Summary Small molecules inhibiting hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are the focus of drug development efforts directed toward the treatment of ischemia and metabolic imbalance. A cell-based reporter produced by fusing HIF-1α oxygen degradable domain (ODD) to luciferase was shown to work as a capture assay monitoring stability of the overexpressed luciferase-labeled HIF PHD substrate under conditions more physiological than in vitro test tubes. High throughput screening identified novel catechol and oxyquinoline pharmacophores with a “branching motif” immediately adjacent to a Fe-binding motif that fits selectively into the HIF PHD active site in in silico models. In accord with their structure-activity relationship in the primary screen, the best “hits” stabilize HIF1α, upregulate known HIF target genes in a human neuronal line, and exert neuroprotective effects in established model of oxidative stress in cortical neurons.

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.

Astrocytes are critical for the antioxidant support of neurons. Recently, we demonstrated that low level hydrogen peroxide (H 2 O 2 ) facilitates astrocyte-dependent neuroprotection independent of the antioxidant transcription factor Nrf2, leaving the identity of the endogenous astrocytic Nrf2 activator to question. In this study, we show that an endogenous electrophile, 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2), non-cell autonomously protects neurons from death induced by depletion of the major antioxidant glutathione. Nrf2 knockdown in astrocytes abrogated 15d-PGJ2's neuroprotective effect as well as 15d-PGJ2 facilitated Nrf2-target gene induction. In contrast, knockdown of the transcription factor peroxisome proliferator activated-receptor gamma (PPARc), a well-characterized 15d-PGJ2 target, did not alter 15d-PGJ2 non-cell autonomous neuroprotection. In addition, several PPARc agonists of the thiazolidinedione (TZD) family failed to induce neuroprotection. Unexpectedly, however, the TZD troglitazone (which contains a chromanol moiety found on vitamin E) induced astrocyte-mediated neuroprotection, an effect which was mimicked by the vitamin E analogs alpha-tocopherol or alpha-tocotrienol. Our findings lead to two important conclusions: (i) 15d-PGJ2 induces astrocyte-mediated neuroprotection via an Nrf2 but not PPARc mediated pathway, suggesting that 15d-PGJ2 is a candidate endogenous modulator of Nrf2 protective pathways in astrocytes; (ii) selective astrocyte treatment with analogs or compounds containing the chromanol moiety of vitamin E facilitates non-cell autonomous neuroprotection. Keywords: astrocytes, neuroprotection, Nrf2, PPARc, vitamin E. J. Neurochem. (2013) 124, 536-547.Astrocytes play an integral role in the CNS by regulating blood flow in response to neuronal activity, modulating synaptogenesis and synaptic activity, and providing metabolic intermediates to neurons (Dringen et al. 2000;Barres 2008;Sofroniew and Vinters 2010). In stroke and other CNS disorders, astrocytes contribute to neuroprotection in part by providing antioxidant support, removing excess extracellular levels of the excitotoxic neurotransmitter glutamate and providing a barrier to inflammation (Sofroniew and Vinters 2010). However, astrocytes may also play a detrimental role by contributing to cytokine and chemokine release and forming a barrier to neuronal regeneration (Sofroniew and Vinters 2010 Abbreviations used: 15d-PGJ2, 15-deoxy-D12,14-prostaglandin J2; ARE, antioxidant response element; CNS, central nervous system; COX, cyclooxygenase; H 2 O 2 , hydrogen peroxide; HMOX or HO-1, heme oxygenase-1; L-PGDS, lipocalin prostaglandin D2 synthase; Nrf2, nuclear factor erythroid 2 related factor 2; PPARc, peroxisome proliferator activated-receptor gamma; siRNA, short interfering RNA; TZD, thiazolidinedione.