Studying astrocyte borders using injectable biomaterials

Astrocyte border formation involving proliferative reactive changes in local astrocytes is a conserved feature of many CNS disorders including neurotraumatic, neurodegenerative, neuroinflammatory, and neoplastic diseases. Across disorders, astrocyte borders form against a non-neural niche compartment, containing

stromal and peripherally derived myeloid cells, whose cellular composition varies across disorders and stages of disease. Despite targeted loss of function studies revealing the neuroprotective roles of astrocyte borders, the context-specific functions of astrocyte borders at different non-neural niches are still poorly understood.

The development of new tools and techniques to dissect the complex biology at astrocyte borders in a simplified, focal, and reproducible model system is needed to address important knowledge gaps. Injecting biomaterials into mouse brain promotes spatiotemporally controlled astrocyte border formation and we see an

opportunity to use biomaterials to generate a new bioassay to study astrocyte borders. The main objective of this project is to advance a new bioassay to stimulate and characterize astrocyte borders and use this bioassay to profile several different functional states of astrocyte borders conferred by interactions with diverse

non-neural cell populations. Our overall hypothesis is that by locally injecting cellulose-based biomaterials with different physiochemical properties into the mouse striatum we will stimulate distinct non-neural niches that will confer temporal- and biomaterial-dependent changes in astrocyte states during the transition into astrocyte

borders which we can profile. In aim 1, we will optimize the bioassay to study astrocyte borders by combining injectable, non-resorbable methyl cellulose hydrogels with viral vectors (AAVs) enabling astrocyte specific expression of RiboTag. At 7, 14, 28, and 70 days after hydrogel injection into the mouse striatum, mRNA from

the formed astrocyte borders will be recovered by RiboTag immunoprecipitation and processed by RNA-Seq. Cohorts of mice taken at the same timepoints will be processed by IHC to visualize changes in astrocyte morphology, ribosome trafficking, and multicellular interactions. To validate capacity to detect altered astrocyte

border states we will disrupt astrocyte border formation by controlled release of molecular inhibitors. In aim 2, we will evaluate astrocyte border states at non-neural niches with different immune cell compositions that will be created by chemically modifying cellulose-based hydrogels to display non-fouling, immunosuppressive, or

immunostimulatory functional groups as well as through local delivery of small molecule immunoregulators from hydrogels. Through this project, we will make important technical innovations to develop a new bioassay that will allow us to identify conserved- and context-dependent mechanisms involved in astrocyte border

formation and function. This work will provide the foundations on which to explore therapeutic targeting of astrocyte borders to direct CNS disorder outcomes and improve medical device biocompatibility in future work.