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

基本信息

批准号：
10045701
负责人：
Rachel Battaglia
金额：
$ 3.11万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2021-08-06
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10045701
关键词：
Affect Age Aging Alexander Disease Alzheimer&apos s Disease Alzheimer&apos s disease brain Alzheimer&apos 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 Gap Junctions 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 therapeutics prevent programs protein aggregation proteostasis response to injury small molecule inhibitor synaptogenesis targeted treatment

项目摘要

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 crosstalk 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 astrocytes 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.

反应性星形胶质细胞（RA）是正常衰老和神经退行性变的一个特征。 RA 极大地改变了它们的形态和基因表达，特别是在损伤或炎症反应中增加了胶质纤维酸性蛋白 (GFAP) 的表达。 GFAP 是成熟星形胶质细胞的主要中间丝蛋白。 GFAP 的常染色体显性突变会导致罕见且致命的脑白质营养不良，即亚历山大病 (AxD)。在 AxD 患者中，星形胶质细胞积聚病理性 GFAP 聚集体（罗森塔尔纤维；RF）并变得活跃。然而，将超过 70 种不同的 GFAP 突变与 AxD 中 RF 形成和其他疾病相关表型联系起来的机制仍然未知。我的大量初步数据表明，异常磷酸化会促进 GFAP 聚集，并且这种修饰是 AxD 严重程度的标志，与疾病突变无关。此外，我还表明位点特异性 GFAP 磷酸化与 caspase-6 的蛋白水解作用增强相关，但两者是否直接相关尚不清楚。我推测酪蛋白激酶 (CK2) 和 caspase-6 之间的协调串扰会促进有缺陷的 GFAP 蛋白稳态，从而加剧 AxD 星形胶质细胞的反应表型。对于 F99 阶段，我建议使用药理学和遗传策略来抑制 CK2 和 caspase-6 活性，以便使用我开发的星形胶质细胞模型（目标 1.1）在体外表征它们的作用，并使用 AxD 小鼠模型在体内表征它们的作用（目标 1.2)。我将掌握使用 CRISPR/Cas9 进行 iPSC 基因编辑，以生成 CK2 和 caspase-6 敲除以及 iPSC 处理和分化为星形胶质细胞和神经元（目标 1.1），并将这些技术应用于我的博士后项目（目标 2）。对于K00阶段，我将在Mel Feany博士的实验室研究RA在阿尔茨海默病中的功能。蛋白多糖 (PG) 是衰老和 RA 中表达最高度上调的基因之一。 Feany 博士实验室的初步数据确定了果蝇中 PG 与神经退行性变模型之间的遗传相互作用。我假设 RA 会导致细胞外基质中 PG 失衡，从而创造一个抑制神经元生长和重塑的环境。为了模拟 AD 大脑中已知发生的机械变化，我将开发一种新模型，通过在不同硬度的基质上培养 iPSC 星形胶质细胞来研究 RA。此外，我将在 iPSC 中敲除候选 PG，并将它们区分为反应性和非反应性星形胶质细胞。我将使用体内果蝇模型以及 iPSC-星形胶质细胞和神经元的共培养物来检查 PG 在 RA 毒性中的作用。我的论文项目和未来的博士后研究将提供丰富的培训经验，使我能够成为一名独立研究者，领导衰老和神经胶质生物学关系的严格研究项目。