GSK3b mediates radiation-induced cytotoxicity in hippocampal neurons

描述(申请人提供)：治疗原发和转移性脑癌，尤其是儿童脑转移癌，面临的一个巨大挑战是颅脑照射(IR)诱导海马神经细胞凋亡所导致的长期神经认知缺陷。我们的实验室发现了代谢激酶GSK3与修复DNA双链断裂(DSB)的非同源末端连接(NHEJ)途径之间的新的功能联系。此外，我们的初步数据显示，NHEJ介体53BP1直接被GSK3β磷酸化；同时，经典的GSK3底物β-catenin的表达增加与IR诱导的DSB的修复和海马神经元的存活有关。因此，我们假设GSK3？调节NHEJ介导的DSB修复，并通过抑制53BP1和？-catenin功能来确定IR后神经元的细胞毒性。此外，含有异常GSK3活性的肿瘤细胞不会表现出GSK3介导的保护作用，使其免受IR诱导的细胞毒作用。我们提出了一系列的体外和体内实验来验证我们的假设：目的1将确定53BP1中的GSK3？磷酸化位点，并确定GSK3？特异的磷酸化是否直接在NHEJ和照射后的海马神经元中发挥作用。目的2将确定GSK3是通过抑制可能通过增加53BP1转录来促进NHEJ活性的连环蛋白，还是通过直接与53BP1相互作用来调节53BP1。目的3将确定GSK3活性异常是否决定了脑瘤细胞对预防性GSK3抑制介导的辐射细胞毒性保护的抵抗力。 公共卫生相关性：为了努力降低神经毒性和提高患者的生活质量，人们正在严格研究具有神经保护作用的药理药物。因此，本研究的目的是确定保护神经元免受辐射诱导的海马神经元凋亡的作用机制，以期预防颅脑IR的神经认知毒性。

DESCRIPTION (provided by applicant): A formidable challenge in the treatment of primary and metastatic brain cancers, especially in children, is the long-term neurocognitive deficiencies resulting from cranial irradiation (IR)-induced hippocampal neuronal apoptosis. Our laboratory has discovered a novel functional connection between the metabolic kinase GSK3¿ and the Non- homologous End-joining (NHEJ) pathway that repair DNA double-strand breaks (DSBs). Furthermore, our preliminary data revealed that the NHEJ mediator 53BP1 is directly phosphorylated by GSK3¿; meanwhile, increased expression of the classic GSK3¿ substrate ¿-catenin is associated with enhanced repair of IR-induced DSBs and survival in hippocampal neurons. Thus, we hypothesize that GSK3¿ regulates NHEJ-mediated repair of DSBs and determines neuron cytotoxicity following IR via suppression of 53BP1 and ¿-catenin function. In addition, tumor cells which contain abnormal GSK3 ¿ activity will not exhibit GSK3¿-mediated protection from IR-induced cytotoxicity. A series of in vitro and in vivo experiments are proposed to test our hypotheses: Aim 1 will identify the GSK3 ¿ phosphorylation sites in 53BP1 and determine whether GSK3 ¿ -specific phosphorylation direct 53BP1 function in NHEJ and in survival of irradiated hippocampal neurons. Aim 2 will determine whether GSK3 ¿ regulates 53BP1 through suppressing ¿ - catenin that may promote NHEJ activity by increasing 53BP1 transcription, or by directly interacts with 53BP1. Aim 3 will determine if abnormal GSK3 ¿ activity determine brain tumor cell resistance to the prophylactic GSK3 ¿ -inhibition mediated protection from radiation induced cytotoxicity. PUBLIC HEALTH RELEVANCE: In an effort to decrease neurotoxicity and improve patient quality of life, pharmacologic agents which exhibit neuroprotective effects are being rigorously investigated. Thus, the goal of this proposal is to determine the mechanisms of action that protect neurons from irradiation-induced hippocampal neuronal apoptosis with a view toward prevention of neurocognitive toxicity from cranial IR.