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

Abstract

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.

Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes

“…Primary astrocyte cultures were prepared from the cerebral cortices of Sprague-Dawley rat pups (P1-3) as described in Haskew-Layton et al 1 In brief, astrocyte cultures were grown for about 2 weeks until reaching confluency in minimal essential medium (Invitrogen) supplemented with 10% horse serum and 25 U∕mL penicillin plus 25 g∕mL streptomycin. Once confluent, the astrocytes were treated with 8 μM cytosine-D-arabinofuranoside (Ara-C), a mitotic inhibitor for ∼3 days, to kill off contaminating cells.…”

“…[1][2][3][4][5][6][7] The aberrant production or accumulation of H 2 O 2 within cellular mitochondria over time due to oxidative stress or genetic mutation is connected to serious pathological conditions including cancer, 8 diabetes, 9 and neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases, as well as stroke. [10][11][12] In addition, H 2 O 2 is involved in therapeutic processes such as wound healing, stem cell proliferation, and an adaptive response in astrocytes leading to neuronal protection.…”

“…[10][11][12] In addition, H 2 O 2 is involved in therapeutic processes such as wound healing, stem cell proliferation, and an adaptive response in astrocytes leading to neuronal protection. 1,2,7,13 A substantial challenge in elucidating the diverse roles of H 2 O 2 in complex biological environments is the lack of methods to determine the spatial and temporal dynamics of this reactive oxygen metabolite in living systems. For the detection of ROS production in vitro, several fluorescent probes have been developed based on small molecules, fluorescent proteins, and nanoparticles.…”

How Much H2O2 Is Produced by Recombinant D-Amino Acid Oxidase in Mammalian Cells?

“…highly specific to D-amino acids, has relatively low affinity to D-alanine (D-Ala) (about 1 mM) and high enzymatic activity in comparison with known DAO from other organisms (8). DAO can serve as a convenient genetically encoded source for controlled D-Ala-dependent H 2 O 2 production in mammalian cells (3). However, the estimation of the intracellular H 2 O 2 production requires extracellular measurements of the released oxidant (3).…”

Astrocyte NMDA receptors' activity sustains neuronal survival through a Cdk5–Nrf2 pathway

“…13 However, whether Nrf2 has a function in astrocyte-mediated neuronal protection is a matter of debate. [14][15][16][17][18] Like postsynaptic neurons, astrocytes express ionotropic glutamate-receptor subtype NMDAR, 19 although their precise function in these glial cells is still enigmatic. In view that excess NMDAR activity, by inhibiting APC/C-Cdh1 in neurons, 6 leads to oxidative damage, 7 we sought to investigate whether astrocyte NMDAR, by sensing neurotransmission, would support neuronal survival.…”