Structure/function of Nerve Growth Factor/Neurotrophins

描述（由申请人提供）：神经营养因子家族，与神经生长因子（NGF）相关，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，以支持响应性周围神经元（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存活提供信号。该建议旨在进一步开发神经营养蛋白突变体（'muteins'），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比野生型神经营养因子更有效。受体选择性神经营养蛋白“突变蛋白”正处于神经退行性疾病动物模型测试的阶段。该提案的具体目的是：(i)利用受体选择性神经营养蛋白异源二聚体来测试特定受体激活模型。新型神经营养素异二聚体将被表征，以确定与每种受体的结合亲和力，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依赖和独立的机制表征细胞凋亡的信号通路。神经营养蛋白将筛选那些区分p53依赖和不依赖途径与p53温度敏感的PC12细胞。几种神经细胞系和原代培养物也将用选择的突变蛋白进行研究。（iii）检测信号选择性神经营养蛋白经鼻吸入给药到啮齿动物中枢神经系统的治疗潜力。使用正常小鼠、p53缺失小鼠、p75NTR缺失小鼠、正常大鼠和老年大鼠。信号通路，包括TrkA磷酸化、MAP激酶和DNA片段化，将在治疗大鼠和小鼠的大脑中进行免疫组织化学和生化比较。合理设计由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系统疾病的新疗法——反应选择性NGF突变体最终可能比野生型NGF更有效地治疗神经退行性疾病。

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.