Abstract
Within the central nervous system, astrocytes play a critical role in neural circuit formation by modulating synaptic plasticity and maintaining synaptic health. These functions are enabled by their unique morphology, in which fine processes closely associate with synapses, sense changes in the local microenvironment, and generate dynamic responses. As a single astrocyte can contact up to 100,000 synapses in mice, a key question emerges: how do individual astrocyte processes produce localized responses independent of signals received by other processes despite sharing a common nucleus? Local translation is hypothesized to enable quasi-independent activity of peripheral/perisynaptic astrocyte processes (PAPs) by enabling spatiotemporal control of gene expression. Previous work in our lab characterized the local translatome in astrocytes, providing key insight into the functions of locally translated genes. However, the regulatory mechanisms underlying mRNA localization remain unclear. Specifically, which cis-regulatory sequences direct mRNA localization and what trans-regulatory factors mediate this process? This dissertation presents complimentary studies aimed at defining the regulatory logic of local translation in astrocytes. Chapter 1 provides the historical and conceptual foundation for this dissertation, highlight astrocyte function, the need for in vivo approach high-throughput methods, and current models of local translation regulation. In Chapter 2, I present the Synaptoneurosomal Massively Parallel Reporter Assay (SN-MPRA), the first in vivo MPRA designed to identify cis-regulatory sequences sufficient to drive mRNA localization in astrocytes. By tiling the 3’ UTRs of two locally translated genes, Glt1a and Sparc, in 20 nucleotide steps, this approach provides comprehensive coverage of the UTR to directly assess which sequences are sufficient to drive localization. Coupling the SN-MPRA with astrocyte Translating Ribosome Affinity Purification (TRAP) further enables simultaneous measurement of local translation. Follow up experiments using a single nucleotide mutagenesis SN-MPRA library provided higher resolution insight into the specific UTR features driving RNA localization. These studies demonstrate that astrocytes likely employ multiple mechanisms to establish distinct sub-cellular translatomes. Importantly, this methodology establishes a framework for future studies to directly investigate sequence determinants of sub-cellular localization in other nervous system cell types in vivo. In Chapter 3, I evaluated three strategies for astrocyte-specific, Cre-dependent miRNA affinity purification approaches to identify the local regulatory miRNA profile in astrocyte PAPs. This chapter underscores key challenges in the field of astrocyte biology, specifically precise, genetic targeting of mature astrocytes in vivo. While tamoxifen-inducible Cre and viral delivery of Cre approaches mitigate the loss of astrocyte specificity seen with constitutive GFAP Cre mice, recombination efficiency or transgene expression was below thresholds of detection, limiting their utility. Thus, this chapter highlights the need for development of genetic tools for use in astrocytes, and the importance of rigorous validation of any tool. Finally, in Chapter 4, I used an alternative approach to investigate the profile of miRNAs regulating local translation in astrocytes. I analyzed Cross-linking, Ligation, and Sequencing of Hybrids (CLASH) data for astrocyte soma-retained genes and PAP-enriched genes. This analysis revealed global differences in miRNA targeting, identified miRNAs that preferentially target PAP transcripts, and combine this data with the SN-MPRA data from Chapter 2 to gain insight into which miRNAs might be targeting the UTR elements significantly changing ribosome occupancy. By identifying candidate miRNAs, this chapter provides a foundation for future investigations into miRNA pathways involved in local translation regulation in astrocytes. Collectively, this dissertation establishes a framework for understanding the cis- and trans-regulatory logic of local translation in astrocytes. The SN-MPRA enables in vivo dissection of 3’ UTR features sufficient for localization, revealing multiple mechanisms contribute to this process in astrocytes. Further, analysis of CLASH data identifies candidate miRNAs to further investigate how RNA localization is regulated by this class of RNAs. Together, these findings advance our understanding of how perisynaptic astrocyte processes regulate synaptic function and provide a broadly applicable tool for studying local gene regulation in the nervous system.
Committee Chair
Joseph Dougherty
Committee Members
Hani Zaher; Michael White; Sarah Ackerman; Timothy Miller
Degree
Doctor of Philosophy (PhD)
Author's Department
Biology & Biomedical Sciences (Molecular Cell Biology)
Document Type
Dissertation
Date of Award
6-24-2026
Language
English (en)
DOI
https://doi.org/10.7936/9j23-4k76
Recommended Citation
Koester, Sarah Kathleen, "An In Vivo Framework for Decoding the Regulatory Logic of RNA Localization and Local Translation in Astrocytes: From Cis-Regulatory Sequences to Trans-Acting Factors" (2026). Arts & Sciences Graduate Student Theses and Dissertations. 3843.
The definitive version is available at https://doi.org/10.7936/9j23-4k76