Calreticulin and other candidate Alarmins and Opsonins from Microglia

博士项目战略主题：通过生物科学全面了解健康警报是从受损细胞中释放的可溶分子，可促进免疫细胞的非感染性炎症激活。这种激活对于启动对组织损伤或疾病的早期先天免疫反应很重要。钙网蛋白是内质网的胞内伴侣蛋白，是小胶质细胞的候选蛋白，小胶质细胞是大脑中常驻的巨噬细胞。在旋转项目中，发现钙网蛋白从受损细胞中释放出来，在体外化学吸引小胶质细胞，并刺激它们释放与炎症表型相关的细胞因子肿瘤坏死因子-α和白介素6。因此，钙网蛋白可能在脑组织损伤和小胶质细胞对损伤的免疫反应之间起到中介作用。首先，通过了解涉及的机制途径，包括细胞释放的机制，以及小胶质细胞上钙网蛋白及其下游信号的受体。其次，小胶质细胞在炎症状态和神经毒性方面的后果。第三，将测试钙网蛋白是否在突触、髓鞘和神经元的小胶质细胞吞噬中起调理作用。第四，如果钙网状蛋白起调理蛋白的作用，那么是否会促进抗原呈递给T细胞将会被确定。为了确定是否在体内发生了钙网织蛋白释放，我们将检测有无脑外伤患者脑脊液中的钙网织蛋白和细胞因子水平。同样，将在包括脑损伤在内的各种疾病模型的大鼠和小鼠的脑脊液中测量钙网织蛋白和细胞因子的水平。样本将在损伤后的多个采集时间点采集，以更好地阐明钙网织蛋白释放的生理模式，并将其与炎症情况联系起来。由于小胶质细胞的免疫反应是一个涉及众多复合体和分子的多层过程，因此，除了钙网织蛋白外，还将研究其他可溶性介质作为小胶质细胞警报蛋白和/或调理蛋白的潜力。原代小胶质细胞在各种条件下的分泌体将被获取、纯化，然后通过质谱学分析得到可识别的蛋白质。然后，将评估候选蛋白质作为如上所述的小胶质细胞的警示蛋白或调理蛋白的能力。在这个项目的同时，将对小胶质细胞受体P2Y6进行评估，以测试它是否在发育中的突触吞噬(修剪)中起作用。突触修剪太少与自闭症有关，而太多修剪与精神分裂症有关。布朗实验室的初步研究表明，突触的小胶质细胞吞噬可能需要P2Y6受体；3-4个月大的P2Y6-/-小鼠的行为测试表明它们存在记忆缺陷。该项目将进一步测试一个月大的P2Y6-/-小鼠是否有社交缺陷和/或突触密度增加，以及这是否由于突触的小胶质细胞吞噬功能减少所致。

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.