Molecular Motors in Transport and Signaling by APP

描述（由申请人提供）：拟议研究的长期目标是了解分子马达在组织与人类疾病相关的信号通路中的作用。本研究将探讨淀粉样前体蛋白（APP）的细胞内运输，淀粉样β肽的前体，形成阿尔茨海默病的细胞外老年斑。最近的数据表明，APP被运输到细胞表面的驱动蛋白-I，在那里它可以调节细胞的运动，其蛋白水解裂解产物，C γ逆行运输（推测由细胞质动力蛋白），并在信号细胞核的功能。APP的顺行运输和C γ的逆行运输是高度调节的过程，并且可能与应激激活的c-Jun NH 2-末端激酶（JNK）信号传导复合物和支架蛋白Fe 65一起发生，所述支架蛋白Fe 65可能参与阿尔茨海默病的发病机制。将在CAD细胞（源自中枢神经系统的儿茶酚胺能细胞系）中研究这种调节，CAD细胞显示出正常神经元和阿尔茨海默病患者脑中存在的退化神经元的特征。第一个SpecificAim将阐明激酶Cdk 5和JNK对APP的磷酸化在调节驱动蛋白I向APP的募集中的作用，从而将APP转运到轴突中。实时成像将用于分析荧光标记的APP在阻断或不阻断其磷酸化的条件下的运动性。为了确定Cdk 5和JNK在APP转运中的作用，将在其活性被抑制的条件下分析运动性，并与来自测试APP与驱动蛋白I相互作用的生化实验的数据相关联。通过使用类似的方法，以及亚细胞分级分离和超微结构定位，第二个特定目的将研究APP和另一种驱动蛋白-I货物，JNK信号传导复合物，是否独立转运（即，在单独的囊泡上），或一起，作为同一货物的一部分。第三个具体目标将解决参与的驱动蛋白-I和细胞质动力蛋白在运输的信号复合物，containAPP或C γ，和支架蛋白，Fe 65。整个研究期间获得的信息将用于体外重构APP/JIP-1和APP/Fe 65运动性。这项研究应该揭示了涉及APP，JNK和Cdk 5的信号通路之间可能的串扰的关键见解，并对大脑发育和神经退行性变产生影响。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand the role of molecular motors in organizing signaling pathways relevant to human disease. This study will investigate the intracellular trafficking of amyloid precursor protein (APP), the precursor of amyloid beta peptide, which forms the extracellular senile plaques in Alzheimer's disease. Recent data suggest that APP is transported to the cell surface by kinesin-I, where it may regulate cell movement, and that its proteolytic cleavage product, Cgamma is transported retrogradely (presumably by cytoplasmic dynein), and functions in signaling to the nucleus. The anterograde transport of APP and the retrograde transport of Cgamma are highly regulated processes, and may occur in conjunction with the stress-activated, c-Jun NH2-terminal kinase (JNK) signaling complex, and the scaffolding protein, Fe65 that may be involved in the pathogenesis of Alzheimer's disease. This regulation will be studied in CAD cells (a catecholaminergic cell line derived from the central nervous system), which show features of both normal neurons, and of degenerating neurons present in the brains of Alzheimer's disease patients. The first SpecificAim will address the role of phosphorylation ofAPP by the kinases, Cdk5 and JNK in regulating recruitmentof kinesin-I to APP and, thereby transport of APP into axons. Real-time imaging will be used to analyze the motility of fluorescently-taggedAPP in conditions that do or do not block its phosphorylation. To determine the role of Cdk5 and JNK inAPP transport, motility will be analyzed in conditions in which their activity is suppressed, and correlated with data from biochemical experiments that test the interactionof APP with kinesin-I. By using a similar methodology, as well as subcellular fractionation and ultrastructural localization, the second Specific Aim will investigate whether APP and another kinesin-I cargo, the JNK signaling complex, are transported independently (i.e., on separate vesicles), or together, as part of the same cargo. The third Specific Aim will address the participation of kinesin-I and cytoplasmic dynein in the transport of signaling complexes that containAPP or Cgamma, and the scaffolding protein, Fe65. Information gained throughout the study will be used to reconstitute APP/JIP-1 and APP/Fe65 motility in vitro. This study should reveal key insights into a possible cross-talk between signaling pathways involving APP, JNK, and Cdk5, with implications for brain development and neurodegeneration.