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（Ca2+）是通用且多样的第二使者，对于一般和特定的细胞功能，其细胞内（IC）浓度通过泵，通道和缓冲液的细胞特异性工具包进行了细腻维持。这些细胞和上下文的特定表达模式可在细胞内Ca2+浓度上在空间和时间上异质性变化，用于多种表型输出。在神经科学中，Ca2+信号传导对于通过动作电位传播和电触发神经递质的信息处理至关重要。然而，细胞内Ca2+变化也与中枢神经系统（CNS）的非驱动细胞（尤其是小胶质细胞）的激活有关，CNS常驻监测的先天性免疫细胞在神经炎症过程中的激活。神经炎症广泛定义为对细胞损伤的CNS局部和协调的免疫血管反应，并且与小胶质细胞形态的明显变化以及“促炎”表型以及诸如细胞因子和趋化因子和趋化因子的分泌等谱系以及吞噬，吞噬作用，吞噬作用和炎性症。尽管已经在体外和体内测量了小胶质细胞中钙信号的增加，并且与其他小胶质细胞和神经元表型相关，并与刺激特定的阈值和机械细节相关，包括精确的钙动员机制和下降症状的事件，涉及该型号的现象。通过使用高内容想象（HCI）方法生成小胶质细胞“指纹”，对IC钙信号如何与小胶质细胞激活状态联系起来有了新的了解。这些指纹将在不假定哪些特性相关的情况下将细胞内钙信号传导与其他物理，化学和功能读数进行多重报告。将在人类小胶质细胞（HMC3）和神经元（SH-SY5Y）永生的细胞系中进行一系列新颖的体外测定套件，并并行开发和优化。最初，测定将基准基准与小胶质细胞激活相关的广泛特征的“ Pro”和“抗”炎症刺激，涵盖了广泛的小胶质细胞表达受体，以及用Madden Lab中化学家在房屋中合成的新工具化合物的处理。一些最有效，最有力的表型测定将被扫描，以使用HCI（自动显微镜成像（Lilly，2018））筛选预先注销的化合物库，以识别新型神经毒素调节剂和目标，而无需涉及分子途径。图像采集可能会使用细胞发现终点，并将其用于动力学测定。然后将确定的命中率向下游靶向反卷积前进，并有可能发现用于治疗神经炎症相关疾病的新靶标。神经炎症的失调涉及一系列脑部疾病的病因和/或发病机理，包括痴呆症，神经精神病学和创伤性脑损伤并发症。