Analysis and manipulation of astrocyte heterogeneity after stroke

描述（由申请人提供）：中风是死亡和残疾的主要原因，但几乎没有治疗方案可用于促进中风恢复。虽然人们早就知道中风后星形胶质细胞形态以分级方式变化，但对星形胶质细胞经历的确切变化或这些反应可被操纵以促进恢复的方式知之甚少。反应性星形胶质细胞被认为是中枢神经系统损伤的简单瘢痕形成细胞。然而，在一些损伤模型中，星形胶质细胞对恢复具有明显的作用，这取决于时间、位置和星形胶质细胞表型，但这在中风中尚未研究。在这里，我建议确定星形胶质细胞的表型子集和操纵这些子集内的关键星形胶质细胞分泌的蛋白质，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将研究星形胶质细胞的形态学、表型和转录组学变化，以鉴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，将在梗死周围皮质中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，清楚地描绘了星形胶质细胞的完整结构，并允许详细分析星形胶质细胞长度，极性，复杂性和结构域大小的变化。表型分析将使用特定的星形胶质细胞标志物进行，这些标志物揭示了与组织修复相关的星形胶质细胞功能的不同方面。最后，不同区域的星形胶质细胞所经历的不同转录组学变化将使用一系列小鼠来确定，其中标记的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异性的方式改变。促进神经修复的星形胶质细胞分泌蛋白是一种新发现的和不断增长的蛋白质类，对中风知之甚少;它们的区域特异性可能在它们的作用中起着至关重要的作用。已知有三种主要的蛋白质从星形胶质细胞分泌并影响突触形成和神经元修复：1）帮助形成突触的蛋白质，2）拮抗第一类蛋白质的蛋白质，和3）帮助突触变得电活性的蛋白质。初步结果表明，这些类别的差异调节不同的星形胶质细胞亚群后， 中风我将首先全面分析这些分子在中风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，因此将外源性蛋白局部递送至那些在中风后不表达它们的星形胶质细胞亚群可能改善恢复。同时，降低2类蛋白的表达可能允许1类和3类蛋白的修复介导作用被揭示。这些修饰将使用星形胶质细胞特异性递送范例的局部递送来进行。

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.