Structure/function of Nerve Growth Factor/Neurotrophins

描述(申请人提供)：与神经生长因子(NGF)相关的神经营养因子家族与两种类型的受体(Trk酪氨酸激酶受体和常见的神经营养因子受体p75NTR)相互作用，支持反应灵敏的外周神经元(感觉、交感)和中枢神经元(胆碱能、多巴胺能)的神经元发育，并为神经退行性疾病中的神经元提供生存信号。这项建议旨在进一步开发神经营养因子突变体(‘突变体’)，旨在产生优先受体特异性。一个主要的假设是，受体和/或信号选择性神经营养因子突变体在某些疾病中将比野生型神经营养因子更有效的治疗试剂。受体选择性神经营养因子“突变体”正处于一个阶段，将在神经退行性疾病的动物模型中进行测试。该提案的具体目的是：(I)利用受体选择性神经营养素突变体异源二聚体来测试特定的受体激活模型。新的神经营养因子异二聚体的特征是确定与每个受体的结合亲和力、主要的细胞反应(存活、凋亡或分化)和关键信号通路中的酶；(Ii)通过p53肿瘤抑制蛋白/Capase-3依赖和非依赖的机制来表征细胞凋亡的信号通路。将对神经营养因子突变体进行筛选，以寻找那些区分P53依赖和非依赖通路与P53温度敏感的PC12细胞的突变体。一些神经细胞系和原代培养细胞也将用精选突变蛋白进行研究。(Iii)通过鼻腔吸入将信号选择性神经营养因子突变体输送到啮齿动物的中枢神经系统，以测试其治疗潜力。将使用正常小鼠、p53缺失小鼠、p75NTR缺失小鼠、正常大鼠和老龄大鼠。信号通路，包括TrkA磷酸化、MAP激酶和DNA片段化，将在治疗的大鼠和小鼠的大脑中进行免疫组织化学和生化比较。合理设计神经营养因子衍生的治疗试剂，选择性地刺激信号通路，可能会导致阿尔茨海默病、帕金森病和相关神经疾病的新疗法-反应选择性神经生长因子突变体在治疗神经退行性疾病方面最终可能比野生型神经生长因子更有效。

DESCRIPTION (provided by applicant): The family of neurotrophins, related to nerve growth factor (NGF), interact with two types of receptors, the Trk tyrosine kinase receptor and the common neurotrophin receptor p75NTR, to support neuronal development in responsive peripheral neurons (sensory, sympathetic) and central neurons (cholinergic, dopaminergic) and to provide signals for survival of neurons in neurodegenerative disorders. This proposal is aimed at further development of neurotrophin mutants ('muteins') designed to produce preferential receptor specificity. A major hypothesis being addressed is that receptor- and/or signal- selective neurotrophin mutants would be more effective therapeutic reagents in certain disorders than wild type neurotrophins. The receptor-selective neurotrophin 'muteins' are at a stage to be tested in animal models of neurodegenerative diseases. The Specific Aims of the proposal are: (i) To utilize receptor-selective neurotrophin mutein heterodimers to test specific receptor activation models. Novel neurotrophin heterodimers will be characterized to determine the binding affinity to each receptor, the main cellular responses (survival, apoptosis, or differentiation) and enzymes in key signaling pathways, (ii) To characterize the signaling pathway to apoptosis through p53 tumor suppressor protein/capase-3 -dependent and -independent mechanisms. Neurotrophin muteins will be screened for those that discriminate between p53 -dependent and -independent pathways with p53 temperature sensitive PC12 cells. Several neuronal cell lines and primary cultures will also be studied with select muteins. (iii) To test the therapeutic potential of signal- selective neurotrophin muteins by intranasal inhalation delivery to the central nervous system of rodents. Normal mice, p53 null mice, p75NTR null mice, normal rats, and aged rats will be used. Signaling pathways, including TrkA phosphorylation, MAP kinase, and DNA fragmentation, will be compared immunohistochemically and biochemically in the treated rat and mice brains. The rational design of therapeutic reagents derived from neurotrophins that selectively stimulate signaling pathways may lead to novel treatments for Alzheimer's Disease, Parkinson's Disease, and related neurological disorders - response-selective NGF mutants may ultimately be more effective than wild type NGF in treating neurodegenerative diseases.