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

钙（Ca2+）是一种普遍和多样的第二信使，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。这些细胞和环境特异性表达模式使细胞内Ca2+浓度在空间和时间上发生异质变化，用于多种表型输出。在神经科学中，Ca2+信号是至关重要的信息处理通过动作电位传播和神经传递在电兴奋的神经元细胞，加上电压的变化。然而，细胞内Ca2+的变化也与中枢神经系统（CNS）的不可兴奋细胞的激活有关，特别是小胶质细胞，中枢神经系统驻留监视先天免疫细胞的激活发生在神经炎症期间。神经炎症被广泛定义为一组中枢神经系统局部和协调的免疫血管对细胞损伤的反应，并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、吞噬和炎性体激活）相关。尽管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中钙信号的增加，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的信号事件）仍然不明确。总的来说，这个博士项目旨在通过使用高含量想象（HCI）方法产生小胶质细胞“指纹”，对IC钙信号如何与小胶质细胞激活状态联系产生新的理解。这些指纹将使细胞内钙信号与其他物理、化学和功能读数的报告复合，而不需要假设哪些特性将相关。一套新的人小胶质细胞（HMC3）和神经元（SH-SY5Y）永生化细胞系的体外检测将被开发和优化。最初，测试将以广谱的“亲”和“抗”炎症刺激为基准，这些炎症刺激与小胶质细胞激活有关，涵盖了广泛的小胶质细胞表达受体，以及由Madden实验室的化学家在室内合成的新型工具化合物的治疗。一些最适用和最强大的表型分析将被升级，用于使用HCI（多端点的自动显微镜成像（Lilly, 2018））筛选预注释的化合物文库，目的是在不事先了解所涉及的分子途径的情况下识别新的神经炎症调节剂和靶标。图像采集将可能使用CellDiscover作为终点，使用IncuCyte进行动力学分析。确定的靶点将被用于下游靶标反褶积，潜在地发现治疗神经炎症相关疾病的新靶点。神经炎症的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.