Structure/function of Nerve Growth Factor/Neurotrophins

描述（由申请人提供）：与神经生长因子（NGF）相关的神经营养蛋白家族与两种类型的受体（Trk酪氨酸激酶受体和常见神经营养蛋白受体p75 NTR）相互作用，以支持反应性外周神经元（感觉、交感神经）和中枢神经元（胆碱能、多巴胺能）中的神经元发育，并为神经退行性疾病中的神经元存活提供信号。该提议旨在进一步开发设计用于产生优先受体特异性的神经营养因子突变体（“突变蛋白”）。正在解决的一个主要假设是，受体和/或信号选择性神经营养蛋白突变体在某些疾病中将是比野生型神经营养蛋白更有效的治疗试剂。受体选择性神经营养素“突变蛋白”正处于神经退行性疾病动物模型的测试阶段。该提案的具体目的是：（i）利用受体选择性神经营养素突变蛋白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将被表征以确定对每个受体的结合亲和力、主要的细胞应答（存活、凋亡或分化）和关键信号传导途径中的酶。（ii）表征通过p53肿瘤抑制蛋白/半胱天冬酶-3依赖性和非依赖性机制的凋亡信号传导途径。神经营养因子突变蛋白将被筛选，以区分p53依赖性和p53非依赖性途径与p53温度敏感性PC 12细胞。几个神经元细胞系和原代培养物也将与选择突变蛋白进行研究。(iii)测试信号选择性神经营养素突变蛋白通过鼻内吸入递送至啮齿动物中枢神经系统的治疗潜力。将使用正常小鼠、p53缺失小鼠、p75 NTR缺失小鼠、正常大鼠和老年大鼠。将在处理的大鼠和小鼠脑中比较信号通路，包括TrkA磷酸化、MAP激酶和DNA片段化。从神经营养因子衍生的选择性刺激信号传导途径的治疗试剂的合理设计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治疗-反应选择性NGF突变体可能最终比野生型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.