Mechanisms of Neuronal Death and Neuroprotection

描述(申请人提供)：铂化合物在癌症治疗中的使用正在增加。顺铂、卡铂和奥沙利铂会产生剂量相关和剂量受限的感觉神经病。我们已经证明顺铂与神经元DNA结合，激活DNA损伤识别通路，启动异常细胞周期进入，并在体外和体内诱导细胞凋亡。我们现在建议验证一个假设，即铂化合物通过一个共同的机制产生神经元损伤，该机制涉及单独的核(n-DNA)和线粒体DNA(mt-DNA)结合，然后协同激活平行的死亡途径。通过抑制聚合酶链式反应，发展了一种测量mt-DNA平台化的新方法。我们将确定背根神经节神经元mt-DNA中的铂加合物数量是否足以阻止线粒体基因组的复制和转录。将测量呼吸链组件的功能。无法修复mt-DNA中的铂-DNA损伤会导致线粒体磨损和慢性神经元死亡，这解释了停药后神经病变的临床现象或进展。我们将使用Bax和Cyclin D1基因敲除小鼠的DRG神经元来确定铂诱导的线粒体功能抑制是否足以导致神经元死亡。我们将使用线粒体DNA合成抑制剂双脱氧胞苷(DdC)来确定抑制线粒体DNA复制是否独立足以导致细胞死亡。线粒体基因组将通过选择性地增加线粒体谷胱甘肽来保护。谷氨酸半胱氨酸连接酶(GCL)和谷胱甘肽合成酶(GS)的修饰和催化亚基将靶向于腺病毒载体中的线粒体，以减少mt-DNA加合物的形成。神经生长因子(NGF)和色素上皮源性生长因子(PEDF)对顺铂诱导的DRG细胞凋亡有一定的保护作用。我们将确定阻断n-DNA诱导的细胞凋亡和保护mt-DNA的联合治疗策略是否能在体外促进顺铂治疗的DRG的长期存活。如果联合策略有效，我们将通过开发方法在动物模型中进行测试，以提供体内生长因子的长期输送和GCL和GS的病毒靶向DRG。我们的目标是开发一种基于机制的疗法，以防止铂化合物的主要剂量限制副作用。

DESCRIPTION (provided by applicant): The use of platinum compounds is increasing in the treatment of cancer. Cisplatin, carboplatin and oxaliplatin produce a dose-related and dose-limiting sensory neuropathy. We have demonstrated that cisplatin binds to neuronal DNA, activates DNA-damage recognition pathways, initiates aberrant cell cycle entry, and induces apoptosis in vitro and in vivo. We now propose to test the hypothesis that platinum compounds produce neuronal injury by a common mechanism that involves separate nuclear (n-DNA) and mitochondrial DNA (mt-DNA) binding followed by synergistic activation of parallel death pathways. A new approach to measuring mt-DNA platination has been developed using inhibition of the polymerase chain reaction. We will determine whether the number of platinum adducts in mt-DNA of DRG neurons is sufficient to prevent replication and transcription of the mitochondrial genome. Function of respiratory chain components will be measured. Inability to repair Pt-DNA lesions in mt-DNA would result in attrition of mitochondria and chronic neuronal death explaining the clinical phenomenon of "coasting" or progression of neuropathy after drug cessation. We will use DRG neurons from Bax and cyclin D1 knockout mice to determine whether platinum-induced inhibition of mitochondrial function is sufficient to cause neuronal death. We will use the mitochondrial DNA synthesis inhibitor dideoxycytidine (ddC) to determine whether inhibition of mitochondrial DNA replication is independently sufficient to cause cell death. The mitochondrial genome will be protected by selectively increasing mitochondrial glutathione. The modifier and catalytic subunits of glutamate cysteine ligase (GCL) and glutathione synthetase (GS) will be targeted to mitochondria in an adeno-viral vector to reduce formation of mt-DNA adducts. Both nerve growth factor (NGF) and pigment epithelium derived growth factor (PEDF) have been demonstrated to partially protect DRG from cisplatin-induced apoptosis. We will determine whether a combination of therapeutic strategies that block n-DNA induced apoptosis and protect mt-DNA promote long-term survival of cisplatin treated DRG in vitro. If the combination strategy is effective, we will test it in an animal model by developing methods to provide long-term delivery of growth factors and viral targeting of GCL and GS to DRG in vivo. The goal is to develop a mechanism-based therapy that will prevent the major dose-limiting side effect of the platinum compounds.