A nuclear-localized fluorescent hydrogen peroxide probe for monitoring sirtuin-mediated oxidative stress responses in vivo

Abstract

Hydrogen peroxide (H(2)O(2)) can serve as a beneficial signaling agent or toxin depending on its concentration and location within a cell or organism. Methods to measure the localized accumulation of H(2)O(2) in living specimens remain limited. Motivated to meet this need, we have developed a nuclear-localized fluorescent probe for H(2)O(2), Nuclear Peroxy Emerald 1 (NucPE1), to selectively interrogate ROS fluxes within this sensitive organelle. NucPE1 selectively accumulates in the nuclei of a variety of mammalian cell lines as well as in whole model organisms like Caenorhabditis elegans, where it can respond to subcellular changes in H(2)O(2) fluxes. Moreover, in vivo NucPE1 imaging reveals a reduction in nuclear H(2)O(2) levels in worms overexpressing sir-2.1 compared with wild-type congeners, supporting a link between this longevity-promoting sirtuin protein and enhanced regulation of nuclear ROS pools.

Figure 1. Synthesis and in vitro characterization of NucPE1

(A) Synthesis and activation of NucPE1. (B) Fluorescence response of 5 μM NucPE1 to H₂O₂. Time points represent 0, 5, 15, 30, 45, and 60 min after the addition of 100 μM H₂O₂. (C) Fluorescence responses of NucPE1 to various reactive oxygen species (ROS). Bars represent relative responses at 0, 5, 15, 30, 45, and 60 min after addition of each ROS.

Figure 2. Localization and turn-on of NucPE1 in cell culture

(A) HEK 293 or HeLa cells loaded with 5 μM NucPE1 and 1 μM Hoechst 33342 for 15 min in DPBS, washed with DPBS, incubated 20 minutes in DPBS, and imaged. 25 μm scale bar shown. (B) HEK 293 cells loaded with 5 μM NucPE1 and 1 μM Hoechst 33342 for 15 min in DPBS, washed with DPBS, incubated 20 minutes in DPBS with carrier or 100 μM H₂O₂, and imaged. 20 μm scale bar shown. (C) Quantification of NucPE1 fluorescence for experiment as shown in (B). Statistical analysis were performed with a Student’s t-test (n = 4) and error bars are ± s.e.m. See also Figures S1 and S2.

Figure 3. Localization of NucPE1 in C. elegans

(A) C. elegans loaded with 150 μM Hoechst 33342 and 50 μM NucPE1 for 8 h and imaged. 20 μm scale bar shown. (B) The portion illustrated by a square in (A) is enlarged. 10 μm scale bar shown. See also Figure S3.

Figure 4. Sir-2.1 overexpression provides nuclear redox protection

(A) Wild-type (N2) or Sir-2.1-overexpressing C. elegans loaded with 150 μM Hoechst 33342 and 50 μM NucPE1 for 8 h, stimulated with either carrier or 10 mM H₂O₂ for 30 min, and imaged. Representative images of NucPE1 and a bright field overlay are displayed for one worm from each condition. 20 μm scale bar shown. (B) Quantification of NucPE1 fluorescence for experiment as shown in (A). Error bars are ± s.e.m. See also Figure S4.