Abstract TP281: Astrocytic Regulation of Neuronal Ferroptosis Through Nrf2 Signaling in Ischemia-Reperfusion Injury

Oxidative stress-induced ferroptosis, iron-dependent cell death, results from the accumulation of lipid peroxidation caused by the free radicals produced during ischemia-reperfusion injury (IRI). The Nrf2 antioxidant pathway has been implicated in improvement of stroke outcomes by alleviating oxidative stress and reducing ferroptosis. During IRI, neurons rely on astrocyte support for survival, but the mechanism of astrocyte-protection of neurons is not clear. In this study, we investigated astrocyte regulation of neuronal ferroptosis. Adult male and female rats (n=8) underwent 2h MCAo followed by 6h reperfusion. We evaluated spatially resolved transcriptomic changes of astrocytes and neurons in the peri-infarct, marginal, and contralateral regions with Nanostring GeoMx DSP. Deseq2 analyzed differential expression. Ingenuity pathway analysis and gene set enrichment analysis analyzed pathway enrichnment. Oxygen-glucose deprivation (OGD)-induced astrocyte conditioned media (oACM) was generated from OGD-astrocytes. Reperfusion-induced astrocyte conditioned media (rACM) was generated from reperfused OGD-astrocytes. Neurons were treated with oACM or rACM. Lipid peroxidation was measured with C11 BODIPY. NRF2 regulators (GSK3B, AKT) and downstream enzymatic antioxidants (HO-1, NQO1) were measured by chemiluminescent western blots. Penumbral astrocytes and neurons upregulated pro-survival pathways, including Nrf2-mediated oxidative stress response pathway, NGF-signaling, and glutamate-glutamine metabolism. Astrocytes upregulated anti-ferroptosis genes (Gpx4, Slc7a11, Fth1) and neurons upregulated ferroptosis sensitivity genes (ninj1, sqstm1, slc39a7). In vitro, astrocytes decreased GSK3B (0.63 vs 1.04, p < 0.01), AKT (0.29 vs 0.61, p = 0.1), and NRF2 (0.08 vs 0.13, p < 0.05), and increased HO-1 (0.81 vs 0.25, p < 0.01). There was no difference in NQO1 (1.04 vs 1.07, p = 0.9). Neurons treated with oACM increased NRF2 (2.46 FC vs 0.91 FC, p < 0.05) but did not change NQO1 (2.01 FC vs 2.02 FC, p = 0.9) and HO1 (1.60 FC vs 1.14 FC, p = 0.52). Neuron lipid peroxidation increased after OGD (3.22 vs 15.1, p < 0.01). Lipid peroxidation decreased with oACM (15.1 vs 4.11, p < 0.05) and rACM (15.1 vs 2.35, p < 0.05). Regulation of oxidative stress and nrf2 antioxidant response differs between astrocytes and neurons. Our data suggests that astrocytes are protecting neurons through alleviating lipid peroxidation and regulating neuronal nrf2 pathway activation.