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。

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.