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: 10705621
• 负责人: Carla J Shatz
• 金额: $ 25.48万
• 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/10705621
• 关键词: Alleles; Alzheimer' s Disease; Amblyopia; Astrocytes; Atlases; 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; Proteins; 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; neocortical; neural; neural circuit; novel; receptor; 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.项目摘要/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 发展关键期。在大脑皮层，突触被修剪或稳定与水平和 活动的模式，通常这一过程在关键时期广泛发生，具有高度的特殊性。在……里面 疾病和炎症，胶质细胞的激活可以驱动过度修剪和扰乱突触的可塑性。 治疗疾病中的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H2-T23；人类人类白细胞抗原-E)，是 在大脑皮层L6中高表达，可能与视皮层的活动依赖性可塑性有关。QA- 1已经在外周免疫细胞中进行了研究，但到目前为止还不知道它的表达或功能。 健康的大脑。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Qa-1/HLA-E 疾病。这里提出的实验验证了这样的假设，即QA-1在神经元中的表达限制了感觉- 关键期视皮层的驱动可塑性。提出了两个具体目标：1)鉴定细胞类型 QA-1mRNA在大脑皮层的表达、活性调节及亚细胞定位。表达Qa-1的细胞类型 MRNA将通过RNAScope原位杂交结合神经元或神经胶质特异性标记物进行鉴定。 视觉体验将被操纵来测试Qa-1的表达是否受到调控，就像许多已知基因所发生的那样 来调节依赖活动的可塑性。将使用RiboTag细胞类型特定基因图谱来检测Qa-1 特异性地从神经元、小胶质细胞和突触体中分离出的mRNA。QA-1的存在和浓缩 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 关键时期。2)构建Qa-1条件等位基因，探索Qa-1功能的细胞类型特异性 依赖活性的可塑性。为了分析神经元和神经胶质细胞的Qa-1功能，EASI-CRISPR方法(Quadros et 艾尔2017)将用于将loxP位点插入到H2-T23基因中。一旦产生了稳定的品系，Qa-1fl/fl小鼠将 与表达Cre的品系杂交，产生神经细胞和神经胶质细胞群体中缺乏Qa-1的小鼠。这些 然后将研究LINE的依赖于活动的表型，包括眼睛优势可塑性和活动- 依赖性小胶质细胞反应。结果应该揭示Qa-1是作用于神经元，还是作用于神经胶质细胞，还是两者兼而有之，以实现完整的活动- 视皮层的依赖可塑性。如果QA-1在L6神经元中是特定需要的，结果将暗示一个关键角色 这个MHCI位于连接丘脑和皮质的主要回路中。这些对视皮层的研究应该有广泛的 意义是因为Qa-1在新皮质中表达。此外，还表达了Qa-1同源基因人类白细胞抗原-E 因此，我们的研究可能会阐明这种MHCI在人脑中的正常功能，以及 也有助于理解疾病的基础，在这些疾病中，活动依赖性突触可塑性和 修剪会出错，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。