Function of reactive astrocytes in aging and neurodegenerative disease

反应性星形胶质细胞在衰老和神经退行性疾病中的功能

• 批准号: 10480931
• 负责人: Rachel Battaglia
• 金额: $ 8.59万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10480931
• 关键词: Affect; Age; Aging; Alexander Disease; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease model; Antibodies; Astrocytes; Biological Assay; Biology; Brain; CASP6 gene; CRISPR/Cas technology; Calcium Signaling; Cells; Coculture Techniques; Cytoskeleton; Data; Development; Disease; Disease Progression; Drosophila genus; Environment; Extracellular Matrix; Fiber; Functional disorder; Future; Gene Expression; Generations; Genes; Genetic; Glial Fibrillary Acidic Protein; Glycoproteins; Glypican; Goals; Heparitin Sulfate; Heterogeneity; Human; In Vitro; Inflammation; Intermediate Filament Proteins; Knock-out; Lead; Link; Maintenance; Mass Spectrum Analysis; Measures; Mechanics; Mediating; Missense Mutation; Modeling; Modification; Molecular; Morphology; Mus; Mutate; Mutation; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neuroglia; Neurons; Pathologic; Pathway interactions; Patients; Pharmacology; Phase; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Proteins; Proteoglycan; Proteolysis; Proteome; Proteomics; Research; Research Personnel; Research Project Grants; Role; Secretory Component; Severity of illness; Signal Transduction; Site; System; Techniques; Testing; Toxic effect; Training; Treatment Efficacy; Work; autosomal dominant mutation; axon growth; base; career; casein kinase; casein kinase II; cell type; central nervous system injury; disorder control; experience; experimental study; fly; genetic approach; in vitro Model; in vivo; induced pluripotent stem cell; leukodystrophy; mouse model; mutant; nervous system development; neurogenesis; neuronal growth; normal aging; novel; novel therapeutic intervention; prevent; programs; protein aggregation; proteostasis; response to injury; small molecule inhibitor; synaptogenesis; targeted treatment

ABSTRACT/PROJECT SUMMARY Reactive astrocytes (RAs) are a feature of normal aging and neurodegeneration. RAs drastically change their morphology and gene expression, notably increasing the expression of glial fibrillary acidic protein (GFAP) in response to injury or inflammation. GFAP is the major intermediate filament protein of mature astrocytes. Autosomal dominant mutations in GFAP cause the rare and fatal leukodystrophy, Alexander Disease (AxD). In AxD patients, astrocytes accumulate pathological GFAP aggregates (Rosenthal fibers; RFs) and become reactive. However, the mechanisms linking >70 different GFAP mutations to RF formation and other disease- relevant phenotypes in AxD remain unknown. My extensive preliminary data show that aberrant phosphorylation promotes GFAP aggregation, and that this modification is a marker of AxD severity, independently of the disease mutation. Further, I show that site-specific GFAP phosphorylation is associated with increased proteolysis by caspase-6, but whether the two are directly linked is unknown. I hypothesize that coordinated cross-talk between casein kinase (CK2) and caspase-6 promotes defective GFAP proteostasis to exacerbate the reactive phenotype of AxD astrocytes. For the F99 phase, I propose to use pharmacological and genetic strategies to inhibit CK2 and caspase-6 activity in order to characterize their roles in vitro using the astrocyte model that I developed (Aim 1.1), and in vivo utilizing an AxD mouse model (Aim 1.2). I will master iPSC gene editing with CRISPR/Cas9 to generate CK2 and caspase-6 knockouts and iPSC handling and differentiation to astrocyte and neurons (Aim 1.1), and I will apply these techniques to my postdoctoral project (Aim 2). For the K00 phase, I will investigate the functions of RAs in Alzheimer's disease in the lab of Dr. Mel Feany. Proteoglycans (PGs) are among the most highly upregulated genes in aging and RAs. Preliminary data from Dr. Feany's lab identified genetic interactions between PGs and models of neurodegeneration in the fly. I hypothesize that RAs produce an imbalance of PGs in the extracellular matrix, which creates an environment that is inhibitory to neuronal growth and remodeling. To model the mechanical changes known to occur in AD brain, I will develop a novel model to study RAs by culturing iPSC-astrocytes on substrates of different stiffness. Additionally, I will generate knockouts of candidate PGs in iPSCs and differentiate them to reactive and non-reactive astrocytes. I will use in vivo fly models and co-cultures of iPSC- astrocytes and neurons to examine the role of PGs in toxicity of RAs. My thesis project and my future postdoctoral studies will provide a rich training experience that will prepare me for a career as an independent investigator leading a rigorous research program at the nexus of aging and glial biology.

摘要/项目摘要 反应性星形胶质细胞（RAS）是正常衰老和神经变性的特征。拉斯急剧改变他们的 形态和基因表达，特别是增加了神经纤维纤维酸性蛋白（GFAP）在中的表达 对伤害或炎症的反应。 GFAP是成熟星形胶质细胞的主要中间细丝蛋白。 GFAP中的常染色体显性突变会导致稀有和致命的白细胞营养不良，亚历山大病（AXD）。在 AXD患者，星形胶质细胞积累病理GFAP骨料（Rosenthal纤维； RFS），并成为 反应性。但是，将> 70种不同GFAP突变与RF形成和其他疾病的机制联系起来 AXD中的相关表型仍然未知。我广泛的初步数据表明异常 磷酸化促进GFAP聚集，并且这种修饰是AXD严重程度的标志， 独立于疾病突变。此外，我表明特定地点的GFAP磷酸化是相关的 caspase-6的蛋白水解增加，但是两者是否直接链接是未知的。我假设 酪蛋白激酶（CK2）和caspase-6之间的协调串扰促进了GFAP缺陷 蛋白毒酸加剧AXD星形胶质细胞的反应性表型。对于F99阶段，我建议使用 抑制CK2和caspase-6活性的药理和遗传策略，以表征其角色 在体外使用我开发的星形胶质细胞模型（AIM 1.1）和使用AXD鼠标模型的体内（AIM 1.2）。我将使用CRISPR/CAS9掌握IPSC基因编辑，以生成CK2和CASPASE-6淘汰赛和IPSC 对星形胶质细胞和神经元的处理和差异化（AIM 1.1），我将将这些技术应用于我的 博士后项目（AIM 2）。对于K00阶段，我将研究RAS在阿尔茨海默氏病中的功能 梅尔·费尼博士的实验室。蛋白聚糖（PGS）是衰老和 拉斯。 Feany博士实验室的初步数据确定了PGS与模型之间的遗传相互作用 神经退行性变化。我假设RA在细胞外基质中产生PG的不平衡， 它创造了一种抑制神经元生长和重塑的环境。建模机械 在AD大脑中已知发生的变化，我将开发一种新型模型来研究RAS，通过在 不同刚度的底物。此外，我将在IPSC中生成候选PG的淘汰 将它们区分为反应性和非反应性星形胶质细胞。我将使用IPSC-的体内飞行模型和共同培养 星形胶质细胞和神经元检查PG在RAS毒性中的作用。我的论文项目和未来 博士后研究将提供丰富的培训经验，这将使我成为独立的职业 研究人员领导了衰老和神经胶质生物学联系的严格研究计划。