Investigating CaMKII regulation of extracellular vesicle trafficking to promote synaptic plasticity

项目摘要 细胞外囊泡（EV）是在细胞之间交换物质的小的膜结合区室， 在神经系统的细胞通讯中起着重要的作用。神经元EV介导活动- 依赖性突触可塑性，以及与之相关的有毒蛋白聚集体的扩散和清除。 神经退行性疾病，如阿尔茨海默病和帕金森病。EV生物发生和 分泌通过内体运输发生，但神经元活动调节这些细胞的机制 生物过程尚未得到很好的表征。通过识别活动依赖机制， 神经元EV动力学，我们将获得更深入的了解在神经系统的基本和病理过程。 大多数EV研究利用体外非神经元细胞模型来分离异源EV群体， 这使得直接测量神经元EV生物发生和运输机制具有挑战性。果蝇 神经肌肉接头（NMJ）作为一种强大的体内模型，其非常易于操作， 可视化电动车货物我在这个系统中的初步发现表明，钙/钙调素依赖蛋白 激酶II（CaMKII）是一种参与突触可塑性结构和功能形式的关键突触蛋白， 在电动汽车交易中扮演的角色。具体而言，CaMKII无效突变体显示EV货物的水平显著降低， 突触前神经元和突触后分泌的EV中的EV前体中的Syt 4。Syt 4调节NMJ 突触生长和神经递质的释放是以活动依赖的方式进行的，这导致了我的假设， CaMKII调节Syt 4 EV信号以促进结构和功能可塑性。在目标1中，我会问CaMKII是否 控制EV货物水平的内吞运输途径，我们以前确定介导EV货物 贩卖人口在第二个目标中，我将阐明CaMKII和Syt 4之间的关系，并测试它们是否在一个特定的时间内起作用。 共同的途径来促进结构和功能的可塑性。在目标3中，我将分离特定的CaMKII 涉及电动汽车货物和贩运监管的职能。这些目标将提供先进成像方面的培训， 定量图像分析、果蝇遗传学和电生理学。从这获得的信息 该项目将为CaMKII如何调节EV运输以促进突触可塑性提供新的见解， 确定神经系统中调节EV的新的活性依赖性机制。

PROJECT SUMMARY Extracellular vesicles (EVs) are small membrane-bound compartments that exchange materials between cells, and play an important role in cell communication in the nervous system. Neuronal EVs mediate activity- dependent synaptic plasticity, as well as the spread and clearance of toxic protein aggregates associated with neurodegenerative disorders such as Alzheimer’s Disease and Parkinson’s Disease. EV biogenesis and secretion occurs via endosomal trafficking, but the mechanisms by which neuronal activity regulates these cell biological processes have not been well characterized. By identifying activity-dependent mechanisms influencing neuronal EV dynamics, we will gain greater insight into basic and pathological processes in the nervous system. The majority of EV research utilizes in vitro non-neuronal cell models to isolate heterogenous EV populations, making direct measurements of neuronal EV biogenesis and trafficking mechanisms challenging. The Drosophila neuromuscular junction (NMJ) serves as a powerful in vivo model that is highly amenable to manipulating and visualizing EV cargo. My preliminary findings in this system suggest that Calcium/calmodulin-dependent protein kinase II (CaMKII), a key synaptic protein involved in structural and functional forms of synaptic plasticity, plays a role in EV trafficking. Specifically, CaMKII null mutants showed significantly decreased levels of the EV cargo Syt4 in both EV precursors in the presynaptic neuron and postsynaptically secreted EVs. Syt4 modulates NMJ synaptic growth and neurotransmitter release in an activity-dependent manner, leading to my hypothesis that CaMKII regulates Syt4 EV signaling to promote structural and functional plasticity. In Aim 1, I will ask if CaMKII controls EV cargo levels in an endocytic trafficking pathway that we previously identified to mediate EV cargo trafficking. In the second aim, I will elucidate the relationship between CaMKII and Syt4 and test if they act in a shared pathway to promote structural and functional plasticity. In Aim 3, I will isolate the specific CaMKII function(s) involved in EV cargo and trafficking regulation. These aims will provide training in advanced imaging, quantitative image analysis, Drosophila genetics, and electrophysiology. The information obtained from this project will provide new insight into how CaMKII regulates EV trafficking to facilitate synaptic plasticity, and identify new activity-dependent mechanisms regulating EVs in the nervous system.