Reactive astrocytes (RAs) are a feature of normal aging and neurodegeneration of varying etiologies, suggesting a potential common mechanism. However, the role of RAs in neurodegeneration is complex because they can be triggered by many different stimuli and can have both beneficial and harmful effects to the Central Nervous System (CNS). Thus, a better understanding of the causes and functions of different reactive astrocytes is necessary to design therapeutics that can selectively bolster protective functions and inhibit toxic effects. There is also a vital need to expand the tools available to generate reactive astrocytes and to describe diverse reactive astrocyte functions. The long-term research goal of this application is to gain deep mechanistic understanding of the factors that elicit reactive astrocytes and the functions of different types of reactive astrocytes. For the K00 phase, the formation and the role of RAs in Alzheimer’s Disease (AD) and aging will be interrogated in the lab of Dr. Mel Feany. Dramatic changes in the extracellular matrix (ECM) and brain stiffness are observed in various CNS injuries, including Alzheimer’s disease (AD), cancer, and aging. However, while growing astrocytes on stiff substrates is known to promote a reactive astrocyte phenotype, the physiological effects of mechanically activated astrocytes have not been explored. In Aim 1, a novel model will be developed to study RAs by culturing iPSCastrocytes on substrates of different stiffness to understand the effects of mechanical changes on astrocytes. This model will be utilized to examine potential mechanisms of RA-induced toxicity. 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 ECM, which creates an environment that is neurotoxic and inhibitory to neuronal growth and remodeling. To test this, candidate PGs will be knocked out in iPSCs, which will be differentiated to reactive and non-reactive astrocytes and co-cultured with neurons to determine the role of each PG in astrocyte-mediated neurotoxicity. Proteomics will be performed on wild-type reactive and non-reactive iPSC-astrocytes to define the reactive astrocyte secretome of mechanically activated astrocytes and identify new candidate genes. In Aim 2, in vivo fly models will be used to examine the role of PGs in toxicity of RAs in the contexts of aging and neurodegeneration. To model aging, PG candidates will be selectively knocked down in wild-type flies, which will be aged up to 2 months. Additionally, a dual bipartite expression system will be used to selectively knockdown PG candidates in glial cells while manipulating neuronal cells independently to model AD. Neurodegeneration will be assessed upon PG knockdown to determine the role of PGs in each context. The proposed studies will r...