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蛋白前体蛋白（APP）的细胞内运输，APP是淀粉样蛋白β肽的前体，在阿尔茨海默病中形成细胞外老年斑。最近的数据表明，APP通过激酶i转运到细胞表面，在那里它可以调节细胞运动，并且其蛋白水解裂解产物cγ逆行转运（可能是通过细胞质动力蛋白），并在向细胞核发送信号中起作用。APP的顺行转运和Cgamma的逆行转运是高度调控的过程，可能与应激激活的c-Jun nh2末端激酶（JNK）信号复合物以及可能参与阿尔茨海默病发病机制的支架蛋白Fe65一起发生。这种调节将在CAD细胞（一种源自中枢神经系统的儿茶酚胺能细胞系）中进行研究，CAD细胞显示出阿尔茨海默病患者大脑中正常神经元和退化神经元的特征。第一个特异性目标将讨论激酶、Cdk5和JNK在调节激酶i向APP的募集以及APP向轴突运输中的作用。实时成像将用于分析荧光标记的dapp在阻断或不阻断其磷酸化的条件下的运动性。为了确定Cdk5和JNK在APP运输中的作用，将分析其活性被抑制的条件下的运动性，并与测试APP与激酶i相互作用的生化实验数据相关联。通过使用类似的方法，以及亚细胞分离和超微结构定位，第二个特异性目标将研究APP和另一种激酶i货物，JNK信号复合物，是单独运输（即在单独的囊泡上），还是作为同一货物的一部分一起运输。第三个特定目标将讨论激酶i和细胞质动力蛋白在含有app或Cgamma的信号复合物和支架蛋白Fe65的运输中的参与。在整个研究过程中获得的信息将用于在体外重建APP/JIP-1和APP/Fe65的运动。这项研究将揭示APP、JNK和Cdk5信号通路之间可能的串扰，对大脑发育和神经变性具有重要意义。

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.