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+) 是一种通用且多样化的第二信使，对一般和特定细胞功能至关重要，其细胞内 (IC) 浓度由泵、通道和缓冲液组成的细胞特异性工具包精细维持。这些细胞和上下文特定的表达模式使得细胞内 Ca2+ 浓度在空间和时间上发生异质性变化，用于不同的表型输出。在神经科学中，Ca2+ 信号传导对于通过电兴奋神经元细胞中的动作电位传播和神经传递进行信息处理至关重要，耦合到电压的变化。然而，细胞内 Ca2+ 的变化也与中枢神经系统 (CNS) 的非兴奋性细胞的激活有关，特别是小胶质细胞、CNS 常驻监视先天免疫细胞的激活，其激活发生在神经炎症期间。神经炎症被广泛定义为一组针对细胞损伤的中枢神经系统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一系列“促炎症”表型相关，例如细胞因子和趋化因子的分泌、吞噬作用和炎症小体激活。尽管在体外和体内测量了一系列特征性炎症刺激下小胶质细胞中钙信号传导的增加，并确定了与其他小胶质细胞和神经元表型的相关性，但刺激特异性阈值和机制细节，包括精确的钙动员机制和下游表型相关信号传导所涉及的事件仍然不明确。总的来说，这个博士项目旨在对 IC 钙信号如何通过生成小胶质细胞与小胶质细胞激活状态联系起来产生新的理解使用高内涵想象（HCI）方法的“指纹”。这些指纹将多重报告细胞内钙信号传导与其他物理、化学和功能读数，而不假设哪些特性将相互关联。将同时开发和优化人类小胶质细胞 (HMC3) 和神经元 (SH-SY5Y) 永生化细胞系的一套新型体外测定。最初，检测将对与小胶质细胞激活相关的广泛特征“促”和“抗”炎症刺激进行基准测试，涵盖广泛的小胶质细胞表达受体，以及由Madden实验室化学家内部合成的新型工具化合物的治疗。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（Lilly，2018））筛选预先注释的化合物库，目的是在不事先了解分子的情况下识别新型神经炎症调节剂和靶点涉及的途径。图像采集可能会使用 CellDiscover 进行终点分析，并使用 IncuCyte 进行动力学分析。然后，确定的命中将被用于下游靶点解卷积，有可能发现治疗神经炎症相关疾病的新靶点。神经炎症失调与一系列脑部疾病的病因和/或发病机制有关，包括痴呆、神经精神疾病和创伤性脑损伤并发症。