Calreticulin and other candidate Alarmins and Opsonins from Microglia

“警报器”是从受损细胞释放的可溶性分子，可促进免疫细胞的非感染性炎症激活。这种激活对于启动组织损伤或疾病的早期先天免疫反应是重要的。钙网蛋白是一种内质网细胞内的伴侣蛋白，是脑内巨噬细胞小胶质细胞的一种候选警报蛋白。在旋转项目中，发现钙网蛋白从受损细胞、体外化学吸引的小胶质细胞中释放，并刺激其释放肿瘤坏死因子- α和白细胞介素-6；细胞因子与炎症表型相关。因此，钙调蛋白可能在大脑组织损伤和小胶质细胞免疫反应之间起着中间作用。博士项目将以这些发现为基础。首先，通过了解所涉及的机制途径，包括细胞释放机制，以及小胶质细胞上钙调蛋白的受体和下游信号传导。其次，炎症和神经毒性对小胶质细胞的影响。第三，将测试钙调蛋白是否作为突触、髓磷脂和神经元小胶质吞噬的调理素。第四，如果钙调蛋白作为一种调理蛋白，那么这是否会促进抗原向T细胞的递呈将被确定。为了确定钙调钙蛋白是否在体内释放，将测量创伤性脑损伤或非创伤性脑损伤患者脑脊液中钙调钙蛋白和细胞因子的水平。同样，将测量来自各种疾病模型（包括脑外伤）的大鼠和小鼠脑脊液中的钙调钙蛋白和细胞因子水平。将在损伤后的多个采集时间点采集样本，以更好地阐明钙调钙蛋白释放的生理模式，并将其与炎症特征联系起来。由于小胶质细胞免疫反应是一个涉及许多复合物和分子的多层过程，因此除钙调蛋白外，其他可溶性介质作为小胶质细胞警报剂和/或调理素的潜力将被进一步研究。在不同条件下的初级小胶质细胞分泌组将被获得，纯化，然后通过质谱法分裂成可识别的蛋白质。候选蛋白将被评估其作为小胶质细胞的警报或调理素的能力。与此同时，本项目将评估小胶质受体P2Y6，以测试其是否在发育性突触吞噬（修剪）中发挥作用。突触修剪过少与自闭症有关，而修剪过多则与精神分裂症有关。Brown实验室的初步研究表明P2Y6受体可能是小胶质细胞吞噬突触所必需的；对3-4个月大的P2Y6-/-小鼠的行为测试表明它们有记忆缺陷。本项目将进一步测试1月龄P2Y6-/-小鼠是否存在社交缺陷和/或突触密度增加，以及这是否由于突触小胶质吞噬减少所致。

PhD project strategic theme: Biosciences for an integrated understanding of healthAlarmins are soluble molecules released from damaged cells that promote the non-infectious inflammatory activation of immune cells. This activation is important for initiating the early stage innate immune response to tissue damage or disease. Calreticulin, an intracellular chaperone of the endoplasmic reticulum, is a candidate alarmin to microglia - the resident macrophage of the brain. During the rotation project, it was found that calreticulin was released from damaged cells, chemoattracted microglia in vitro, and stimulated their release of tumour necrosis factor-alpha and interleukin-6; cytokines associated with an inflammatory phenotype. Thus, calreticulin may act as an intermediate between tissue damage in the brain and the microglial immune response to that damage.The PhD project would build on these findings. Firstly, by understanding the mechanistic pathways involved, including the mechanism of cellular release, and the receptor (or receptors) on microglia for calreticulin and downstream signalling. Secondly, the consequences for microglia in terms of the inflammatory profile and neurotoxicity. Thirdly, it will be tested whether calreticulin acts as an opsonin for microglial phagocytosis of synapses, myelin and neurons. Fourthly, if calreticulin acts as an opsonin, then whether this promotes antigen presentation to T cells will be determined. To determine whether calreticulin release occurs in vivo, calreticulin and cytokine levels will be measured in the cerebrospinal fluid of human patients that have either suffered from traumatic brain injury or not. Similarly, calreticulin and cytokine levels will be measured in the cerebrospinal fluid of rats and mice from various disease models, including brain trauma. Samples would be acquired across multiple acquisition timepoints post-injury to better elucidate the physiological pattern of calreticulin release and relate this to the inflammatory profile. As the microglial immune response is a multi-layered process involving numerous complexes and molecules, further to calreticulin other soluble mediators will be investigated for their potential as microglial alarmins and/or opsonins. The secretome of primary microglia under various conditions will be acquired, purified, and then fragmented into identifiable proteins by mass spectrometry. Candidate proteins will then be assessed for their ability to act as alarmins or opsonins for microglia as above. Concurrently with this project, the microglial receptor P2Y6 will be assessed to test whether it has a role in developmental synaptic phagocytosis (pruning). Too little synaptic pruning is implicated in autism, and too much pruning in schizophrenia. Preliminary research in the Brown lab indicates that the P2Y6 receptor may be required for microglial phagocytosis of synapses; and behavioural testing of 3-4 month old P2Y6-/- mice indicates they have memory deficits. This project will further test whether one-month-old P2Y6-/- mice have social deficits and/or increased synaptic density, and whether this is due to reduced microglial phagocytosis of synapses.