Function of reactive astrocytes in aging and neurodegenerative disease

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。RAS彻底改变了他们的 胶质纤维酸性蛋白(GFAP)的表达显著增加 对损伤或炎症的反应。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导致罕见和致命的脑白质营养不良，亚历山大病(AxD)。在……里面 AxD患者，星形胶质细胞积聚病理性GFAP聚集体(Rosenthal纤维；RFs)并成为 被动的。然而，将&gt；70种不同的GFAP突变与RF形成和其他疾病- AxD的相关表型尚不清楚。我大量的初步数据显示，反常 磷酸化促进GFAP聚集，这种修饰是AxD严重程度的标志， 不受疾病突变的影响。此外，我还证明了特定部位的GFAP磷酸化与 随着caspase-6对蛋白质的降解增加，但这两者是否直接相关尚不清楚。我假设 酪蛋白激酶(CK2)和Caspase-6之间的协同信号促进GFAP缺陷 蛋白抑制作用加剧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活性的药物和遗传策略，以确定它们的作用 在体外使用我开发的星形胶质细胞模型(AIM 1.1)，在体内使用AxD小鼠模型(AIM 1.2)。我将掌握使用CRISPR/Cas9进行IPSC基因编辑以生成CK2和Caspase-6基因敲除和IPSC 处理和分化星形胶质细胞和神经元(目标1.1)，我将把这些技术应用到我的 博士后项目(目标2)。对于K00期，我将研究RAS在阿尔茨海默病中的功能 梅尔·费尼博士的实验室。蛋白多糖(PG)是衰老和衰老过程中表达最高的基因之一。 拉斯。来自Feany博士实验室的初步数据确定了PGs和模型之间的遗传交互作用 果蝇的神经退化。我假设RAS会在细胞外基质中产生不平衡的PG， 这创造了一种抑制神经元生长和重塑的环境。建立机械模型的步骤 已知在AD脑中发生的变化，我将开发一种新的模型，通过培养IPSC-星形胶质细胞来研究RAS 不同硬度的衬底。此外，我将在iPSC中生成候选PG的淘汰赛 将它们区分为反应性和非反应性星形胶质细胞。我将使用体内苍蝇模型和IPSC的共培养- 观察PGs在RAS毒性中的作用。我的论文项目和我的未来 博士后学习将提供丰富的培训经验，为我的独立职业生涯做好准备 在衰老和神经胶质生物学的结合部领导着一项严格的研究计划的研究员。

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.