novel areas that are potential targets for preventative therapeutic development.