Function of reactive astrocytes in aging and neurodegenerative disease

反应性星形胶质细胞(RAS)是正常衰老和神经变性的特征。RAS显著改变其形态和基因表达，显著增加损伤或炎症反应中胶质纤维酸性蛋白(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导致罕见和致命的脑白质营养不良，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(Rosenthal纤维；RFs)并变得活跃。然而，将&gt；70种不同的GFAP突变与AxD的RF形成和其他疾病相关表型联系起来的机制仍不清楚。我的大量初步数据显示，异常的磷酸化促进了GFAP的聚集，这种修饰是AxD严重程度的一个标志，与疾病突变无关。此外，我还证明了位点特异性GFAP的磷酸化与caspase-6促进蛋白分解有关，但这两者是否直接相关尚不清楚。我推测酪蛋白激酶(CK2)和caspase-6之间的协同信号促进了有缺陷的GFAP蛋白平衡，从而加剧了AxD星形胶质细胞的反应表型。对于F99期，我建议使用药理学和遗传学策略来抑制CK2和caspase-6的活性，以便使用我开发的星形胶质细胞模型(Aim 1.1)在体外和体内使用AxD小鼠模型(Aim 1.2)来表征它们的作用。我将掌握使用CRISPR/Cas9进行IPSC基因编辑，以生成CK2和caspase-6基因敲除以及iPSC对星形胶质细胞和神经元的处理和分化(目标1.1)，并将这些技术应用于我的博士后项目(目标2)。在K00阶段，我将在梅尔·费尼博士的实验室里研究RAS在阿尔茨海默病中的功能。蛋白多糖(PG)是衰老和RAS中表达最高的基因之一。来自费尼博士实验室的初步数据证实了前列腺素和果蝇神经变性模型之间的遗传相互作用。我假设RAS在细胞外基质中产生PG的失衡，这创造了一个抑制神经元生长和重塑的环境。为了模拟AD脑中已知的机械变化，我将开发一种新的模型，通过在不同硬度的底物上培养IPSC-星形胶质细胞来研究RAS。此外，我将在IPSCs中产生候选PG的敲除，并将它们区分为反应性和非反应性星形胶质细胞。我将使用体内苍蝇模型以及IPSC-星形胶质细胞和神经元的共培养来研究PGs在RAS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，为我的职业生涯做好准备，成为一名独立调查员，领导一项关于衰老和神经胶质生物学联系的严格研究计划。

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.