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

钙(Ca~(2+))是一种普遍而多样的第二信使，对一般和特定的细胞功能至关重要，其细胞内(IC)浓度由泵、通道和缓冲液组成的细胞特有的工具箱很好地维持。这些细胞和上下文特定的表达模式使得细胞内钙离子浓度在空间和时间上的异质性变化，被用于不同的表型输出。在神经科学中，钙离子信号在信息处理中至关重要，通过动作电位的传播和神经传递，在电兴奋的神经元细胞，再加上电压的变化。然而，细胞内钙离子的变化也与中枢神经系统(CNS)中不可兴奋的细胞，特别是小胶质细胞的激活有关，小胶质细胞是中枢神经系统固有免疫细胞在神经炎症过程中被激活的监控者。神经炎被广泛地定义为一组中枢神经系统对细胞损伤的局部和协调的免疫血管反应，与小胶质细胞形态的明显变化以及一系列促炎症表型有关，如细胞因子和趋化因子的分泌、吞噬和炎性小体激活。虽然已经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钙信号的增加，并与其他小胶质细胞和神经元表型识别的相关性，但刺激的特定阈值和机制细节，包括精确的钙动员机制和下游涉及的表型相关信号事件仍未确定。总的来说，该PHD项目旨在通过使用高含量成像(HCI)方法产生小胶质细胞的指纹，以产生对IC钙信号如何与小胶质细胞激活状态相联系的新的理解。这些指纹将细胞内钙信号的报告与其他物理、化学和功能读数多路传输，而不假设哪些特性将相关。在人类小胶质细胞(HMC3)和神经元(SH-SY5Y)永生化细胞系中，一套新的体外分析方法将被开发和优化。最初，化验将以一系列与小胶质细胞激活相关的广泛的“亲”和“抗炎”刺激为基准，涵盖广泛的小胶质细胞表达的受体，以及使用由马登实验室的化学家在室内合成的新型工具化合物进行治疗。一些最具适应性和最强大的表型分析将被扩大，用于使用HCI(多个端点的自动显微成像(Lilly，2018))筛选预先注释的化合物文库，目的是在不事先了解所涉及的分子通路的情况下识别新的神经炎性调节剂和靶点。图像采集可能会使用CellDiscover作为终点，IncuCyte用于动力学分析。然后，确定的靶点将被用于下游靶点去卷积，潜在地发现治疗神经炎性相关疾病的新靶点。神经炎症调节失调与一系列脑部疾病的病因学和/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。

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.