Determining cell-type specificity for a nonclassical MHC class I during an activity-dependent cortical critical period.

确定活动依赖性皮质关键期非经典 MHC I 类的细胞类型特异性。

• 批准号: 10426738
• 负责人: Carla J Shatz
• 金额: $ 21.61万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10426738
• 关键词: Alleles; Alzheimer' s Disease; Amblyopia; Astrocytes; Axon; Brain; Brain Diseases; CRISPR/Cas technology; Cell surface; Cells; Cerebral cortex; Development; Disease; Gene Family; Genes; Goals; Histocompatibility; Homologous Gene; Human; Immune; Immune system; Immunologic Receptors; Impairment; In Situ Hybridization; Inflammation; Knockout Mice; Light; MHC Class I Genes; Mediating; Messenger RNA; Microglia; Molecular; Monitor; Morphology; Mus; Names; Natural regeneration; Neocortex; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neuroglia; Neurons; Ocular Dominance; Orthologous Gene; Pattern; Peripheral; Phenotype; Population; Process; Qa-1 Antigen; Regulation; Research; RiboTag; Role; Schizophrenia; Sensory; Shapes; Signal Transduction; Site; Specificity; Structure; Synapses; Synaptic plasticity; Synaptosomes; Testing; Thalamic structure; Visual; Visual Cortex; Work; autism spectrum disorder; cell type; critical developmental period; critical period; dark rearing; experience; experimental study; hippocampal pyramidal neuron; interest; member; monocular; neural circuit; novel; receptor; relating to nervous system; response; synaptic pruning; transcriptome sequencing; visual deprivation

7. Project Summary/ Abstract The long-term goal of this research is to understand how neural activity sculpts brain circuits during developmental critical periods. In cerebral cortex, synapses are pruned or stabilized in relation to levels and patterns of activity and normally this process occurs extensively during critical periods and is highly specific. In disease and inflammation, activation of glia can drive excessive pruning and perturb synaptic plasticity. Systematic approaches to treating pruning disorders in disease require understanding cell and molecular mechanisms normally engaged in synapse plasticity and remodeling. In healthy brain, we have discovered that Qa-1, a nonclassical Major Histocompatibility Class I (MHCI) molecule (gene name H2-T23; human HLA-E), is highly expressed in L6 of cerebral cortex and may contribute to activity-dependent plasticity in visual cortex. Qa- 1 has been studied in peripheral immune cells, but until now nothing was known about expression or function in healthy brain. However, Qa-1/HLA-E has been detected in neurons and glia in inflammation and Alzheimer's disease. Experiments proposed here test the hypothesis that Qa-1 expression in neurons restricts sensory- driven plasticity in visual cortex during the critical period. Two specific aims are proposed: 1) Identify cell type expression, activity-regulation and subcellular localization of Qa-1 mRNA in cortex. Cell types expressing Qa-1 mRNA will be identified by RNAScope in situ hybridization in combination with neuronal or glial-specific markers. Visual experience will be manipulated to test if Qa-1 expression is regulated, as occurs for many genes known to mediate activity-dependent plasticity. RiboTag cell type specific gene profiling will be used to detect Qa-1 mRNA isolated specifically from neurons, microglia and synaptosomes. The presence and enrichment of Qa-1 mRNA in L6 cortical neurons and synaptosomes will provide clues about how Qa-1 regulates plasticity during the critical period. 2) Generate a conditional allele of Qa-1 and explore cell-type specificity of Qa-1 function in activity-dependent plasticity. To dissect neuronal vs glial Qa-1 function, the Easi-CRISPR method (Quadros et al. 2017) will be used to insert loxP sites into the H2-T23 gene. Once a stable line is generated, Qa-1fl/fl mice will be crossed to Cre-expressing lines to generate mice lacking Qa-1 in neuronal vs glial cell populations. These lines will then be studied for activity-dependent phenotypes including ocular dominance plasticity and an activity- dependent microglial response. Results should reveal if Qa-1 acts in neurons, in glia or both for intact activity- dependent plasticity in visual cortex. If Qa-1 is required specifically in L6 neurons, results would imply a key role for this MHCI in a major circuit connecting thalamus and cortex. These studies of visual cortex should have broad significance because Qa-1 is expressed throughout neocortex. Moreover, the Qa-1 ortholog HLA-E is expressed in human cerebral cortex, so our studies may illuminate how this MHCI normally functions in human brain, and also contribute to understanding the basis for disorders in which activity-dependent synaptic plasticity and pruning go awry, as are thought to occur in Amblyopia, Autism, Schizophrenia and Alzheimer’s disease.

7.项目总结/摘要 这项研究的长期目标是了解神经活动如何塑造大脑回路， 发展的关键时期。在大脑皮层，突触被修剪或稳定的水平， 这一过程通常在关键时期广泛发生，并且具有高度的特异性。在 在疾病和炎症中，神经胶质的激活可以驱动过度修剪并扰乱突触可塑性。 治疗疾病中修剪障碍的系统方法需要了解细胞和分子 通常参与突触可塑性和重塑的机制。在健康的大脑中，我们发现 Qa-1是一种非经典的主要组织相容性I类（MHCI）分子（基因名称H2-T23;人HLA-E）， 在大脑皮层L 6区高表达，可能与视皮层的活动依赖性可塑性有关。啊- 1已经在外周免疫细胞中进行了研究，但直到现在还不知道在细胞中的表达或功能。 健康的大脑然而，Qa-1/HLA-E已在炎症和阿尔茨海默病的神经元和神经胶质中检测到。 疾病这里提出的实验验证了神经元中的Qa-1表达限制感觉神经元的假设。 在关键期视觉皮层的可塑性。提出了两个具体目标：1）识别细胞类型 Qa-1 mRNA在皮层的表达、活性调节和亚细胞定位。表达Qa-1的细胞类型 将通过RNAScope原位杂交结合神经元或神经胶质特异性标志物鉴定mRNA。 将操纵视觉体验来测试Qa-1表达是否受到调节，就像许多已知基因一样 来调节活动依赖的可塑性。RiboTag细胞类型特异性基因谱分析将用于检测Qa-1 专门从神经元、小胶质细胞和突触体中分离的mRNA。Qa-1的存在和富集 L 6皮层神经元和突触体中的mRNA将提供关于Qa-1如何在L 6皮层神经元和突触体中调节可塑性的线索。 关键时期。2)产生Qa-1的条件等位基因并探索Qa-1功能的细胞类型特异性， 活动依赖性可塑性为了剖析神经元与神经胶质Qa-1功能，使用了CRISPR方法（Quadros et al. 2017）将用于将loxP位点插入H2-T23基因中。一旦产生稳定系，Qa-1fl/fl小鼠将 与表达Cre的细胞系杂交，以产生在神经元细胞群与神经胶质细胞群中缺乏Qa-1的小鼠。这些 然后将研究品系的活动依赖性表型，包括眼优势可塑性和活动- 依赖性小胶质细胞反应。结果应该揭示Qa-1是否在神经元、神经胶质或两者中起作用以保持完整的活性- 视皮层的依赖可塑性如果Qa-1在L 6神经元中是特异性需要的，那么结果将暗示Qa-1在L 6神经元中的关键作用。 连接丘脑和皮层的主要回路中的MHCI。这些视觉皮层的研究应该有广泛的 因为Qa-1在整个新皮层中表达。此外，Qa-1直系同源物HLA-E表达为 因此，我们的研究可能会阐明这种MHCI在人脑中的正常功能， 也有助于理解活动依赖性突触可塑性和 修剪出错，就像被认为发生在弱视、自闭症、精神分裂症和阿尔茨海默氏症中一样。