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Hydrogen peroxide (H 2 O 2 ) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H 2 O 2 dynamics in live cells within a limited field of view. The visualization of H 2 O 2 within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the H 2 O 2 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 H 2 O 2 , 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 (p K a of 5.9 vs. ≥7.6) and oxidized states (p K a 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible H 2 O 2 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.

While irradiation can effectively treat brain tumors, this therapy also causes cognitive impairments, some of which may stem from the disruption of hippocampal neurogenesis. To study how radiation affects neurogenesis, we combine phenotyping of subpopulations of hippocampal neural stem and progenitor cells with double- and triple S-phase labeling paradigms. Using this approach, we reveal new features of division, survival, and differentiation of neural stem and progenitor cells after exposure to gamma radiation. We show that dividing neural stem cells, while susceptible to damage induced by gamma rays, are less vulnerable than their rapidly amplifying progeny. We also show that dividing stem and progenitor cells that survive irradiation are suppressed in their ability to replicate 0.5-1 day after the radiation exposure. Suppression of division is also observed for cells that entered the cell cycle after irradiation or were not in the S phase at the time of exposure. Determining the longer term effects of irradiation, we found that 2 months after exposure, radiation-induced suppression of division is partially relieved for both stem and progenitor cells, without evidence for compensatory symmetric divisions as a means to restore the normal level of neurogenesis. By that time, most mature young neurons, born 2-4 weeks after the irradiation, still bear the consequences of radiation exposure, unlike younger neurons undergoing early stages of differentiation without overt signs of deficient maturation. Later, 6 months after an exposure to 5 Gy, cell proliferation and neurogenesis are further impaired, though neural stem cells are still available in the niche, and their pool is preserved. Our results indicate that various subpopulations of stem and progenitor cells in the adult hippocampus have different susceptibility to gamma radiation, and that neurogenesis, even after a temporary restoration, is impaired in the long term after exposure to gamma rays. Our study provides a framework for investigating critical issues of neural stem cell maintenance, aging, interaction with their microenvironment, and post-irradiation therapy.

The modes of stem cell divisions (e.g., symmetric vs. asymmetric) can have a profound impact on the number of progeny and tissue growth, repair, and function. This is particularly relevant for adult neural stem cells, since stem cell-derived neurons affect cognitive and mental states, resistance to stress and disease, and response to therapies. Here we show that although dividing stem cells in the adult hippocampus display a certain bias towards paired distribution (which could imply the prevalence of symmetric divisions), this bias already exists in the distribution of the general population of stem cells and may be responsible for the perceived occurrence of symmetric stem cell divisions. Remarkably, the bias in the distribution of stem cells decreases with age. Our results argue that the preexisting bias in stem cell distribution may affect current assumptions regarding stem cell division and fate as well as conjectures on the prospects of brain repair and rejuvenation.