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。这项研究将调查淀粉样前体蛋白(APP)在细胞内的转运，APP是淀粉样β蛋白的前体，它形成阿尔茨海默病的细胞外老年斑。最近的数据表明，APP通过Kinesin-I运输到细胞表面，在那里它可能调节细胞的运动，其蛋白分解产物CGamma被逆行运输(可能是通过细胞质动力蛋白)，并在向细胞核发出信号中发挥作用。APP的顺行转运和CGamma的逆行转运都是高度调控的过程，可能与应激激活的c-jun NH2末端激酶(JNK)信号复合体和支架蛋白Fe65一起发生，可能参与阿尔茨海默病的发病机制。这一调节将在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，这种细胞既表现出正常神经元的特征，也表现出阿尔茨海默病患者大脑中存在的退化神经元的特征。第一个特指目的将阐述APP的磷酸化在调节激动素-I到APP的招募中的作用，从而将APP运输到轴突中。实时成像将被用来分析荧光标记的APP在阻止或不阻止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，我们将在它们的活性受到抑制的条件下分析它们的运动性，并将其与测试APP与Kinesin-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，以及亚细胞分级和超微结构定位，第二个具体目标将调查APP和另一个kinesin-I货物，JNK信号复合体，是独立运输(即，在单独的小泡上)，还是作为同一货物的一部分一起运输。第三个具体目标将涉及动蛋白-I和细胞质动力蛋白在含有APP或CGamma的信号复合体以及支架蛋白Fe65的运输中的参与。整个研究过程中获得的信息将用于重建APP/JIP-1和APP/Fe65的体外运动能力。这项研究应该会揭示APP、JNK和CDK5信号通路之间可能存在的相互作用，并对大脑发育和神经退化产生影响。

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.