Developing new tools and technologies to study calcium signalling in the brain's immune system

开发新工具和技术来研究大脑免疫系统中的钙信号传导

• 批准号: 2815099
• 金额: --
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2815099
• 关键词: Developing; new; tools; technologies; study

Calcium (Ca2+) is a universal and diverse second messenger critical for general and specific cellular function, with its intracellular (IC) concentration finely maintained by a cell-specific toolkit of pumps, channels, and buffers. The cell and context specific expression pattern of these enables spatially and temporally heterogeneous changes in intracellular Ca2+ concentration, exploited for diverse phenotypic outputs.In neuroscience, Ca2+ signalling is vital for information processing via action potential propagation and neurotransmission in electrically excitable neuronal cells, coupled to changes in voltage. However, intracellular Ca2+ changes are also associated with activation of non-excitable cells of the central nervous system (CNS), particularly microglia, CNS resident surveillant innate immune cells activation of which occurs during neuroinflammation. Neuroinflammation is broadly defined as the set of CNS-localised and coordinated immunovascular responses to cell damage, and is associated with distinct changes in microglial morphology alongside a spectrum of "pro-inflammatory" phenotypes such as secretion of cytokines and chemokines, phagocytosis, and inflammasome activation. Although increases in calcium signalling in microglia with a range of characterised inflammatory stimuli have been measured in vitro and in vivo, and correlation with other microglial and neuronal phenotypes identified, stimuli specific thresholds and mechanistic details including the precise calcium mobilisation mechanisms and downstream phenotypically relevant signalling events involved remain ill-defined.Broadly, this PhD project aims to develop to generate a new understanding of how IC calcium signalling links to microglial activation states via generating microglial 'fingerprints' using high content imagine (HCI) approaches. These fingerprints will multiplex reports of intracellular calcium signalling with other physical, chemical, and functional readouts without assumption of which properties will correlate. A suite of novel in vitro assays in human microglia (HMC3) and neuronal (SH-SY5Y) immortalised cell lines will be developed and optimised in parallel. Initially, assays will benchmark of a broad spectrum of characterised "pro" and "anti" inflammatory stimuli associated with microglial activation covering a broad range of microglial expressed receptors, as well as treatment with novel tool compounds synthesised in house by chemists in the Madden lab. Some of the most amenable and powerful phenotypic assays will be upscaled for screening pre-annotated compound libraries using HCI (automated microscopy imaging of multiple endpoints (Lilly, 2018)) with the aim of identifying novel neuroinflammatory modulators and targets without prior knowledge of the molecular pathways involved. Image acquisition will likely use the CellDiscover for end-point and the IncuCyte for kinetic assays. Identified hits will then be taken forward for downstream target deconvolution, potentially discovering novel targets for treatment of neuroinflammatory associated diseases. Dysregulation of neuroinflammation is implicated in aetiology and/or pathogenesis of a range of brain disorders including dementias, neuropsychiatric conditions, and traumatic brain injury complications.

钙（Ca 2+）是一种通用和多样的第二信使，对一般和特定的细胞功能至关重要，其细胞内（IC）浓度由细胞特异性的泵，通道和缓冲液工具包精细维持。这些细胞和上下文特定的表达模式，使空间和时间异质性变化的细胞内Ca 2+浓度，利用不同的表型outputs.In神经科学，Ca 2+信号是至关重要的信息处理通过动作电位传播和电兴奋的神经元细胞中的神经传递，加上电压的变化。然而，细胞内Ca 2+变化也与中枢神经系统（CNS）的非兴奋性细胞（特别是小胶质细胞）的活化相关，小胶质细胞是CNS驻留的监视性先天免疫细胞，其活化发生在神经炎症期间。神经炎症被广义地定义为一组CNS定位和协调的免疫血管对细胞损伤的反应，并且与小胶质细胞形态的明显变化以及一系列“促炎”表型如细胞因子和趋化因子的分泌、吞噬作用和炎性小体活化相关。尽管已经在体外和体内测量了具有一系列特征性炎症刺激的小胶质细胞中钙信号传导的增加，并且鉴定了与其他小胶质细胞和神经元表型的相关性，但是刺激特异性阈值和机制细节（包括精确的钙动员机制和涉及的下游表型相关信号传导事件）仍然不明确。这个博士项目旨在通过使用高内容想象（HCI）方法生成小胶质细胞“指纹”，对IC钙信号传导如何与小胶质细胞激活状态联系产生新的理解。这些指纹将细胞内钙信号与其他物理，化学和功能读数的多重报告，而无需假设哪些属性将相互关联。将平行开发和优化一套新的人小胶质细胞（HMC 3）和神经元（SH-SY 5 Y）永生化细胞系体外试验。最初，检测将对与小胶质细胞活化相关的广泛的特征性“促”和“抗”炎症刺激进行基准测试，这些刺激涵盖了广泛的小胶质细胞表达受体，以及用化学家在Madden实验室内部合成的新型工具化合物进行治疗。一些最适合和最强大的表型测定将被升级，用于使用HCI（多个终点的自动显微镜成像（礼来公司，2018））筛选预先注释的化合物文库，目的是在没有所涉及的分子途径的先验知识的情况下鉴定新型神经炎症调节剂和靶标。图像采集可能会使用CellDiscover进行终点检测，使用IncuCyte进行动力学检测。然后将识别的命中用于下游靶点去卷积，从而可能发现用于治疗神经炎性相关疾病的新靶点。神经炎症的失调涉及一系列脑疾病的病因学和/或发病机制，包括痴呆、神经精神病症和创伤性脑损伤并发症。