The roles of microglial phagocytosis in ageing-induced synaptic, neuronal and memory loss

在老鼠和人类的衰老过程中，大脑都会失去突触、白质以及学习和记忆能力，这可能会随着年龄的增长严重限制工作、功能和生活质量。小胶质细胞是在特定情况下吞噬(吞噬)突触和神经元的脑巨噬细胞。这个项目测试了年龄引起的突触和记忆的丧失是否是由于小胶质细胞吞噬这些突触所致。该项目还可能调查突触发育迟缓丧失的原因(人类的青春期，老鼠的1-4个月)。该项目将汇集布朗实验室的小胶质信号通路和功能方面的专业知识，并以礼来神经科学小组为基础，对大脑功能随年龄变化的适应性产生浓厚兴趣。P2Y6是神经元吞噬小胶质细胞所需的受体，布朗教授的实验室发现，在炎症、帕金森氏症和阿尔茨海默病模型中，P2Y6受体基因敲除的小鼠受到保护。P2Y12受体是一种相关受体，需要将小胶质细胞招募到应激神经元。TREM2是一种小胶质细胞受体，需要吞噬各种靶标，可能包括突触。小鼠从大约12个月大的时候就失去了海马区突触、神经元和记忆，文献中有证据表明这是由突触和神经元的小胶质细胞吞噬所介导的。在初步研究中，布朗教授的实验室发现，在培养中，突触的小胶质细胞吞噬需要P2Y6受体，而老年小鼠(16个月)会失去对新物体和新位置的长期记忆，这在P2Y6受体基因敲除小鼠中是被阻止的。因此，我们现在想知道阻断P2Y6或其他小胶质细胞受体是否能防止与衰老相关的突触和/或神经元的丢失。这项建议的目标是：1)确定P2Y6受体敲除或抑制对培养的突触的小胶质细胞吞噬功能的影响；2)确定P2Y6受体敲除或抑制对衰老引起的突触、神经元和记忆丧失的影响。3)研究其他小胶质细胞受体(包括TREM2和P2Y12)对小胶质细胞吞噬突触的作用。项目工作可以包括：a)对wt和P2Y6受体敲除小鼠(可能还有TREM2和P2Y12受体敲除小鼠)的大脑进行组织分析，以分析1、4、12和/或16月龄不同脑区突触、神经元和髓鞘的密度，以及b)在12至16个月龄时阻断P2Y6受体，无论是使用P2Y6R抑制剂还是诱导基因敲除，并测量：13和16个月时的学习和记忆，随后进行突触、神经元和髓鞘密度的组织学分析D)WT和转基因小鼠的海马脑切片，用于炎症时突触、神经元和髓鞘的小胶质细胞吞噬的成像，和/或)体外调节分离突触的小胶质细胞吞噬的因素的分析。该项目将包括固定脑切片的图像分析，以及活细胞和切片培养工作，以及调节分离突触的小胶质细胞吞噬的特征信号的特征。小胶质细胞将从老化的大脑中分离出来，并以RNAseq为特征，需要生物信息学和数据处理。学生还将学习如何描述老年老鼠的行为特征。这个项目解决了BBSRC的战略主题：生物科学促进健康，通过揭示正常大脑生理背后的生物学机制，以维持一生的健康。

During ageing of both mice and humans, the brain loses synapses, white matter and the ability to learn and remember, which can severely limit work, function and quality of life with age. Microglia are brain macrophages that eat (phagocytose) synapses and neurons in specific circumstances. This project tests whether age-induced lose of synapses and memory is due to microglial phagocytosis of these synapses. The project may also investigate the causes of the late developmental loss of synapses (adolescence in humans, 1-4 months in mice). The project will bring together expertise on microglial signalling pathways and function, based in the Brown lab, with a strong interest in adaptations in brain function as humans' age, based in the Eli Lilly Neuroscience group.P2Y6 is a receptor required for microglial phagocytosis of neurons, and Prof. Brown's lab have found that P2Y6 receptor knockout mice are protected in models of inflammation, Parkinson's and Alzheimer's disease. The P2Y12 receptor is a related receptor required to recruit microglia to stressed neurons. TREM2 is a microglial receptor required for phagocytosis of a variety of targets, possibly including synapses. Mice lose hippocampal synapses, neurons and memory from about 12 months of age, and there is evidence in the literature that this is mediated by microglial phagocytosis of synapses and neurons. In preliminary research, Prof. Brown's lab have found that the P2Y6 receptor is required for microglial phagocytosis of synapses in culture, and that aged mice (16 months) lose long- term memory of novel objects and novel locations, which is prevented in the P2Y6 receptor knockout mice. So we now want to find out whether blocking the P2Y6 or other microglial receptors prevents the synaptic and/or neuronal loss associated with aging.The goals of this proposal are to:1) Determine the effects of P2Y6 receptor knockout or inhibition on microglial phagocytosis of synapses in culture,2) Determine the effects of P2Y6 receptor knockout or inhibition on aging-induced loss of synapses, neurons and memories, and3) Investigate the role of other microglial receptors (including TREM2 and P2Y12) on microglial phagocytosis of synapses.The project work may consist of:a) histological analysis of brains from wt and P2Y6 receptor knockout mice (and potentially also TREM2 and P2Y12 receptor knockout mice) to analyse density of synapses, neurons and myelin in various brain regions at 1, 4, 12 and/or 16 months old, andb) blocking the P2Y6 receptor from 12 to 16 months of age , either using a P2Y6R inhibitor or induced-knockout of the gene, and measuring: learning and memory at 13 and 16 months, followed by histological analysis of density of synapses, neurons and myelin, andc) analysis of wild-type and transgenic microglia at 4 and 16 months for: synaptic, neuronal and myelin markers, and general RNA expression by RNAseq.d) hippocampal brain slices from wt and transgenic mice, imaged for microglial phagocytosis of synapses, neurons and myelin when inflamed, and/ore) analysis of factor regulating microglial phagocytosis of isolated synapses in vitro.The project would involve image analysis of fixed brain sections, but also live cell and slice culture work, and characterising signals mediating microglial phagocytosis of isolated synapses. Microglia will be sorted from the aged brain and characterised by RNAseq, requiring bioinformatics and data handling. The student will also learn how to behaviourally characterise aged mice.This project addresses the BBSRC Strategic Theme: Bioscience for Health, by revealing the biological mechanisms underlying normal brain physiology, in order to sustain wellbeing throughout life.