Investigating CaMKII regulation of extracellular vesicle trafficking to promote synaptic plasticity

项目总结 细胞外小泡(EVS)是细胞间交换物质的膜结合的小隔间， 在神经系统的细胞通讯中扮演着重要的角色。神经元EVS调节活动- 依赖突触的可塑性，以及与以下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氏病。EV的生物发生和 分泌是通过内体运输发生的，但神经元活动调节这些细胞的机制 生物过程还没有得到很好的描述。通过确定活动依赖的机制来影响 神经EV动力学，我们将对神经系统中的基本和病理过程有更深入的了解。 大多数EV研究利用体外非神经细胞模型来分离异源EV群体， 这使得对神经元EV的生物发生和运输机制的直接测量具有挑战性。果蝇 神经肌肉接头(NMJ)是一种功能强大的体内模型，具有高度的可操作性和可操作性。 可视化电动汽车货物。我在这个系统中的初步发现表明，钙/钙调蛋白依赖蛋白 激酶II(CaMKII)是一种关键的突触蛋白，参与突触可塑性的结构和功能形式 在电动汽车贩运中扮演的角色。具体地说，CaMKII零突变体显示EV Cargo水平显著降低 Syt4在突触前神经元和突触后分泌的EV前体中均有表达。Syt4调制NMJ 突触生长和神经递质的释放依赖于活动，这导致了我的假设 CaMKII调节Syt4 EV信号，促进结构和功能的可塑性。在目标1中，我会问CaMKII 控制EV货物在我们先前确定的介导EV货物的内吞运输途径中的水平 贩卖人口。在第二个目标中，我将阐明CaMKII和Syt4之间的关系，并测试它们是否在 促进结构和功能可塑性的共同途径。在目标3中，我将分离特定的CaMKII 职能(S)参与电动汽车货运和贩运监管。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。从这一点上获得的信息 该项目将为CaMKII如何调控EV运输以促进突触可塑性提供新的见解，以及 确定调节神经系统中EVS的新的活动依赖机制。

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.