Development of genetically encoded sex hormone biosensors to understand neuroactive steroid signaling dynamics in vivo

ABSTRACT/SUMMARY Understanding the role of sex steroids like estrogens and androgens within the brain is crucial for elucidating their impact on various brain functions, including mood, cognition, and reproductive behavior. However, our current understanding of how these steroids modulate neuronal physiology and behavior in vivo remains limited, largely due to the absence of technologies capable of monitoring these hormones in real time within the brain. The goal of this project is to bridge this gap by developing a suite of genetically encoded fluorescent biosensors for steroid hormones. These biosensors will enable cell-specific monitoring of steroid hormone levels in awake and behaving animals, providing unprecedented insights into the dynamic roles of neuroactive steroids derived from endocrine tissues or synthesized within the brain. Our central goal is to engineer green fluorescent biosensors for estrogens and androgens based on their nuclear receptors. By incorporating fluorescent reporters into receptor regions that change conformation upon ligand binding, we aim to create sensors with enhanced ligand affinity and signal strength through structure-guided engineering and high-throughput screening. Additionally, we plan to develop far-red shifted sensors for multiplexed applications, extending our innovative approach to a broader spectrum of steroid hormones. Our project is innovative because it employs structure-guided design and a high-throughput engineering platform to rapidly screen thousands of protein variants. This approach significantly accelerates the development of biosensors capable of real-time steroid hormone monitoring in vivo. Furthermore, by applying these sensors to study the rapid effects of steroid signaling on neural activity in the hypothalamus of behaving mice, we aim to dissect the temporal dynamics of steroid action beyond their genomic effects. This research is significant because it addresses a critical gap in our ability to study steroid hormones in live animals. By providing tools for real-time monitoring of steroid hormone signaling within genetically specified brain cell types, we pave the way for a deeper understanding of how steroids influence brain circuit function and behavior. The successful completion of this project will not only advance our knowledge of steroid hormone dynamics but also offer new avenues for therapeutic intervention in steroid-related disorders.