Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway

Neurons rely on their metabolic coupling with astrocytes to combat oxidative stress. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) appears important for astrocyte-dependent neuroprotection from oxidative insults. Indeed, Nrf2 activators are effective in stroke, Parkinson disease, and Huntington disease models. However, key endogenous signals that initiate adaptive neuroprotective cascades in astrocytes, including activation of Nrf2-mediated gene expression, remain unclear. Hydrogen peroxide (H ₂O₂) plays an important role in cell signaling and is an attractive candidate mediator of adaptive responses in astrocytes. Here we determine (i) the significance of H₂O₂ in promoting astrocyte-dependent neuroprotection from oxidative stress, and (ii) the relevance of H₂O₂ in inducing astrocytic Nrf2 activation. To control the duration and level of cytoplasmic H₂O₂ production in astrocytes cocultured with neurons, we heterologously expressed the H₂O₂-producing enzyme Rhodotorula gracilis D-amino acid oxidase (rgDAAO) selectively in astrocytes. Exposure of rgDAAO-astrocytes to D-alanine lead to the concentration-dependent generation of H ₂O₂. Seven hours of low-level H₂O₂ production (∼3.7 nmol·min·mg protein) in astrocytes protected neurons from oxidative stress, but higher levels (∼130 nmol· min·mg protein) were neurotoxic. Neuroprotection occurred without direct neuronal exposure to astrocyte-derived H₂O₂, suggesting a mechanism specific to astrocytic intracellular signaling. Nrf2 activation mimicked the effect of astrocytic H₂O₂ yet H ₂O₂-induced protection was independent of Nrf2. Astrocytic protein tyrosine phosphatase inhibition also protected neurons from oxidative death, representing a plausiblemechanism for H₂O₂-induced neuroprotection. These findings demonstrate the utility of rgDAAO for spatially and temporally controlling intracellular H₂O₂ concentrations to uncover unique astrocyte-dependent neuroprotective mechanisms.