Slowly Reducible Genetically Encoded Green Fluorescent Indicator for In Vivo and Ex Vivo Visualization of Hydrogen Peroxide.

Grigori N Enikolopov,Yulia G Ermakova,Oksana M Subach,Dmitri V Bezryadnov,Tatiana A Kunitsyna,Olga A Mineyeva,Vsevolod V Belousov,Konstantin V Anokhin,Dmitry S Bilan,Kiryl D Piatkevich,Natalia V Barykina,Alexander A Lazutkin,Fedor V Subach

doi:10.3390/ijms20133138

Abstract

Hydrogen peroxide (H2O2) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H2O2 dynamics in live cells within a limited field of view. The visualization of H2O2 within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the H2O2 concentration over desired time scales. This would enable post hoc optical readouts in chemically fixed samples. Herein, we report the development and characterization of NeonOxIrr, a genetically encoded green fluorescent indicator, which rapidly increases fluorescence brightness upon reaction with H2O2, but has a low reduction rate. NeonOxIrr is composed of circularly permutated mNeonGreen fluorescent protein fused to the truncated OxyR transcription factor isolated from E. coli. When compared in vitro to a standard in the field, HyPer3 indicator, NeonOxIrr showed 5.9-fold higher brightness, 15-fold faster oxidation rate, 5.9-fold faster chromophore maturation, similar intensiometric contrast (2.8-fold), 2-fold lower photostability, and significantly higher pH stability both in reduced (pKa of 5.9 vs. ≥7.6) and oxidized states (pKa of 5.9 vs.≥ 7.9). When expressed in the cytosol of HEK293T cells, NeonOxIrr demonstrated a 2.3-fold dynamic range in response to H2O2 and a 44 min reduction half-time, which were 1.4-fold lower and 7.6-fold longer than those for HyPer3. We also demonstrated and characterized the NeonOxIrr response to H2O2 when the sensor was targeted to the matrix and intermembrane space of the mitochondria, nucleus, cell membranes, peroxisomes, Golgi complex, and endoplasmic reticulum of HEK293T cells. NeonOxIrr could reveal endogenous reactive oxygen species (ROS) production in HeLa cells induced with staurosporine but not with thapsigargin or epidermal growth factor. In contrast to HyPer3, NeonOxIrr could visualize optogenetically produced ROS in HEK293T cells. In neuronal cultures, NeonOxIrr preserved its high 3.2-fold dynamic range to H2O2 and slow 198 min reduction half-time. We also demonstrated in HeLa cells that NeonOxIrr preserves a 1.7-fold ex vivo dynamic range to H2O2 upon alkylation with N-ethylmaleimide followed by paraformaldehyde fixation. The same alkylation-fixation procedure in the presence of NP-40 detergent allowed ex vivo detection of H2O2 with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible H2O2 indicator NeonOxIrr can be used for both the in vivo and ex vivo visualization of ROS. Expanding the family of fixable indicators may be a promising strategy to visualize biological processes at a single cell resolution within an entire organism.

Highlights

Small signaling molecules define and coordinate changes in the brain that mediate experience-dependent neuronal plasticity or underlie neurological disorders
Based on the amino acids’ alignment for mNeonGreen and enhanced yellow fluorescent protein (EYFP), we identified the circular permutation site to be between residues 144 and 145, analogous to that in circularly permutated YFP protein used in HyPers [5]
We examined the action of apoptosis-inducing drug staurosporine [27] in HeLa cells transiently expressing Mito-NeonOxIrr indicator or its C199S mutant version in the matrix of mitochondria

Summary

Introduction

Small signaling molecules define and coordinate changes in the brain that mediate experience-dependent neuronal plasticity or underlie neurological disorders. Depending on the intracellular concentration, H2O2 can act as a signaling molecule or can cause oxidative stress followed by adaptation or apoptosis of the affected cell. A new highly-sensitive family of yeast peroxiredoxin-based indicators has recently been reported [10]; it is not functional in mammalian cells. For ex vivo studies of H2O2 generation, CAG-NeonOxIrr-P2A-mCherry rAAV particles were bilaterally injected into the barrel fields of the somatosensory cortex perpendicular to the surface of the skull. The suspension of the viral particles (total volume of 0.2 μL) was injected into one brain hemisphere at the rate of 0.2 μL over 12 min. Oxidation of the Indicator with H2O2 Followed by its Fixation ex vivo

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