INHIBITION OF APOPTOSIS IN NEURAL GRAFTS

帕金森病移植治疗临床成功的关键 (PD)是移植DA神经元存活的方法的发展 宿主纹状体的再神经支配明显增加。这个 幼年大鼠移植的DA神经元存活率为5-10%， 在移植到老年纹状体上就更差了。这件事的重点是 修订后的KO1指导研究奖申请是为了继续之前的 候选人的研究表明纹状体靶细胞可以 通过补充营养物质提高同时移植DA神经元的存活 可扩散的营养因子。本申请中提出的研究 寻求定义一些增强的细胞和分子介质 DA神经元存活，通过与导师的密切合作 使用细胞培养技术的实验室，并将研究结果应用于 细胞培养对老年神经系统的治疗干预作用。 根据文献中的建议，纹状体很可能 少突胶质细胞2型-星形胶质细胞(0-2A)祖细胞是一个细胞 纹状体来源的营养活性DA神经元。其中一个目标就是 应用将是发展细胞培养技术和专业知识 培养中的生长因子处理选择性地丰富纹状体0-2A 研究细胞在多巴胺神经营养效应中的作用 神经元存活率和细胞凋亡率。第二个目标是 拟议的培训将是发展神经生物学方面的经验 老化的动物，这是导师实验室的另一个重点。近期 导师实验室中的证据表明，形态和 多巴胺移植物对老年大鼠长期损伤后行为的影响 与幼年大鼠的移植物相比大大减少。 从最初的一组培养实验中学到的信息将是 应用于幼年和老年大鼠的移植研究，以便 确定DA神经元与0-2A共移植是否富含纹状体 细胞可以为老年人提供更好的形态和行为结果 研究对象。这些研究是否表明增强的DA神经元 生存与抑制细胞凋亡有关，然后直接 还将研究抑制细胞凋亡的方法。因此， 该培训计划的最终目标将是获得以下方面的经验 利用病毒载体在细胞培养中过表达bc1-2和 鉴定出DA神经元中的“存活”基因，并检测其对 在两种培养物和幼体移植物中对细胞凋亡的转导 和衰老的老鼠。这项研究的组织培养方面将在 导师的实验室和病毒载体将由霍华德博士提供 费德洛夫大学罗切斯特医学院。此外， 候选人将参观费德洛夫实验室了解病毒载体 转导技术。这项提议的总体假设是 通过抑制移植的DA神经元的凋亡细胞死亡，两者 通过提供营养因子间接和直接通过干扰 基因死亡计划，将有可能增加DA移植 老年纹状体的存活和随后的再神经支配。这 培训将促进应聘者全面发展 神经细胞培养领域的独立研究员，老龄化 帕金森病的DA系统、细胞凋亡和神经移植。

Critical to clinical success of transplantation for Parkinson's disease (PD) is the development of methods whereby grafted DA neuron viability and reinnervation of the host striatum are markedly increased. The survival rate of DA neurons in grafts to young rats is 5-10 percent, and is even poorer in grafts to the aged striatum. The focus of this revised KO1 Mentored Research Award application is to pursue previous work by the candidate which demonstrated that striatal target cells can increase the survival of simultaneously grafted DA neurons by supplying diffusible trophic factors. The studies proposed in this application seek to define some of the cellular and molecular mediators of enhanced DA neuron survival, through close collaboration with the mentor's laboratory in the use of cell culture techniques, and apply findings in cell culture to therapeutic intervention in the aged nervous system. Based on suggestions in the literature, it is likely that the striatal oligodendrocyte type 2-astrocyte (0-2A) progenitor cell is one cellular source of striatal-derived trophic activity DA neurons. One goal of this application will be to develop expertise in cell culture techniques and growth factor treatments in culture to selectively enrich striatal 0-2A cells for investigation of their role in neurotrophic effects on DA neuron survival and apoptotic death rates. A second goal of the proposed training will be to develop experience in the neurobiology of aging animals, another focus of the mentor's laboratory. Recent evidence in the mentor's laboratory indicates that morphological and behavioral effects of DA grafts in aged rats with long-term lesions is greatly diminished as compared to grafts in young rats. Information learned from the initial set of culture experiments will be applied to transplantation studies in young and aged rats, in order to determine whether DA neurons co-grafted with 0-2A enriched striatal cells can provide superior morphological and behavioral outcomes in aged subjects. Should these studies indicate that enhanced DA neuron survival is related to the inhibition of apoptosis, then a direct apoptosis inhibition approach also will be studied. Therefore, the final goal of this training proposal will be to gain experience in utilizing viral vectors in cell culture to overexpress bcl-2, an identified "survival" gene, in DA neurons and to examine the effect of this transduction on apoptosis in both cultures and in grafts to young and aged rats. Tissue culture aspects of this study will be learned in the mentor's laboratory, and viral vector will-be provided by Dr. Howard Federoff, Univ. of Rochester School of Medicine. In addition, the candidate will visit the Federoff laboratory learn viral vector transduction techniques. The overall hypothesis of this proposal is that by inhibiting apoptotic cell death of grafted DA neurons, both indirectly by supplying trophic factors and directly by interfering with the genetic death program, it will be possible to increase DA graft viability and subsequent reinnervation of the aged striatum. This training will promote the candidate's development into a fully independent investigator in the fields of neural cell culture, aging in the DA system, apoptosis and neural grafting for PD.