Cells in the brain change over time. These changes, such as responses to activity, stress, and injury, are central to both healthy brain function and neurological disease. In disease, molecular changes within neurons accompany and may drive progression. However, existing methods for studying the brain’s molecular composition require destroying tissue and therefore provide only snapshots at single time points rather than a continuous view of how cells evolve. Therefore, which early molecular changes predict or cause dysfunction remains unknown. By contrast, the identification of these molecular markers and drivers of disease could transform early diagnosis and intervention.

We propose to address this gap by creating a new technology that enables longitudinal molecular monitoring of neurons in the living brain. Our approach combines two capabilities, which were invented by the co-investigators: (1) engineered molecular sensors that detect key RNA transcripts inside neurons and record their activity as chemical modifications in a reporter RNA, and (2) RNA secretion systems that package these reporter molecules into protective vesicles and release them from neurons into the surrounding cerebrospinal fluid. By sampling this fluid over time in a minimally invasive manner and sequencing the collected RNA, we can repeatedly read out the molecular states of neurons without harming the brain. In this work, we will build and optimize molecular sensors for neural activity, stress, and resilience pathways (Aim 1), engineer efficient RNA secretion from neurons (Aim 2), and apply this integrated system to track how neurons change during a stress paradigm in mice, linking molecular trajectories to behavioral outcomes (Aim 3). This technology will provide, to our knowledge, the first longitudinal, molecularly resolved view of how neural cell states evolve and predict neurological disease in a living animal. By bringing together expertise in RNA sensors, cellular secretion systems, and neuroscience models, this work will establish a platform with broad applications across neuroscience and neurological disease.