Single-Cell and Spatial Transcriptomic Dissection of Brain Cellular Responses to Nutritional and Injury Perturbations

Abstract

Single-cell and spatial transcriptomic technologies have transformed our ability to resolve the cellular composition of complex tissues, such as the brain, and provide single-cell-level expression profiles. Yet interpreting these data requires computational approaches that move beyond cataloging discrete cell types to capture the continuous, context-dependent transcriptional states that cells adopt in response to perturbation. In this work, we develop and apply single-cell, single-nucleus, and spatial transcriptomic approaches to characterize how the developing and injured brain responds to two classes of perturbation-nutritional and injury-reading out each challenge as a shift in cell-type composition and cell-state programs. In Chapter 2, we apply spatial transcriptomics together with single-nucleus multi-omics (paired snRNA-seq and snATAC-seq) to investigate how excess maternal folic acid supplementation affects offspring brain development. We find that maternal folic acid excess alters gene programs governing neurogenesis and axon myelination in a region-specific manner, and identify maturing excitatory neurons of the hippocampal dentate gyrus as particularly vulnerable, exhibiting coupled changes in gene expression and chromatin accessibility within ribosomal biogenesis pathways critical for synaptic formation. In Chapter 3, we extend the nutritional perturbation to postnatal timing by modeling an abrupt prenatal-to-postnatal drop in folate availability—a "folate cliff"—in the developing cerebellum. Combining behavioral assays with bulk transcriptomic analysis, we observe a nonlinear behavioral dose-response and a convergent disruption of glial and myelination programs, marked by upregulation of Gfap and downregulation of Pdgfra and Mbp. We interpret these changes as a coordinated glial/myelin disruption signature, while noting the limited sample size and the cell-type resolution deferred to future work. In Chapter 4, we develop StateCommute, a computational framework to present cells in pathway space and integrate non-negative matrix factorization, compositional (differential-abundance) analysis, trajectory inference, and cell-cell communication modeling to resolve transcriptional states across distinct brain injuries. Applying it to a harmonized atlas of viral encephalitis (VEEV), organophosphorus nerve-agent (OPNA) exposure, and traumatic brain injury (TBI), we identify cellular states and pathway-level programs that are shared across injuries as well as those specific to each-recovered without reference to condition labels yet anchored in genuine, injury-driven transcriptional change. Together, these chapters demonstrate how single-cell and spatial transcriptomic approaches can be used to characterize cellular responses to diverse biological perturbations during brain development and injury. In addition to providing biological insights into the mechanisms, this work introduces and applies computational methods for integrating multimodal and spatial transcriptomic data to better define cellular states and their interactions. Collectively, these findings provide a framework for studying dynamic cellular responses in the brain and establish a foundation for future investigations of brain development, injury, and neurological disease.