Mechanisms of Neuronal Death and Neuroprotection

铂化合物在癌症治疗中的使用越来越多。顺铂、卡铂和 奥沙利铂可引起剂量相关和剂量受限的感觉神经病变。我们已经证明了 顺铂与神经元DNA结合，激活DMA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。我们现在建议检验这样一个假设：铂 化合物通过一种共同的机制产生神经元损伤，这种机制涉及分离的核(n-DNA)和 线粒体DNA(mt-DNA)结合后，平行死亡途径的协同激活。一种新的 通过抑制聚合酶链发展了一种测量mt-dna平台化的方法。 反应。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够 以防止线粒体基因组的复制和转录。呼吸链的功能 将测量组件。无法修复mt-DNA中的铂-DNA损伤将导致 线粒体和慢性神经元死亡解释临床上“滑行”或进展的现象 停药后的神经病。我们将使用Bax和Cyclin D1基因敲除小鼠的DRG神经元 确定铂引起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复制的数量独立地足以导致细胞死亡。 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。这个 谷氨酸半胱氨酸连接酶(GCL)和谷胱甘肽合成酶(GS)的修饰物和催化亚基将被 靶向于腺病毒载体中的线粒体以减少mt-DNA加合物的形成。两种神经生长 神经生长因子(NGF)和色素上皮源性生长因子(PEDF)已被证实部分 保护DRG免受顺铂诱导的细胞凋亡。我们将确定是否联合使用治疗药物 阻断n-DNA诱导的细胞凋亡和保护mt-DNA的策略促进顺铂的长期存活 体外处理背根神经节。如果联合策略有效，我们将在动物模型中测试它，方法是开发 方法在体内向DRG提供生长因子的长期传递和GCL和GS的病毒靶向。 我们的目标是开发一种基于机制的疗法，以防止 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.