Mechanisms of Neuronal Death and Neuroprotection

描述（由申请人提供）：铂化合物在癌症治疗中的使用越来越多。顺铂，卡铂和奥沙利铂产生剂量相关和剂量限制的感觉神经病变。我们已经证明顺铂与神经元DNA结合，激活DNA损伤识别途径，启动异常细胞周期进入，并诱导体外和体内细胞凋亡。我们现在提议测试铂化合物通过一种共同机制产生神经元损伤的假设，该机制涉及分离的核（n-DNA）和线粒体DNA （mt-DNA）结合，然后协同激活平行死亡途径。一种新的方法来测量mt-DNA铂化已开发利用抑制聚合酶链反应。我们将确定DRG神经元mt-DNA中铂加合物的数量是否足以阻止线粒体基因组的复制和转录。测量呼吸链各成分的功能。不能修复mt-DNA中的Pt-DNA损伤会导致线粒体的磨损和慢性神经元死亡，这解释了停药后神经病变“滑行”或进展的临床现象。我们将使用Bax和cyclin D1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合成抑制剂二脱氧胞苷（ddC）来确定线粒体DNA复制的抑制是否足以独立导致细胞死亡。选择性地增加线粒体谷胱甘肽可以保护线粒体基因组。谷氨酸半胱氨酸连接酶（GCL）和谷胱甘肽合成酶（GS）的修饰剂和催化亚基将在腺病毒载体中靶向线粒体，以减少mt-DNA加合物的形成。神经生长因子（NGF）和色素上皮衍生生长因子（PEDF）已被证明部分保护DRG免受顺铂诱导的细胞凋亡。我们将在体外确定阻断n-DNA诱导的细胞凋亡和保护mt-DNA的治疗策略是否能促进顺铂治疗DRG的长期生存。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，以提供生长因子的长期递送和GCL和GS对DRG的病毒靶向。目标是开发一种基于机制的治疗方法，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.