Analysis and manipulation of astrocyte heterogeneity after stroke

描述(由申请人提供)：中风是导致死亡和残疾的主要原因，但几乎没有可用的治疗方案来促进中风的康复。虽然人们早就知道中风后星形胶质细胞的形态会以分级的方式发生变化，但对星形胶质细胞所经历的确切变化或这些反应可能被操纵以促进恢复的方式知之甚少。反应性星形胶质细胞在中枢神经系统损伤中被认为是单纯的瘢痕形成细胞。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的表型，但这在中风中尚未被研究。在这里，我建议识别星形胶质细胞的表型亚群，并操纵这些亚群中星形胶质细胞分泌的关键蛋白，以确定星形胶质细胞对中风的反应的分子机制，以及它们对组织修复的影响。目的1研究星形胶质细胞的形态、表型和转录改变，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的形态，几种驱动荧光团表达的星形胶质细胞特异性慢病毒载体之一将被注射到梗死灶周围的皮质中。这些荧光蛋白遍布整个细胞，清楚地勾勒出星形胶质细胞的完整结构，并允许对星形胶质细胞的长度、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，揭示与组织修复相关的星形细胞功能的不同方面。最后，将使用标记核糖体以星形胶质细胞特有的方式结合激光捕获显微镜表达的小鼠品系来确定星形胶质细胞不同区域经历的不同转录变化。在目标2中，影响突触形成和神经修复的星形胶质细胞分泌的分子将以亚群特有的方式改变。促进神经修复的星形胶质细胞分泌的蛋白质是一类新发现的、不断增长的蛋白质，在中风中知之甚少；它们的区域特异性可能在它们的作用中发挥关键作用。从星形胶质细胞分泌的影响突触形成和神经元修复的蛋白质主要有三类：1)那些帮助形成突触的蛋白质，2)那些拮抗第一类蛋白质的蛋白质，以及3)那些帮助突触变得电活跃的蛋白质。初步结果表明，这些类别在不同的星形细胞亚群中有不同的调节 卒中。我将首先全面分析这些分子在卒中后不同星形细胞亚群中的表达模式。这些信息将为干预性研究提供信息。由于1类和3类蛋白在作用上是互补的，局部将外源蛋白输送到卒中后不表达它们的星形细胞亚群可能会促进恢复。同时，减少2类蛋白的表达可能允许1类和3类蛋白的修复中介作用被揭示出来。这些修改将使用星形胶质细胞特定的递送范例的局部递送来执行。

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.