Investigating the role of MAP2 in chemotherapy-induced peripheral neurotoxicity

项目总结/摘要 化疗引起的周围神经病变（CIPN）是一种严重的副作用，导致发病率 并限制了治疗癌症的化疗剂量。在大多数种类的神经毒性 在化疗药物中，CIPN表现为对背根神经节感觉神经元的损伤。的 神经元损伤被认为是由于未阐明的功能失调的微管的组合， 轴突运输和线粒体，最终导致程序性轴突变性，通过 烟酰胺单核苷酸腺苷酰转移酶2（NMNAT 2）和无菌α和TIR基序 含1（SARM 1）通路。然而，这些过程之间的联系仍然完全 未知 使用人诱导多能干细胞（iPSC）衍生的背根神经节感觉神经元， 在iSN细胞培养系统中，我们鉴定出微管相关蛋白2（MAP 2）是一个关键的 CIPN的行列式当暴露于临床相关剂量的硼替佐米、紫杉醇或 长春新碱，MAP 2水平下降，iSN中存在亚细胞MAP 2错误定位，发生在 轴突变性重要的是，MAP 2的过表达对硼替佐米诱导的肿瘤细胞凋亡具有保护作用。 iSN的神经毒性。 在这项资助申请中，我们建议通过利用一个新的研究方法，进一步剖析MAP 2在CIPN中的作用。 创新的CRISPR-ErCas 12 a系统开发基因工程iPSC系， 荧光标记MAP 2或NMNAT 2。这些新的iPSC系将允许详细分析 改变亚细胞定位和轴突运输的时间过程，以及这些过程如何联系 SARM 1激活和轴突变性。此外，我们将研究重叠 CIPN中硼替佐米、紫杉醇和长春新碱的作用机制， 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 磷酸化蛋白质组学技术拟议的具体目标是建立在现有的理解， CIPN的病理机制，同时创新到未探索的新领域，这些领域是CIPN的潜在目标。 预防性治疗发展。

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.