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.
Reactive astrocytes are common features of neurodegenerative diseases and normal aging, but their role is still poorly understood and can encompass both beneficial and harmful effects. Understanding the molecular mechanisms that lead to astrocyte activation and defining the heterogeneity of astrocyte activation will lead to novel astrocyte-targeted therapies. This training proposal aims to understand the causes and effects of astrocyte reactivity in the context of Alexander disease (Aim 1) and aging and neurodegeneration (Aim 2).
