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。

基本信息

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
项目类别：
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&apos 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

项目摘要

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. 项目概要/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在大脑皮层的发育关键期，突触的水平和水平被修剪或稳定。活动模式，通常这个过程主要发生在关键时期并且是高度具体的。疾病和炎症、神经胶质细胞的激活会导致过度修剪并扰乱突触可塑性。治疗疾病修剪失调的系统方法需要了解细胞和分子我们发现，在健康的大脑中，通常参与突触可塑性和重塑的机制。 Qa-1 是一种非经典主要组织相容性 I 类 (MHCI) 分子（基因名称 H2-T23；人类 HLA-E），是在大脑皮层 L6 中高度表达，可能有助于视觉皮层 Qa- 的活动依赖性可塑性。 1 已在外周免疫细胞中进行了研究，但到目前为止，人们对它的表达或功能还一无所知。然而，在炎症和阿尔茨海默病的神经元和神经胶质细胞中检测到了 Qa-1/HLA-E。这里提出的实验检验了神经元中 Qa-1 表达限制感觉的假设。提出了关键时期视觉皮层驱动可塑性的两个具体目标：1）识别细胞类型。皮质中 Qa-1 mRNA 的表达、活性调节和亚细胞定位表达 Qa-1 的细胞类型。 mRNA 将通过 RNAScope 原位杂交结合神经或神经胶质特异性标记进行鉴定。将操纵视觉经验来测试 Qa-1 表达是否受到调节，正如许多已知基因所发生的那样介导活性依赖性可塑性，RiboTag 细胞类型特异性基因分析将用于检测 Qa-1。从神经元、小胶质细胞和突触体中特异性分离的 mRNA Qa-1 的存在和富集。 L6 皮质神经元和突触体中的 mRNA 将为 Qa-1 如何调节可塑性提供线索 2) 生成 Qa-1 的条件等位基因并探索 Qa-1 功能的细胞类型特异性为了剖析神经与神经胶质 Qa-1 功能，Easi-CRISPR 方法（Quadros 等人） al. 2017）将用于将 loxP 位点插入 H2-T23 基因中，一旦生成稳定的品系，Qa-1fl/fl 小鼠将被使用。与表达 Cre 的细胞系杂交，产生神经细胞和神经胶质细胞群中缺乏 Qa-1 的小鼠。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- 结果应该揭示 Qa-1 是否在神经元、神经胶质细胞或两者中发挥完整的活性。如果 L6 神经元特别需要 Qa-1，那么结果将意味着关键作用。对于连接丘脑和皮层的主要回路中的 MHCI，这些对视觉皮层的研究应该具有广泛的意义。意义在于，Qa-1 在整个新皮质中表达，此外，Qa-1 直系同源物 HLA-E 也得到表达。在人类大脑皮层中，所以我们的研究可能会阐明这种 MHCI 在人类大脑中的正常功能，以及也有助于理解活动依赖性突触可塑性和修剪出现问题，弱视、自闭症、精神分裂症和阿尔茨海默病都会出现这种情况。