Analysis and manipulation of astrocyte heterogeneity after stroke

中风后星形胶质细胞异质性的分析和处理

• 批准号: 9207802
• 负责人: Amy J. Gleichman
• 金额: $ 6.1万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9207802
• 关键词: Affect; Architecture; Astrocytes; Astrocytosis; Automobile Driving; Blood Vessels; Brain; Cause of Death; Cells; Cessation of life; Characteristics; Cicatrix; Clinical Trials; Complementary DNA; Development; Diffuse; Gene Expression; Glial Fibrillary Acidic Protein; Heterogeneity; Immunoprecipitation; Infarction; Inflammation; Inflammatory; Injectable; Injury; Intervention Studies; Lasers; Length; Lentivirus Vector; Location; Maps; Mediating; Messenger RNA; Microscopy; Mitosis; Modeling; Modification; Molecular; Molecular Profiling; Morphology; Mus; Neuroimmune; Neurons; Pattern; Phenotype; Play; Population; Process; Proteins; Recovery; Ribosomes; Role; Signal Transduction; Small Interfering RNA; Specificity; Stroke; Synapses; System; Time; Tissues; Translating; United States; Viral Vector; Virus; Work; base; central nervous system injury; disability; experimental study; fluorophore; improved; injury and repair; laser capture microdissection; neuron loss; novel; post stroke; prevent; public health relevance; relating to nervous system; repaired; response; response to injury; stroke recovery; synaptogenesis; targeted delivery; tissue repair; tool; transcriptome; transcriptome sequencing; transcriptomics; white matter

DESCRIPTION (provided by applicant): Stroke is a leading cause of death and disability, yet there are few treatment options available to enhance stroke recovery. While it has long been known that astrocyte morphology changes in a graded fashion after stroke, little is known about the exact changes astrocytes undergo or the ways in which these responses may be manipulated to promote recovery. Reactive astrocytes have been considered as simple scar-forming cells in CNS injury. However, astrocytes have distinct effects on recovery depending on timing, location and astrocyte phenotype in some injury models, but this has not been studied in stroke. Here, I propose to identify phenotypic subsets of astrocytes and manipulate key astrocyte-secreted proteins within those subsets to determine molecular mechanisms of astrocyte responses to stroke, and their impact on tissue repair. Aim 1 will examine the morphologic, phenotypic, and transcriptomic changes astrocytes undergo, in order to identify subpopulations of astrocytes with functional roles in tissue repair. To explore astrocyte morphology, one of several astrocyte-specific lentiviral vectors that drive expression of fluorophores will be injected in the peri-infarct cortex. These fluorescent proteins diffuse throughout the cell, clearly delinating the full architecture of the astrocyte and allowing a detaied analysis of changes in astrocyte length, polarity, complexity, and domain size. Phenotypic analysis will be conducted using specific astrocytic markers that reveal different aspects of astrocytic function that are related to tissue repair. Finally, the different transcriptomic change that different zones of astrocytes undergo will be determined using a line of mice in which tagged ribosomes are expressed in an astrocyte-specific manner combined with laser capture microscopy. In Aim 2, astrocyte-secreted molecules that affect synapse formation and neural repair will be altered in subpopulation-specific fashions. Astrocyte-secreted proteins that promote neural repair are a newly identified and growing class of proteins about which little is known in stroke; their regional specificity is likely to play a crucial role in their action. Thereare three main known classes of protein that are secreted from astrocytes and affect synapse formation and neuronal repair: 1) those that help form synapses, 2) those that antagonize the first class, and 3) those that help synapses become electrically active. Preliminary results suggest that these classes are differentially regulated in different astrocytic subpopulations post stroke. I will first fully analyze the expression patterns of these molecules in different astrocytc subpopulations post-stroke. This information will inform interventional studies. As class 1 and 3 proteins are complementary in action, local delivery of exogenous proteins to those astrocytic subpopulations that do not express them post-stroke may improve recovery. Simultaneously, decreasing expression of class 2 proteins may allow the repair-mediating effects of class 1 and 3 proteins to be revealed. These modifications will be performed using localized delivery of astrocyte-specific delivery paradigms.

描述（由申请人提供）：中风是导致死亡和残疾的主要原因，但很少有治疗方案可以提高中风的恢复。虽然人们早就知道，脑卒中后星形胶质细胞的形态会发生分级变化，但人们对星形胶质细胞发生的确切变化或这些反应可能被操纵以促进康复的方式知之甚少。反应性星形胶质细胞被认为是中枢神经系统损伤中简单的瘢痕形成细胞。然而，在一些损伤模型中，星形胶质细胞对恢复的影响取决于时间、位置和星形胶质细胞表型，但这在中风中尚未得到研究。在这里，我建议鉴定星形胶质细胞的表型亚群，并在这些亚群中操纵关键的星形胶质细胞分泌蛋白，以确定星形胶质细胞对中风反应的分子机制，以及它们对组织修复的影响。目的1将研究星形胶质细胞所经历的形态、表型和转录组变化，以确定在组织修复中具有功能作用的星形胶质细胞亚群。为了探索星形胶质细胞形态，将在梗死周围皮层注射驱动荧光团表达的几种星形胶质细胞特异性慢病毒载体之一。这些荧光蛋白在整个细胞中扩散，清晰地描绘了星形胶质细胞的完整结构，并允许对星形胶质细胞长度、极性、复杂性和结构域大小的变化进行详细分析。表型分析将使用特定的星形细胞标记物进行，这些标记物揭示了与组织修复相关的星形细胞功能的不同方面。最后，星形胶质细胞的不同区域所经历的不同转录组变化将通过一组小鼠来确定，在这些小鼠中，标记核糖体以星形胶质细胞特异性的方式结合激光捕获显微镜来表达。在Aim 2中，星形胶质细胞分泌的影响突触形成和神经修复的分子将以亚群体特异性的方式改变。星形胶质细胞分泌的促进神经修复的蛋白质是一种新发现和不断增长的蛋白质，对中风知之甚少；它们的区域特殊性可能在其行动中发挥关键作用。目前已知的星形胶质细胞分泌的蛋白质主要有三类，它们影响突触的形成和神经元的修复：1)帮助形成突触的蛋白质，2)对抗第一类蛋白质，以及3)帮助突触变得电活性的蛋白质。初步结果表明，这些类别在不同的星形细胞亚群中受到不同的调控