Extracellular vesicles - Characterising the structure-function relationships of extracellular vesicles in cell communication within the neurovascular

“多细胞生物体中细胞通讯的性质可能导致健康或疾病。在中枢神经系统(CNS)内，人们还不完全了解细胞(神经细胞、非神经细胞和免疫细胞)的混合如何交流，也不完全理解所使用的语言。近年来，细胞外小泡(EV)被认为是一种新的细胞间通讯机制。这些膜袋在健康、疾病和细胞死亡期间从细胞中释放出来，携带许多因子到受体细胞，以引发反应(可取和不可取)。有矛盾的数据表明，EV可能既有神经保护作用，又有神经毒性2。这突显了对每种CNS细胞类型进行详细EV分析的必要性，以便我们可以了解EV的摄取、功能以及它们在年龄相关条件下的变化。初步工作表明，EV携带一系列可能支持其活性的因素，包括一系列可能有助于控制炎症和修复反应的活性酶2。该项目旨在确定和描述与电动汽车相关的关键因素，这些因素调解了电动汽车的功能。本项目的目的是：从诱导的多能干细胞中产生神经血管单位的细胞，并在单一和联合培养中评估EV的释放，以评估EV在神经血管单位的其他细胞上诱导反应(例如炎症反应)的能力，通过详细的MS分析来确定功能的分子介质，以确定EV表面蛋白介导的细胞间通讯，以研究EV相关的酶活性来控制炎症。确定EV表面存在的关键蛋白质，这些蛋白质介导EV与巨噬细胞相互作用并调节先天免疫反应的功能。这将从质量谱结果的大型数据集中识别铅分子，以供进一步研究。确定优先蛋白质在肠道病毒活动中的功能。使用各种细胞生物学和分子方法，这将评估蛋白质作为EV结合、摄取或活性功能的关键配体。将在项目期间使用的技术：分析广泛的蛋白质组学数据：确定使用系统生物学方法进行进一步分析的先导靶标。细胞培养：从人外周血中分离出原代细胞；对一系列细胞系和原代细胞进行组织培养。诱导多能干细胞培养，分化为功能性神经元网络、星形胶质细胞和小胶质细胞。囊泡分析：利用可调谐电阻脉冲传感和流式细胞术分离和分析EV的结构和功能。应用趋化试验和定量聚合酶链式反应分析免疫调节能力成像和分析：流式细胞术、光学和荧光显微镜和自动细胞成像。细胞检测：诱导和分析细胞死亡，结合和吞噬实验。参考文献：1.van Niel，G.，D‘Angelo，G.，&Raposo(2018)。揭示了细胞外小泡的细胞生物学。《自然评论》：分子细胞生物学：doi：10.1038/nrm.2017.1252。石明、盛、L.、斯图尔特、T.、扎贝提安、C.P.、张军(2019)。大脑的新窗口：中枢神经系统衍生的细胞外小泡。神经生物学进展：doi：10.1016/j.puroBio.2019.01.0053。戴维特，A.，格里菲斯，H.R.和米利奇一世(2018)。与死人沟通：脂类、脂类介体和细胞外小泡。生物化学学会会刊：DOI：10.1042/BST2016477“

"The nature of cell communication in a multicellular organism may result in health or disease. Within the central nervous system (CNS), it is not fully understood how the mix of cells (neuronal, non-neuronal and immune cells) communicate, nor the language that is used. The change in this communication in age-associated conditions is also poorly understood.Over recent years, extracellular vesicles (EV) have been identified as a novel intercellular communication mechanism1. These membrane bags, released from cells during health, disease and cell death, carry many factors to recipient cells to elicit responses (desirable and non-desirable). There are paradoxical data to suggest EV may be both neuroprotective and neurotoxic2. This highlights the need for detailed EV analysis from each of the CNS cell types so we may understand EV uptake, function and how they change in age-associated conditions. Preliminary work reveals that EV carry a large range of factors that may underpin their activity, including a family of active enzymes which may help to control inflammation and repair responses2. This project seeks to identify and characterise the key factors associated with EV, that mediate their function. The aims of this project are: to generate cells of the neurovascular unit from induced pluripotent stem cells and assess EV release in mono- and co-culture to assess the ability of EV to induce responses on other cells of the neurovascular unit (e.g. inflammatory responses) to define the molecular mediators of function through detailed MS analysis to define EV surface proteins mediating intercellular communication to investigate EV-associated enzyme activity that controls inflammation. identify key proteins present on the surface of EV that mediate EV function in interacting with macrophages and modulating the innate immune response. This will work from a large dataset of mass-spec results to identify lead molecules for further study. to define the function of prioritised proteins in EV activity. Using a variety of cell biological and molecular approaches, this will assess proteins as key ligands for EV binding, uptake or active function.Techniques that will be utilised during the project: Analysis of extensive proteomics data: to identify lead targets for further analysis using systems biology approaches. Cell culture: Isolation of primary cells from human peripheral blood; Tissue culture of a range of cell lines and primary cells. Induced pluripotent stem cell culture to derive functional neuronal networks, astrocytes and microglia. Vesicle analyses: isolation and analysis of EV structure and function using tunable resistive pulse sensing and flow cytometry. Analysis of immune-modulating capability using chemoattraction assays and qPCR. Imaging and analysis: Flow cytometry, light and fluorescence microscopy and automated cell imaging. Cell assays: Induction and analysis of cell death, binding and phagocytosis assays. Mass spectrometry: for the analysis of EV proteome.References:1. Van Niel, G., D'Angelo, G., & Raposo (2018). Shedding light on the cell biology of extracellular vesicles. Nature Reviews: Molecular Cell Biology: DOI: 10.1038/nrm.2017.1252. Shi, M., Sheng, L., Stewart, T., Zabetian, C.P. & Zhang, J. (2019). New Windows into the brain: CNS-derived extracellular vesicles. Progress in Neurobiology: DOI: 10.1016/j.pneurobio.2019.01.0053. Devitt, A., Griffiths, H.R. & Milic I. (2018). Communicating with the dead: lipids, lipid mediators and extracellular vesicles. Biochemical Society Transactions: DOI: 10.1042/BST2016477"