Mechanisms of Neuronal Death and Neuroprotection

铂化合物在癌症治疗中的使用正在增加。顺铂、卡铂和 奥沙利铂产生剂量相关和剂量限制性感觉神经病变。我们已经证明 顺铂与神经元DNA结合，激活DMA损伤识别途径，启动异常细胞周期 进入，并在体外和体内诱导凋亡。我们现在提出检验铂 化合物通过一种共同的机制产生神经元损伤，该机制涉及分离的核（n-DNA）和 线粒体DNA（mt-DNA）结合，随后协同激活平行死亡途径。一个新 利用聚合酶链的抑制作用，发展了一种测量线粒体DNA铂化的方法 反应我们将确定背根神经节神经元线粒体DNA中铂加合物的数量是否足够 以阻止线粒体基因组的复制和转录。呼吸链功能 将对组件进行测量。不能修复线粒体DNA中的Pt-DNA损伤将导致线粒体DNA的磨损。 线粒体和慢性神经元死亡解释了“滑行”或进展的临床现象， 停药后神经病变。我们将使用Bax和细胞周期蛋白D1敲除小鼠的DRG神经元， 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）来确定是否抑制 线粒体DNA复制的独立性足以导致细胞死亡。 线粒体基因组将通过选择性增加线粒体谷胱甘肽来保护。的 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被 靶向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 已经证明，神经生长因子（NGF）和色素上皮衍生生长因子（PEDF）部分 保护DRG免受顺铂诱导的凋亡。我们将确定是否联合治疗 阻断n-DNA诱导的细胞凋亡和保护线粒体DNA的策略促进顺铂的长期存活 体外处理DRG。如果组合策略有效，我们将通过开发 提供生长因子的长期递送以及GCL和GS体内病毒靶向DRG的方法。 目的是开发一种基于机制的治疗方法，以防止主要的剂量限制性副作用， 铂化合物。

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 DMA-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 ofglutamate cysteine ligase (GCL) andglutathione synthetase (GS) willbe 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.