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 2 O 2 ) 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 H 2 O 2 in promoting astrocyte-dependent neuroprotection from oxidative stress, and (ii) the relevance of H 2 O 2 in inducing astrocytic Nrf2 activation. To control the duration and level of cytoplasmic H 2 O 2 production in astrocytes cocultured with neurons, we heterologously expressed the H 2 O 2 -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 2 O 2 . Seven hours of low-level H 2 O 2 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 H 2 O 2 , suggesting a mechanism specific to astrocytic intracellular signaling. Nrf2 activation mimicked the effect of astrocytic H 2 O 2 yet H 2 O 2 -induced protection was independent of Nrf2. Astrocytic protein tyrosine phosphatase inhibition also protected neurons from oxidative death, representing a plausible mechanism for H 2 O 2 -induced neuroprotection. These findings demonstrate the utility of rgDAAO for spatially and temporally controlling intracellular H 2 O 2 concentrations to uncover unique astrocyte-dependent neuroprotective mechanisms.

SUMMARY The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found—nordihydroguaiaretic acid, fisetin, and gedunin—induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.

Mounting evidence supports the role of hydrogen peroxide (H2O2) in physiological signaling as well as pathological conditions. However, the subtleties of peroxide-mediated signaling are not well understood, in part because the generation, degradation, and diffusion of H2O2 are highly volatile within different cellular compartments. Therefore, the direct measurement of H2O2 in living specimens is critically important. Fluorescent probes that can detect small changes in H2O2 levels within relevant cellular compartments are important tools to study the spatial dynamics of H2O2. To achieve temporal resolution, the probes must also be photostable enough to allow multiple readings over time without loss of signal. Traditional fluorescent redox sensitive probes that have been commonly used for the detection of H2O2 tend to react with a wide variety of reactive oxygen species (ROS) and often suffer from photostablilty issues. Recently, new classes of H2O2 probes have been designed to detect H2O2 with high selectivity. Advances in H2O2 measurement have enabled biomedical scientists to study H2O2 biology at a level of precision previously unachievable. In addition, new imaging techniques such as two-photon microscopy (TPM) have been employed for H2O2 detection, which permit real-time measurements of H2O2in vivo. This review focuses on recent advances in H2O2 probe development and optical imaging technologies that have been developed for biomedical applications.Electronic supplementary materialThe online version of this article (doi:10.1186/2045-3701-4-64) contains supplementary material, which is available to authorized users.