Thomas Delgado - PhD Candidate, Advisor: Gail Johnson, PhD
Astrocytes are essential for maintaining neuronal function in resting and disease states. Following injury, astrocytes take on reactive phenotypes that grade from neurotoxic to neuroprotective, which impact subsequent neuronal recovery processes, such as axonal regeneration. Only recently has the heterogeneity of reactive astrocyte populations begun to be described, and little is known about the contextual requirements and molecular inflection points that underlie these graded responses. Accumulating data from my lab suggests that one of these inflection points is transglutaminase 2 (TG2). TG2 is complex in that it regulates signaling of numerous molecular pathways at the cell membrane, in the cytosol, and in the nucleus; thus, it can provide multiple levels of context-dependent input into gene regulation. Importantly, when TG2 is depleted from astrocytes, they better protect neurons in culture from oxygen-glucose deprivation (OGD), improve motor function recovery in a mouse spinal cord injury model, and better facilitate neurite outgrowth in vitro on an injury-relevant, growth-inhibitory matrix. Additionally, TG2 depletion upregulates lipid handling (lipid uptake and formation of lipid droplets) and lipid metabolism pathways in an injury-dependent manner. As stressed neurons accumulate reactive oxygen species and peroxidated lipids, and cannot metabolize these toxic lipids on their own, they export them to astrocytes. Astrocytic lipid uptake and metabolism eliminates these oxidized lipids and recycles them to produce energy substrates for neurons. This mechanism may partly underlie the protective effect of TG2-/- astrocytes after injury.
In aim 1, I will test the hypothesis that depletion of astrocytic TG2 improves axonal regeneration through an inhibitory extracellular matrix (ECM) after CNS injury using an optic nerve crush model in mice. These studies are built on previous spinal cord injury data from my lab, with the unique addition that, in the optic nerve, axonal regeneration can only occur through the inhibitory ECM deposited at the crush site, while in the spinal cord, regrowth may occur via collaterals around the injury site, through permissive matrices.
In aim 2, I will identify the major molecular pathways and mechanisms of gene and protein regulation underlying the unique function of TG2-/- astrocytes. As TG2 is a known interactor of transcription machinery and chromatin regulators in the nucleus, I will use ATAC-seq to analyze differences in chromatin accessibility between TG2-/- and wild type (WT) astrocytes and integrate these data with differentially regulated transcripts identified through RNA sequencing. I will compare differential enrichment in upstream signaling pathways with differential protein expression identified by tandem mass spectrometry to better approximate the functional impact of these molecular changes.
In aim 3, following data that lipid handling and metabolism is differentially regulated in TG2-/- astrocytes, I hypothesize that TG2-/- astrocytes more efficiently uptake and metabolize lipids that are released by stressed neurons (as peroxidated lipids), ultimately providing better control of oxidative stress and increased energy supply to neurons in injury conditions. Therefore, I will measure the ability of WT and TG2-/- astrocytes to take up and metabolize lipids released by neurons, in control and stressed conditions, as indicated by lipid droplet accumulation. Further, I will measure metabolic flux through ketone and cholesterol synthesis pathways, downstream of fatty acid oxidation, in WT and TG2-/- astrocytes by labeling them with C13-palmitate and measuring C13-labeled metabolites by LC-MS. I will then pair neurons with either astrocyte group to track the export of C13-labeled energy substrates to neurons.
Overall, this project will explore the role of TG2 as a key regulator of astrocyte reactivity after CNS injury and, within this scope, better characterize the astrocytic metabolic pathways that are integral for neuronal and functional recovery.
Apr 07, 2023 @ 2:00 p.m.
Medical Center | Lower Adolph Auditorium (1-7619)