INHIBITION OF APOPTOSIS IN NEURAL GRAFTS

帕金森病移植临床成功的关键 (PD)是开发移植DA神经元存活的方法， 和宿主纹状体的神经再支配显著增加。 的 DA神经元在移植到年轻大鼠的存活率为5- 10%， 在移植到老年纹状体的时候就更差了。 的重点 修订后的KO 1指导研究奖申请是为了追求以前的 候选人的工作表明，纹状体靶细胞可以 通过供应增加同时移植的DA神经元的存活 扩散性营养因子 本申请中提出的研究 试图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， 实验室中使用细胞培养技术，并将研究结果应用于 细胞培养到老年神经系统的治疗干预。 根据文献中的建议，很可能纹状体 少突胶质细胞2型-星形胶质细胞（0-2A）祖细胞是一种细胞 来源纹状体衍生的营养活性DA神经元。其中一个目标是 应用将是发展细胞培养技术的专业知识， 选择性富集纹状体0-2A的生长因子处理 细胞，用于研究它们在DA神经营养作用中的作用 神经元存活率和凋亡死亡率。 第二个目标是 拟议的培训将是发展神经生物学的经验， 衰老的动物，导师实验室的另一个重点。 最近 导师实验室的证据表明， DA移植物对长期损伤的老年大鼠的行为影响是 与年轻大鼠中的移植物相比大大减少。 从最初的培养实验中获得的信息将 应用于年轻和老年大鼠的移植研究，以 确定DA神经元是否与0-2A富集的纹状体共移植 细胞可以在老年人中提供上级的形态和行为结果， 科目 如果这些研究表明增强的DA神经元 存活与细胞凋亡的抑制有关，则直接 细胞凋亡抑制方法也将被研究。 因此 本培训提案的最终目标是获得以下方面的经验： 在细胞培养中利用病毒载体过表达bcl-2， 在DA神经元中鉴定出“存活”基因，并检查 这种转导在两种培养物和移植物中对细胞凋亡的影响 老年老鼠 本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 Federoff，罗切斯特大学医学院。 此外该 候选人将参观Federoff实验室学习病毒载体 转导技术。 这一提议的总体假设是 通过抑制移植DA神经元的凋亡细胞死亡， 间接通过提供营养因子和直接通过干扰 遗传死亡计划，将有可能增加DA移植 老年纹状体的活力和随后的神经再支配。这 培训将促进候选人的发展成为一个全面的 神经细胞培养、衰老、 DA系统、细胞凋亡和神经移植治疗PD。

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.