Ultra-fast genetically encoded sensor for precise real-time monitoring of physiological and pathophysiological peroxide dynamics

Ultra-fast genetically encoded sensor for precise real-time monitoring of physiological and pathophysiological peroxide dynamics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ultra-fast genetically encoded sensor for precise real-time monitoring of physiological and pathophysiological peroxide dynamics Andre Berndt, Justin Lee, Woojin Won, Kandace Kimball, Carlie Neiswanger, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4048855/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Hydrogen Peroxide (H 2 O 2 ) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H 2 O 2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity and response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H 2 O 2 dynamics. We successfully tracked real-time transient and steady-state H 2 O 2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse brain ex vivo and in vivo . These applications demonstrate oROS's capabilities to monitor H 2 O 2 as a secondary response to pharmacologically induced oxidative stress and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aβ-putriscine-MAOB axis, highlighting the sensor’s relevance in validating neurodegenerative disease models. Lastly, we demonstrated acute opioid-induced generation of H 2 O 2 signal in vivo which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool, offering a window into the dynamic landscape of H 2 O 2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for understanding diseases associated with oxidative stress, such as cancer, neurodegenerative, and cardiovascular diseases. Biological sciences/Biological techniques/Molecular engineering/Protein design Biological sciences/Biological techniques/Imaging/Fluorescence imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Endogenous Reactive Oxygen Species (ROS) are indispensable components of aerobic metabolism, which hallmarks the rise of complex life 1 , 2 . Due to their damaging impact on biological macromolecules at high concentrations, redox homeostasis is tightly regulated in most aerobic systems, and high-level accumulation of ROS is often viewed as a pathogenic marker in degenerative diseases (e.g. Alzheimer’s disease, Duchenne Muscular Dystrophy), tumorigenesis, and inflammation 3 – 6 . Furthermore, an increasing number of studies report the role of low-level ROS as physiologic mediator in normal cellular signaling processes 7 – 9 . Specifically, H 2 O 2 is a key redox signaling molecule, owing to its relative stability and ability to modify cysteine residues in proteins, enabling selective downstream signaling 10 . On the other hand, excessive H 2 O 2 is a common pathological marker affecting phenotypic and disease progression in various cell types 11 – 13 . Nevertheless, limited analytic tools to spatiotemporally monitor specific oxidants in situ with precision have been a bottleneck to deciphering their specific role in physiology and the cause and effect of their imbalance 14 , 15 . Thus, methods to interrogate the role of H 2 O 2 would be broadly applicable to the study of redox biology 15 . Most synthetic ROS-sensitive dyes are unsuited for these considerations because of their short working time window, low sensitivity, and low specificity 16 . Protein-based peroxide sensors have been engineered to overcome these shortcomings. For example, the roGFP sensor family fuses roGFP, a redox-sensitive green fluorescent protein variant, to H 2 O 2 -specific enzymes like Orp1 (thiol peroxidase), or Tsa2 (typical 2-Cys peroxiredoxin) from yeast to achieve peroxide-specific roGFP fluorescence changes via redox relay 17 , 18 . The HyPer sensor family is based on the direct fusion of circularly permuted fluorescent protein (cpFP) to the regulatory domain of bacterial peroxide sensor protein OxyR for conformational coupling that leads to H 2 O 2 -specific fluorescence change 19 – 24 . Most HyPer sensors use ecOxyR ( Escherichia coli OxyR), the most extensively studied OxyR variant, as their sensing domain. However, existing ecOxyR-based peroxide sensors exhibit low sensitivity and slow oxidation kinetics (seconds under saturation conditions) 21 , 22 , 25 , while studies reported peroxide-dependent oxidation of ecOxyR at a sub-second scale 26 . We hypothesized that the discrepancy stems from the disruption of structural flexibility in the sensors. Through a series of structure-guided engineering steps, we developed oROS-G (optogenetic hydRogen perOxide Sensor, Green), a green fluorescent protein (GFP, excitation: 488 nm, emission: 515 nm) and an ecOxyR-based peroxide sensor that exhibits exceptional sensitivity and kinetics enabling the visualization of peroxide diffusion. We also engineered oROS-Gr, a ratiometric variant of oROS-G by fusing it with mCherry, which allows measurement of the precise sensor oxidation state by normalizing sensor fluorescence intensity for the expression level. Here, we present diverse use cases of oROS sensors to monitor both steady-state and transient H 2 O 2 levels in various model systems. Specifically, we showed how oROS can detect varying H 2 O 2 levels in astrocytes in the context of Alzheimer’s disease models and assessed the efficacy of a drug in reducing aberrant peroxide levels. Also, we investigated how different glucose levels can result in different intracellular oxidative environments in conjunction with mitochondrial respiratory depression. Lastly, we monitored opioid dependent acute H 2 O 2 generation in mouse brain both ex vivo and in vivo , demonstrating potential utility of oROS sensors as a functional downstream reporter for G-protein biased opioid receptor activation. Result Structure-guided engineering strategies for ecOxyR-based H 2 O 2 sensor with improved sensitivity and kinetics. OxyR is a bacterial H 2 O 2 sensor protein that regulates the transcription of antioxidative genes in response to low-level cellular H 2 O 2 . The specificity of OxyR for H 2 O 2 stems from its unique H 2 O 2 binding pocket 27 . Previous studies have shown that binding H 2 O 2 leads to an intermediate state that facilitates the disulfide bridging of two conserved cysteine residues (C199-C208), which triggers the transition into the oxidized conformational state of OxyR. Due to its unique characteristic as an H 2 O 2 sensor with low scavenging capacity 27 , OxyR is an attractive scaffold for building a protein-based H 2 O 2 reporter. Nevertheless, the slow kinetics and low sensitivity of existing ecOxyR sensors 19 , 21 – 23 , 25 deviate from the reported ecOxyR kinetics, prompting us to revisit the sensor design [Fig. 1A, Supp. Figure 1A]. OxyR-based peroxide sensors have circular permuted fluorescent proteins (cpFP) within the loop between residues C199 and C208. However, the crystal structure of oxidized ecOxyR [PDB:1I6A] predicted an evident peak of B-factor [Fig. 1B] indicating this loop region is more flexible than its surroundings. We hypothesized that inserting the bulky cpFP there (e.g. in HyPer sensors) could diminish sensing performance by possibly increasing the conformational entropy of the intermediate state that brings C199 and C208 into proximity 27 . We performed pairwise residue distance analysis between oxidized and reduced ecOxyR structures and found that the region between residues 209–220 goes through noticeable peroxide-dependent conformational change [Supp. Figure 1B]. Therefore, we tested alternative cpGFP insertion within this region. The functional screening for oROS sensors was performed in Human Embryonic Kidney (HEK293) cells to ensure compatibility with other mammalian host systems. cpGFP insertion between residue 211 and 212 elicited a robust response (97.55% increase in ∆F/Fo; confidence interval 95% (ci) = [96.6, 98.52]) to 300 µM extracellular peroxide, which has been reported to induce full oxidation of OxyR-based sensors 22 [Fig. 1B, C]. The 211–212 variant responded immediately (in 25–75% sensor saturation response kinetics, 1.06s; ci = [1.05, 1.07]) which was not observed in other ecOxyR-based sensors 19 , 21 , 22 , 25 . Moreover, the variant showed improved response amplitudes (20.41% in ∆F/Fo; ci = [19.62, 21.17]) to low peroxide levels (10µM) compared to HyperRed (2.8% in ∆F/Fo; ci = [2.61, 3.0]), which incorporates the red fluorescent protein cpmApple between OxyR positions 205 and 206 [Fig. 1D]. Next, based on the guiding principles learned from engineering of the calcium indicator GCaMP5 28 , we introduced large and apolar amino acid tyrosine at the residue sites putatively proximal to the cpGFP predicted opening to reduce solvent access. We found the E215Y mutation increased response amplitude (∆F/Fo) by 2.1-fold at full oxidation (ci = [1.99, 2.26]) and we named this variant oROS-G [Fig. 1E, Supp. Figure 1C] . Characterization of ultrasensitive and fast peroxide sensor, oROS-G. We first characterized the fluorescence response of the oROS-G sensor in HEK293 cells in response to exogenously or endogenously sourced H 2 O 2 . Direct application of exogenous H 2 O 2 increases intracellular H 2 O 2 by diffusion across the plasma membrane through specific aquaporins, which creates an extracellular-to-intracellular gradient of H 2 O 2 29–31 . Under these conditions, the intracellular concentration of H 2 O 2 is reported to be about 2 or 3 magnitudes lower than that of extracellular H 2 O 2 22,32 . On the other hand, the pharmacological agent menadione produces H 2 O 2 intracellularly through various redox cycling mechanisms 33 . The signal amplitude of oROS-G (192.34% in ∆F/Fo; ci = [190.45, 194.23]) at saturation (300 uM H 2 O 2 ) was ≈ 2-fold greater than that of HyPerRed (97.74% in ∆F/Fo; ci = [96.52, 99.06]), Improved sensitivity of oROS-G yielded a ≈ 7.08 times larger response at low-level peroxide stimulation. (oROS-G: 116.22% in ∆F/Fo; ci = [110.85, 121.73] vs HyPerRed: 16.45% in ∆F/Fo; ci = [15.98, 16.95]) [ Fig. 2A ] oROS-G also exhibited significant improvement in on-kinetics compared to HyPerRed with ≈ 38 times faster 25–75% ∆F/Fo kinetics [Fig. 2B]. Intriguingly, the fast oxidation kinetics of the oROS-G sensor captured the H 2 O 2 diffusion across the imaging field of view from the media mixing, in contrast to HyPerRed which exhibited uniform population response [Supp. Figure 2A, B]. Further analysis revealed the speed of peroxide diffusion during media mixing to be ≈ 824µm/s [Supp. Figure 2C, D]. The speed of peroxide travel slows down to ≈ 100µm/s after passing the cell plasma membrane during intracellular diffusion. This potentially represents peroxide travel becoming rate limited by aquaporin-driven passive transmembrane diffusion 30 , 34 [Supp. Figure 2E, F]. Taken together, visualization of bolus H 2 O 2 introduction using oROS-G was only rate-limited by H 2 O 2 travel speed and transmembrane transport rate, allowing real-time observation of intracellular peroxide diffusion in mammalian cells. Thus, oROS-G can be a vital tool for expanding our understanding of the dynamic topological and temporal landscape of peroxide in biological systems. In HEK293 cells, oROS-G also acutely responded to 10µM and 50µM menadione in a dose-dependent manner (∆F/Fo, 10µM: 89.56%; ci = [81.79, 97.57], 50µM: 173.68%; ci = [166.81, 180.35]) [Fig. 2C]. The result was consistent in human primary astrocytes, [Supp. Figure 3A] highlighting the potential robustness of oROS-G expression and functionality in broader biological host systems. In addition, we confirmed the robust expression and function of oROS-G in rat cortical neurons and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) [Fig. 2D]. Next, we confirmed that oROS-G is a fully reversible sensor by directly reducing it using 10 mM Dithiothreitol (DTT) [Fig. 2E] or media washout [Supp. Figure 2B] . Here, we noticed that the endogenous reduction kinetics of the sensor in mammalian cells was faster than other OxyR-based sensors 22 , 24 . For example, both HyPerRed and HyPer7 took 20 ~ 30 minutes for them to return to baseline after the sensor saturation. HyPer7 is the newest green iteration of the HyPer sensor family that was engineered by swapping the sensing domain with a different OxyR domain from Neisseria meningitidis (nmOxyR) 24 with fluorescent reporter insertion contained to the C-C loop region. oROS-G reached ≈ 90% reduction from its maximum saturation in 4.17 minutes, whereas HyPer7 only achieved about ≈ 20% reduction from its full saturation in the same duration, consistent with the previous report. (HyPer7: 0.81; ci = [0.8, 0.82], oROSG: 0.12; ci = [0.1, 0.15]) oROS-G showed 2.63 times faster decay kinetics than HyPer7 based on approximation with reduction time to 85% of saturation, making oROS-G a more compelling candidate for measurement of peroxide transient rise and decay of intracellular peroxide species [Fig. 2F, Supp. Figure 3C] . Lastly, we created a C199S mutant of oROS-G to show that the fluorescence response was specific to peroxide-induced disulfide bridging of C199-C208, which is consistent with other OxyR-based peroxide sensors [Supp. Figure 3D] . Monitoring the effect of antioxidants on intracellular peroxide level in Alzheimer’s model. Next, we explored using oROS-G in the context of antioxidants that target intracellular peroxides. N-acetyl-cystine (NAC) is a cysteine prodrug widely used as a classical “antioxidant”. Although its detailed mechanism of action has not been established, recent studies highlight its antioxidative role via the production of low-level sulfane sulfur species 35 , 36 . Using oROS-G, we measured the effect of NAC-dependent catabolism on cellular H 2 O 2 levels in real-time. With a 1-hour preincubation of 1 mM NAC, oROS-G expressed in HEK293 showed a 73% diminished response to exogenous 10 µM peroxide exposure [Supp. Figure 4A] . Similarly, we incubated oROS-G expressing HEK293 cells to either NAC (10 mM) or Vehicle (DMSO) for 20 minutes before 10 µM menadione exposure. NAC significantly attenuated the response by 72 percent [Supp. Figure 4B] . We next examined the ability of the oROS-G sensor in primary cultured astrocytes to quantitatively detect endogenous H 2 O 2 levels to assess the H 2 O 2 -scavenging effects of molecules. Initially, we expressed oROS-G in the astrocytes and tested H 2 O 2 concentration-dependent functionality [Supp. Figure 4C] . We observed an increase of 21.27 ± 5.3% and 57.74 ± 9.4% in fluorescence with 10 and 100 µM H 2 O 2 , respectively [Supp. Figure 4D-F] , indicating a functional response of the oROS-G sensor in primary cultured astrocytes. Previously, we have shown that aberrant H 2 O 2 production by reactive astrocytes, a pathological form of astrocytes, is a contributing factor to Alzheimer’s disease (AD) pathology 11 , 37 . In detail, upregulated monoamine oxidase B (MAOB) in reactive astrocytes produces H 2 O 2 by breaking down oligomerized amyloid beta (oAβ)-metabolites, such as putrescine, leading to pathological H 2 O 2 generation 38 , 39 [Fig. 3A]. Therefore, we tested whether we could monitor aberrant endogenous H 2 O 2 production in oROS-G transfected astrocytes treated with oAβ (5 µM) or putrescine (180 µM). Over a 40-hour continuous recording [Fig. 3B] , we observed a significant increase in oAβ-induced oROS-G fluorescence, indicating a notable rise in H 2 O 2 . [Fig. 3C, D]. Conversely, the application of KDS2010 (1 µM), a selective MAOB inhibitor, and sodium pyruvate (1 mM), a potential H 2 O 2 scavenger, showed a smaller increase in H 2 O 2 levels. Additionally, incubation with putrescine, a pre-substrate of MAOB, also significantly increased oROS-G sensor fluorescence [Fig. 3E, F] . However, this H 2 O 2 elevation was significantly reduced by KDS2010 and partially reduced by sodium pyruvate. Taken together, these results suggest that the oROS-G sensor in primary cultured astrocytes is a reliable tool for monitoring endogenous H 2 O 2 production under AD-like conditions and evaluating the efficacy of potential H 2 O 2 -scavenging compounds. Then we asked whether we could measure the endogenous H 2 O 2 levels in mouse brains. To test this idea, we bilaterally injected the AAV5-GFAP104-oROS-G virus into the CA1 hippocampus of APP/PS1 mice 39 , 40 , a well-known AD model, to overexpress oROS-G sensor specifically in the astrocytes [Fig. 3G]. Two weeks post-injection, we prepared brain slices and tested the H 2 O 2 concentration-dependent functionality of the sensor. Again, to test the functionality of the sensor, we applied 10 and 100 µM H 2 O 2 through bath application. We found an increase of 13.24 ± 1.2% and 39.46 ± 3.12% in fluorescence with 10 and 100 µM H 2 O 2 , respectively [Fig. 3H, I]. Like in vitro , the oROS-G sensor functions effectively in astrocytes ex vivo . Next, we examined the capability to measure elevated H 2 O 2 levels in astrocytes of APP/PS1 mice. We hypothesized that treatment with DTT would unmask the portion activated by astrocytic H 2 O 2 . Following DTT (10 mM) administration, we observed a reduction in fluorescence below the baseline levels. Notably, we demonstrated that APP/PS1 mice exhibited a greater reduction compared to wild-type, suggesting a potential method for measuring endogenous H 2 O 2 levels [Fig. 3J-L]. Taken together, these results demonstrate that the oROS-G sensor functions effectively ex vivo , presenting a potential method for measuring endogenous H 2 O 2 levels and investigating the antioxidant capacity of various molecules. oROS-Gr for long-term and non-continuous monitoring of intracellular H 2 O 2 . Most ratiometric sensors designed for peroxide response are based on dual-excitation of the green fluorescent sensor proteins at 405 nm and 488 nm 41 , 42 . Although this sensor type allows flexibility in multi-color optogenetic experiments, illumination at 405 nm could contribute to oxidative stress in mammalian cells. 43 We created oROS-Gr, by fusing mCherry to oROS-G, creating an equimolar reference point inert to H 2 O 2 . Flow cytometry analysis confirmed a strong linear correlation between green (Em. 510 nm) and red (Em. 605 nm) emission intensity of oROS-Gr expressed in HEK293 cells (n = 16,326) [Fig. 4A] . Thus, oROS-Gr can be used for long-term and non-continuous monitoring by calculating the green-to-red light emission ratio independent of sensor expression levels. Upon exogenous H 2 O 2 stimulation, the oROS-Gr ratio (Em. 510/605) showed a dose-dependent response in HEK293 cells. (1 µM: 0.06 (n = 327); ci = [0.06, 0.06], 10 µM: 0.11 (n = 306); ci = [0.1, 0.11], 25 µM: 0.14 (n = 405); ci = [0.14, 0.14], 50 µM: 0.15 (n = 469); ci = [0.15, 0.15]) [Fig. 4B] In addition, the oROS-Gr green-to-red ratio predictably followed a sequence of exogenous H 2 O 2 stimulation (100 uM) and DTT (10 mM) reduction [Fig. 4C, Supp. Figure 5] . Menadione treatment has been widely used to model oxidative stress in biological systems 44 – 47 . Still, studies to monitor its intracellular effect have been mostly limited to short time windows or non-continuous snapshots at varying time points. These studies did not provide insights into the real-time impact on redox homeostasis over a longer period. Here, we used to continuously measure (sampling every 5 minutes) the effects of menadione on cellular H 2 O 2 levels over a ten-hour time window using stable oROS-Gr expressing HEK293 cells. Initially, menadione at 0, 1, 10, and 50 uM induced acute dose-dependent elevation of H 2 O 2 . However, within 30 minutes, the H 2 O 2 levels at 10µM were higher than those at 50µM, which returned to a dose-dependent trend within four hours. [Fig. 4D] Further analysis of intracellular redox landscape analysis and functional role of putative cellular antioxidative elements 48 , 49 is required to understand this phenomenon fully. Nevertheless, the temporally perplexing effects of menadione on intracellular H 2 O 2 level gives us a cautionary insight to avoid the assumption that an oxidative agent such as menadione has always a direct dose-dependent effect on the intracellular peroxide level. We also tested if using oROS-Gr can improve the readout precision over oROS-G. We compared the coefficient of variation (CoV) of the ratiometric data (Em. 510/605) against data acquired in single wavelength mode (Em. 510) during long-term menadione exposure [Fig. 4E]. The ratiometric readout showed about 2-fold lower CoV compared to the single wavelength mode, confirming improvements in precision [Ratiometric: 0.27 (n = 484), non-Ratiometric: 0.46 (n = 484)]. We confirmed the robust expression and functionality of oROS-Gr in various human stem cell-derived cells. For example, we measured peroxide levels in hiPSC-derived cortical neurons in response to 24-hour 10 µM and 50 µM Menadione incubation to be 1.77-fold and 2-fold of oROS-Gr ratio observed at vehicle negative control, respectively (Vehicle: 1.0; ci = [0.82, 1.21], 10 µM: 1.77; ci = [1.62, 1.88], 50 µM: 2.01; ci = [1.97, 2.03]) [Fig. 4F] . Next, we used the sarcoendoplasmic reticulum calcium ATPase (SERCA) blocker cyclopiazonic acid (CPA, 10 µM) to elevate Ca 2+ in the cytosol of hiPSC-cardiomyocytes (CM) 50 . When hiPSC-CMs expressed oROS-Gr, we measured increased cytosolic peroxide levels within 2 hours of CPA incubation [Supp. Figure 4]. As previously reported, this confirms a tight coupling between intracellular Ca 2+ and ROS levels 51 – 54 . Glucose-dependent basal oxidation level in mammalian cells. Superoxide and peroxide are continuously generated as byproducts through electron transfers during aerobic metabolism 55 , 56 , 57 . In this context, glucose, as one of the primary substrates of aerobic metabolic pathways, plays a crucial role in modulating cellular metabolic activity 58 . Intriguingly, low as well as high glucose levels were reported to result in depressed respiratory activity in cultured human podocytes 59 . The study also showed that the reduction of metabolic rates in high-glucose conditions can be reversed by incubation with the antioxidant NAC, indicating that respiratory suppression is correlated with oxidative stress. Thus, we hypothesized high glucose (HG = 25 mM) but also low glucose (LG = 1 mM) media would result in higher basal peroxide levels than medium glucose (MG = 10 mM). We incubated HEK293 cells for 48 hours in HG, NG, and LG media and compared the ratiometric oROS-Gr signals. Here, low and high glucose conditions caused higher peroxide levels than MG (MG: 0.38; ci = [0.378, 0.382], LG: 0.402; ci = [0.4, 0.404], HG: 0.392; ci = [0.389, 0.394]). [Fig. 5A]. We directly measured metabolic activities and found that basal and maximum respiratory rates were also the lowest under low and high glucose conditions [Fig. 5B, Supp. Figure 7A, B] , indicating an inverse correlation with increased peroxide levels. Indeed, cells that were pre-incubated with 1 mM of the antioxidant NAC under HG conditions brought the oROS-Gr level 84% closer to MG conditions, indicating modest suppression of oxidative stress [Supp. Figure 7C]. G-protein biased agonists elicit H 2 O 2 generation in κ and µ opioid receptor-expressing neurons in the Ventral Tegmental Area ex vivo and in vivo . We previously reported that peroxide generated by a NADPH oxidase (NOX) mechanism regulated opioid receptor signaling 60 , 61 , which exemplifies intricate functional G-protein biased agonists influence on arrestin-independent inactivation profile of µ and κ opioid receptors. Briefly, G-protein biased opioid receptor activation triggers cJUN N-terminal kinase (JNK) phosphorylation. Phosphorylated JNK then activates peroxiredoxin 6 (PRDX6), producing superoxide (SO) from NOX. SO can quickly oxidize the Gαi protein complex to inactivate the opioid receptors. This event can be captured using H 2 O 2 as a marker of opioid receptor activation because superoxide is readily transformed into H 2 O 2 by superoxide dismutase 62 – 65 [Fig. 6A] . With impressive sensitivity and robust expressibility of oROS-Gr, we sought to monitor transient H 2 O 2 generation in the animal brain in response to G-protein biased agonists. As a proof of concept, we showed that morphine, a potent G-protein biased agonist of µ-opioid receptors (MOR), triggers transient peroxide generation in MOR-expressing neurons in the ventral tegmental area (VTA) of MOR-Cre transgenic mice, which is consistent with our previous findings. The oROS signals were measured using 2-photon microscopy on ex vivo live brain slices after viral delivery of the AAV1-DIO-oROS-Gr into the VTA of MOR-Cre transgenic mice. Expression of oROS-Gr in the VTA was verified with one-photon confocal microscopy of post-mortem fixed brain slices [Fig. 6B] . The VTA in ex vivo brain slices showed an acute increase in sensor fluorescence during bath application of 1µM morphine over 30 minutes of monitoring. This increase was blocked by the opioid receptor antagonist 1µM Naloxone (Normalized ∆F/Fo to first 5 baseline frames, MOR: 3.35; ci = [1.94, 4.82], MOR + NLX: 0.63; ci = [0.42, 0.85]) [Fig. 6C] . κ-opioid receptor (KOR) has emerged as an promising drug target for pain management with less side-effects 66 . We previously showed behavioral and pharmacological evidence on how the oxidative pressure of JNK-PRDX6-PLA2-NOX cascade from KOR results in acute analgesic tolerance as shown in the warm water tail withdrawal assay 60 . Nalfurafine is a functionally selective G-protein biased κ-opioid agonist shown to have therapeutic potential as a non-dysphoric antipruritic analgesic 67 . Here, we explored the use of the oROS sensor to directly monitor acute H 2 O 2 response to Nalfurafine in vivo , confirming activation of JNK-PRDX6-PLA2-NOX in KOR positive neurons in the VTA. KOR-Cre transgenic mice were injected with AAV1-DIO-oROS-Gr, and the sensor fluorescence (ex:488/em:510) was monitored by fiber photometry in the VTA [Fig. 6D] . Intraperitoneal administration of 100µg/kg Nalfurafine led to transient increase of H 2 O 2 . Mice pre-treated 30 min prior to a 100µg/kg Nalfurafine injection with a high dose of naloxone (10 mg/kg), sufficient to block KOR 68 , showed no significant increase in fluorescence compared to mice only treated with Nalfurafine [Fig. 6E] . This confirms that the Nalfurafine induced H 2 O 2 signal in KOR expressing neurons of VTA is opioid receptor specific. Discussion To further improve our understanding of redox biology, we need the ability to monitor oxidative agents in diverse, multi-faceted contexts. Our development of the oROS sensor framework represents a significant step in this direction. As a novel green fluorescent sensor, oROS-G demonstrates unparalleled sensitivity and response kinetics for H 2 O 2 monitoring, surpassing the capabilities of previous ecOxyR-based sensors. This enhancement is largely attributed to our structural refinement of the sensor, where we relocated the cpGFP insertion site to maintain flexibility of C199-C208 loop of ecOxyR. Drawing inspiration from Akerboom et al. 28 , we incorporated bulky residues adjacent to the cpGFP barrel opening, exemplified by the E215Y mutation in oROS-G. Our study has revealed a novel insertion site within ecOxyR, paving the way for the creation of H 2 O 2 sensors that are both fast and sensitive. Our hypothesis suggests that the flexible region in the ligand sensing domain is intrinsically linked to sensor function. These principles could lay the groundwork for future optogenetic sensors tailored to detecting other analytes. Interestingly, the diversity of OxyR variants in nature, each characterized by a conserved peroxide oxidation mechanism, opens avenues for exploring a range of sensor functionalities. Notably, OxyRs from different bacterial strains exhibit distinct reduction mechanisms; ecOxyR predominantly follows a Grx (glutaredoxin)-dependent reduction pathway, where Grx proteins facilitate the reduction of oxidized proteins 69 . In contrast, other variants like nmOxyR ( Neisseria meningitidis ) might employ a Trx (thioredoxin)-dependent reduction mechanism, involving the Trx system known for mitigating cellular oxidative stress (e.g. HyPer7) 41 . This variation necessitates further exploration of these domains for sensors in mammalian systems, where they could serve as complementary tools for dissecting peroxide biology in various redox environments. Our study also demonstrated the practical versatility of oROS sensors in a range of experimental setups. With oROS-G, we successfully monitored H 2 O 2 levels in astrocytes, both in vitro and ex vivo , shedding light on cellular redox states. Moreover, the ratiometric oROS-Gr sensor enabled us to observe the effects of glucose on cytoplasmic peroxide levels, which correlated with known patterns of mitochondrial oxidative stress. Future studies should aim to clarify the sources of peroxide accumulation, considering factors like NADPH oxidase activity and mitochondrial respiration. Additionally, our work highlights the oROS sensor's efficacy in detecting opioid-induced peroxide increase in vivo , further emphasizing its broad applicability. In conclusion, the oROS sensors, exemplified by oROS-G and oROS-Gr, offer a new paradigm for studying peroxide biology. Their application across various model systems has the potential to revolutionize our approach to understanding and monitoring complex redox processes, with significant implications for unraveling the mechanisms underlying various oxidative stress-related diseases. Declarations Acknowledgments J.D.L was supported by 1F31DA056121-01A1 and an ISCRM Fellowship. A.B was supported by the Brain Research Foundation, UW Royalty Research Fund, UW ISCRM IPA, NIGMS R01 GM139850-01, P30 DA048736, NIMH RF1MH130391, NINDS U01NS128537, NIDA R21DA051193 and the McKnight Foundation’s Technologies in Neuroscience Award. K. E. was supported by T32AG066574. The research received additional support from the Lynn and Mike Garvey Imaging Core, the UW NAPE Center, and ISCRM Shared Equipment. We want to thank Dr. Randy Moon for his support. Also, this work was supported by the Institute for Basic Science, Center for Cognition and Sociality (IBS-R001-D2) to C.J.L. We are also grateful to the IBS virus facility for providing a virus packaging service for in vivo experiments. Data Availability Source data will be available via figshare shortly. Code Availability Source code will be available at https://github.com/justindaholee/oROS-G_manuscript shortly. Ethics Statement All animal procedures performed at the University of Washington have been approved by the University of Washington’s Animal Use Committee (protocol #4422-01) and follow the National Institute of Health and the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. Handling and animal care have been performed according to the Institutional Animal Care and Use Committee of the Institute for Basic Science. Material requests Plasmid name: Addgene # pC3.1_CMV_oROS-G: 216111, pC3.1_CMV_oROS-G_LF: 216112, pCAG_oROS-Gr: 216113, pCAG_oROS-Gr-LF: 216114, AAV2_CAG_oROS-G: 216115, AAV2_CAG_oROS-G_LF: 216116 Methods Protein structure analysis Protein structure analysis and plotting were performed using Chimera-X-1.2.1. Oxidized [PDB:1I6A] and reduced [PDB:1I69] crystal structures of ecOxyR were imported from the Protein Data Bank (PDB). Pairwise residue distance between reduced and oxidized ecOxyR structure was achieved by aligning both structures using a matchmaker algorithm that superimposes protein structures by creating a pairwise sequence alignment and then fitting the aligned residue pairs to derive pairwise residue distances. Molecular Biology oROS-HT variants were cloned based on the pC1 plasmid backbone from pC1-HyPer-Red (Addgene ID: 48249). Primers for point mutations or fragment assembly required to generate the oROS-HT screening variants were designed for In Vitro Assembly cloning (IVA) technique 70 , gibson assembly (New England Biolabs; E2611L) or blunt-end amplification for KLD-based site-directed mutagenesis methods. Primers were ordered from Integrated DNA Technologies (IDT). All gene fragment amplifications were done using Seither Q5-polymerase (New England Biolabs; M0492L) or Superfi-II polymerase (Invitrogen; 12368010). Amplification of DNA fragments were verified with agarose gel electrophoresis. 30 minutes of DpnI enzyme treatment were done on every PCR product to remove the plasmid template from PCR samples. For IVA cloning circularization or assembly of the PCR products was achieved by transforming linear DNA products into competent E.Coli cells (DH5ɑ or TOP10) and grown on agar plates that contain either ampicillin or kanamycin selection antibiotic (50 µg/mL). For gibson assembly and KLD cloning, circularized DNA was transformed as above. Upon colony formation, single colonies were picked and grown in 5mL cultures containing LB Broth (Fisher BioReagents; BP9723-2) and selection antibiotic (ampicillin/kanamycin; 50 µg/mL) overnight (37°C, 230 RPM). DNA was isolated using Machery Nagel DNA prep kits (Machery Nagel; 740490.250). Sanger sequencing (Genewiz; Seattle, WA) or Whole plasmid nanopore sequencing (Plasmidsarus; Eugene, OR) of the isolated plasmid DNA was used to confirm the presence of the intended mutation. Genes encoding the final variants were cloned into a CAG-driven backbone, pCAG-Archon1-KGC-EGFP-ER2-WPRE (Addgene; #108423), using the methods above. England Biolabs; E2621L). All subsequences were verified with Sanger sequencing (Genewiz; Seattle, WA) or Whole plasmid nanopore sequencing (Plasmidsarus; Eugene, OR) Chemicals H 2 O 2 working solutions were freshly prepared before every experiment from H 2 O 2 solution 30 % (w/w) in H 2 O (Sigma-Aldrich, H1009). Stock solution of Menadione (VENDOR, CAT) was prepared in 100% DMSO at 50mM. Stock solution of Cyclopiazonic Acid (Tocris, 1235) was prepared in 100% DMSO at 20mM. Chemicals specific to other method sections can be found in their respective sections. Cell culture and transfection Human Embryonic Kidney (HEK293; ATCC Ref: CRL-1573) cells were cultured in Dulbecco’s Modified Eagle Medium + GlutaMAX (Gibco; 10569-010) supplemented with 10% fetal bovine serum (Biowest; S1620). When cultures reached 85% confluency, the cultures were seeded at 150,000/75,000 cells per well in 24/48-well plates, respectively. 24 hours after cell seeding, the cells were transfected using Lipofectamine3000 (Invitrogen; L3000015) at 1000/500 ng of DNA per well of a 24/48-well plate, according to the manufacturer’s instructions. Primary rat neuron isolation Primary cortical neurons were prepared as previously described 47,48 . Briefly, 24-well tissue culture plates were coated with Matrigel (mixed 1:20 in cold-PBS, Corning; 356231) solution and incubated at 4°C overnight prior to use. Sterile dissection tools were used to isolate cortical brain tissue from P0 rat pups (male and female). Tissue was minced until 1mm pieces remained, then lysed in equilibrated (37°C, 5% CO 2 ) enzyme (20 U/mL Papain (Worthington Biochemical Corp; LK003176) in 5mL of EBSS (Sigma; E3024)) solution for 30 minutes at 37°C, 5% CO 2 humidified incubator. Lysed cells were centrifuged at 200xg for 5 minutes at room temperature, and the supernatant was removed before cells were resuspended in 3 mLs of EBSS (Sigma; E3024). Cells were triturated 24x with a pulled Pasteur pipette in EBSS until homogenous. EBSS was added until the sample volume reached 10 mLs prior to spinning at 0.7 rcf for 5 minutes at room temperature. Supernatant was removed, and enzymatic dissociation was stopped by resuspending cells in 5 mLs EBSS (Sigma; E3024) + final concentration of 10 mM HEPES Buffer (Fisher; BP299-100) + trypsin inhibitor soybean (1 mg/ml in EBSS at a final concentration of 0.2%; Sigma, T9253) + 60 µl of fetal bovine serum (Biowest; S1620) + 30 µl 100 U/mL DNase1 (Sigma;11284932001). Cells were washed 2x by spinning at 0.7 rcf for 5 minutes at room temperature and removing supernatant + resuspending in 10 mLs of Neuronal Basal Media (Invitrogen; 10888022) supplemented with B27 (Invitrogen; 17504044) and glutamine (Invitrogen; 35050061) (NBA++). After final wash spin and supernatant removal, cells were resuspended in 10 mLs of NBA++ prior to counting. Just before neurons were plated, matrigel was aspirated from the wells. Neurons were plated on the prepared culture plates at desired seeding density. Twenty-four hours after plating, 1µM AraC (Sigma; C6645) was added to the NBA++ growth media to prevent the growth of glial cells.Plates were incubated at 37°C and 5% CO 2 and maintained by exchanging half of the media volume for each well with fresh, warmed Neuronal Basal Media (Invitrogen; 10888022) supplemented with B27 (Invitrogen; 17504044) and glutamine (Invitrogen; 35050061) every three days. Human primary astrocytes, and stem cell derived cardiomyocytes and neurons Astrocytes: Human primary cortical astrocytes were purchased from ScienCell Research Laboratories (Carlsbad, CA) and were stored, thawed and sub-cultured based on the manufacturer’s protocol. Briefly, the astrocytes were cultured for 72 h in a base medium with an astrocyte growth supplement and fetal bovine serum provided by the same manufacturer. Cultures were maintained in a 37°C/5% CO 2 incubator throughout the culture period, and the astrocytes with low passage numbers (p0-p3) were used to guarantee consistent phenotype expression. When the culture became 70% confluent, the cells were dissociated with TrypLE (Thermo Fisher), followed by passaging on the PDL-coated 24 cover glasses for oROS-G1 transfection. The transfected cells were then cultured for an additional 96 h before H 2 O 2 treatment (10 µM, 100 µM) for recording the fluorescence response upon H 2 O 2 stimulation. Cardiomyocytes: Undifferentiated IMR90 (WiCell) hiPSCs were maintained on Matrigel (Corning) coated tissue culture plates in mTeSR1 (Stemcell Technologies). Cardiomyocyte directed differentiation was performed using a modified small molecule Wnt-modulating protocol using Chiron 99021 and IWP-4 as previously described. 71,72 . Lactate enrichment was performed following differentiation to purify hiPSC-CMs. 73 Cortical neurons: Neurons were generated from the previously characterized wild type CV background human induced pluripotent stem cell line (Young et al. 2015). Neural progenitor cells (NPCs) from this cell line were differentiated from hiPSCs using dual-SMAD inhibition and NPCs were differentiated to neurons as previously described (Knupp et al., 2020; Shin et al., 2023). Briefly, for cortical neuron differentiation from NPCs, NPCs were expanded into 10 cm plates in Basal Neural Maintenance Media (BNMM) (1:1 DMEM/F12 (#11039047 Life Technologies) + glutamine media/neurobasal media (#21103049, GIBCO), 0.5% N2 supplement (# 17502-048; Thermo Fisher Scientific,) 1% B27 supplement (# 17504-044; Thermo Fisher Scientific), 0.5% GlutaMax (# 35050061; Thermo Fisher Scientific), 0.5% insulin-transferrin-selenium (#41400045; Thermo Fisher Scientific), 0.5% NEAA (# 11140050; Thermo Fisher Scientific), 0.2% β-mercaptoethanol (#21985023, Life Technologies) + 20 ng/mL FGF (R&D Systems, Minneapolis, MN). Once the NPCs reached 100% confluence, they were switched to Neural Differentiation Media (BNMM +0.2 mg/mL brain-derived neurotrophic factor (CC# 450–02; PeproTech) + 0.2 mg/mL glial-cell-derived neurotrophic factor (CC# 450–10; PeproTech) + 0.5 M dbcAMP (CC# D0260; Sigma Aldrich). Neural Differentiation Media was changed twice a week for 21 days at which point the differentiation is considered finished. Neurons were replated at a density of 500,000 cells/cm 2 . Imaging Imaging experiments described in this study were performed as follows unless specifically noted. Epifluorescence imaging experiments were performed on a Leica DMI8 microscope (Semrock bandpass filter: GFP ex/em: FF01-474-27/FF01-520-35, RFP ex/em:FF01-578-21/FF01-600-37) controlled by MetaMorph Imaging software, using a sCMOS camera (Photometrics Prime95B) and 20x magnification lens (Leica HCX PL FLUOTAR L 20x/0.40 NA CORR) or 10× objective (Leica HCX PL FLUOTAR L 10x/0.32 NA). Confocal imaging experiments were performed on a Leica SP8 confocal microscope from the Lynn and Mike Garvey Imaging Core at the Institute of Stem Cell and Regenerative Medicine. Cells were imaged in live cell imaging solution with 10mM glucose (LCIS+, Gibco, A14291DJ). Image analysis methods described below. Analysis Analysis of cell fluorescence imaging data was done by FUSE, a custom cloud-based semi-automated time series fluorescence data analysis platform written in Python. First, the cell segmentation quality of the selected Cellpose 74 model was manually verified. For the segmentation of cells expressing cytosolic fluorescent indicators, model ‘cyto’ was selected as our base model. If the selected Cellpose model was low-performing, we further trained the Cellpose model using the Cellpose 2.0 human-in-the-loop system 75 . Using an “optimized” segmentation model, fluorescence time-series data is extracted for each region of interest. This allows for unbiased extraction of change in cellular fluorescence information for a complete set of experimental samples. Extracted fluorescence data is normalized as specified in the text using custom python script. Astrocyte study Primary mouse astrocyte culture: Primary mouse cultured astrocytes were prepared from P1-P3 C57BL/6J mouse pups as previously described. 76 Briefly, 60-mm culture dishes were coated with 0.1 mg/ml poly-D-lysine (PDL, Sigma; P6407) solution prior to use. The hippocampal tissue was isolated, and dissociated into single cell suspension by trituration in Dulbecco’s modified Eagle’s medium supplemented with 4.5 g/L glucose, L-glutamine, sodium pyruvate (DMEM, Corning; 10-013-CV) + 10% heat-inactivated horse serum (Gibco; 26050-088) + 10% heat-inactivated fetal bovine serum (Dawin bio; A0100-010) + 1000 unit/ml penicillin-streptomycin (Gibco; 15140122). Dissociated cells were plated onto the PDL coated dishes. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO 2 incubator. On the third day, cells were vigorously washed with repeated pipetting using medium to get rid of debris and other floating cell types. On the 10th day of culture, cultured primary astrocytes were electrophoretically transfected with oROS-G plasmid with a voltage protocol (1200 V, 20 pulse width, 2 pulses) using the Microporator (Invitrogen Neon Transfection System; MPK5000S) and replated onto coverglass coated with PDL (Sigma; P6407) or µ-Plate 96 Well Black (ibid; 89626). Imaging of cultured primary mouse astrocytes: On the 14th day of culture, the oROS-G transfected cultured primary astrocytes were transferred to a recording chamber which were mounted on an inverted Nikon Ti2-U microscope and continuously perfused with an external solution contained (in mM): 150 NaCl, 10 HEPES, 5.5 glucose, 3 KCl, 2MgCl2, 2 CaCl2, and pH adjusted to pH 7.3. Intensity images of 525 nm wavelength were taken at 485 nm excitation wavelengths using ORCA-Flash4.0 CMOS camera (Hamamatsu; C13440). Imaging workbench (INDEC Biosystem) and ImageJ (NIH) were utilized for image acquisition and ROI analysis of cultured astrocytes. To examine H 2 O 2 -dose dependent responses of oROS-G transfected cultured astrocytes, concentration of 10 and 100 µM of H 2 O 2 (Sigma; 88597) were introduced by bath application. The peak response of the sensor was normalized to its baseline (ΔF/Fo), which was measured 90 seconds before introducing H 2 O 2 . For confocal live-cell imaging and monitoring antioxidant drugs, confocal imaging was performed by using Nikon A1R confocal microscope mounted onto a Nikon Eclipse Ti body with 20x objective lens. A Live-cell imaging chamber and incubation system were used for maintaining environmental conditions at 10% CO2 and 37°C during 40-hour continuous recording. Images were acquired by using NIS-element AR (Nikon). For image analysis, NIS-element (Nikon) and ImageJ (NIH) were used. Animals: All APP/PS1 mice were group-housed in a temperature- and humidity-controlled environment with a 12 h light/dark cycle and had free access to food and water. Virus injection: The AAV5-GFAP104-oROS-G viral vector was cloned and AAV containing GFAP-104-oROS-G was packaged by the IBS virus facility (Daejeon, Korea). Mice were deeply anesthetized via vaporized 1% isoflurane and immobilized in a stereotaxic (RWD Life Science). Following an incision on the midline of the scalp, bilateral craniotomies were performed above the hippocampus CA1 (anterior/posterior, -2 mm; medial/lateral, ±1.6 mm; dorsal/ventral, -1.45 mm from the bregma) using a microdrill. The virus was bilaterally microinjected (0.1 μl/min for 10 min; total 0.8 μl) using a syringe pump (KD Scientific). oROS-G imaging of GFAP-positive astrocytes in the brain slices: A total of 2 weeks after the virus injection into the hippocampus, animals were anesthetized with 1% isoflurane and then decapitated. The brains were submerged in chilled cutting solution that contained (in mM): 250 Sucrose, 26 NaHCO3, 10 D(+)-glucose, 4 MgCl2, 0.1 CaCl2, 2.5 KCl, 2 Sodium Pyruvate, 1.25 NaH2PO4, 0.5 ascorbic acid, and pH adjusted to pH 7.4. Coronal slices (300 μm thick) were prepared with a vibrating-knife microtome D.S.K LinearSlicer pro 7 (Dosaka EM Co. Ltd). For stabilization, brain slices were incubated at room temperature for at least 1 h before imaging. For imaging, the slices were transferred to a recording chamber which were mounted on an upright Zeiss Examiner D1 microscope and continuously perfused with an artificial cerebrospinal fluid (aCSF) solution that contained (in mM): 130 NaCl, 24 NaHCO3, 1.25 NaH2PO4, 3.5 KCl, 1.5 MgCl2, 1.5 CaCl2, D(+)-glucose, and pH adjusted to pH 7.4. All solutions were equilibrated with 95% O2 and 5% CO2. Imaging was acquired at 0.25 frame per second with 60X water-immersion objective lens, a ORCA-Flash4.0 CMOS camera (Hamamatsu; C13440), and a LED (CoolLED) filtered with 485-nm fluorescence was applied. Imaging workbench (INDEC Biosystem) and ImageJ (NIH) were utilized for image acquisition and ROI analysis. To examine H 2 O 2 -dose dependent responses of sensor-expressing astrocytes, concentration of 10 and 100 µM of H 2 O 2 were introduced by bath application. The peak response of the sensor was normalized to its baseline (ΔF/Fo), wich was measured 90 seconds before introducing H 2 O 2 . To measure endogenous H 2 O 2 in astrocytes of APP/PS1 mice and their littermates, we used 10 mM DTT (Thermo; R0861). This method reduced the oROS-G sensor bound to H 2 O 2 , resulting in fluorescence below the baseline levels. These reduced fluorescence responses were normalized to its baseline (ΔF/Fo), suggesting the basal endogenous H 2 O 2 levels. Generation of stable oROS-Gr expressing HEK293 cells. HEK293 cells in a T75 flask were transfected (using lipofection, as described above) with oROS-Gr-P2A-Puromycin plasmid. 3 Days after the transfection, cells were passaged to 2 T75 flasks. 2 Days after, puromycin-based selection was performed for a week using complete DMEM media (as previously described) supplemented with puromycin (1µg/mL). Cells after selection were expanded for 3 passages. Enrichment of cell populations with robust oROS-Gr expression was achieved with BD FACSAria II Cell Sorter at Flow and Imaging Core Lab of University of Washington South Lake Union Campus. Glucose experiment and Seahorse Assay oROS-Gr stable cells cultured in complete DMEM with 10mM glucose were plated at 75,000/well in 24-well plates. oROS-Gr stable cells were plated at 75,000/well in 24-well plates. 1 day post seeding, FBS in the DMEM media was brought down to 2% from 10%. 2 day post seeding cells were in serum-free DMEM with various levels of glucose. Mannose was supplemented as needed to keep osmotic pressure of each media consistent (final total sugar content: 25mM). oROS-Gr ratio (GFP/RFP) were imaged in LCIS media with varying glucose and mannose level. For Seahorse assay, oROS-Gr stable cells mentioned above were plated in a Matrigel-coated 96 well Seahorse plate at a density of 2 × 10 5 cells/well for an equivalent procedure as above. The MitoStress protocol in the Seahorse XF96 Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA) was performed two weeks later. An hour before the assay, the culture media was replaced with media (Agilent Seahorse XF base medium, 103334-100 Agilent Technologies, Santa Clara, CA, USA) supplemented with 25 mM glucose and 1 mM Sodium pyruvate (11360070 Gibco/Thermo Scientific, Waltham, MA, USA). Substrates and select inhibitors of the different complexes were added during the measurement to achieve final concentrations of oligomycin (2.5 μM), FCCP (1 μM), rotenone (2.5 μM) and antimycin (2.5 μM). The oxygen consumption rate (OCR) values were then normalized with readings from Hoechst staining (HO33342 Sigma-Aldrich, St. Louis, MO, USA), which corresponded to the number of cells in the well. Opioid receptor study AAV : An adenovirus associated double floxed inverted (AAV1-DIO) virus was generated containing the oROS-Gr by cloning oROS-Gr into pAAV1-Ef1a-DIO using Nhe1 and Asc1 restriction sites. AAV1 were prepared by the NAPE Molecular Genetics Resources Core as described previously (Gore, et al, 2013). HEK293T cells were transfected with 25 μg AAV1 vector plasmid and 50 μg packaging vector (pDG1) per 15 cm plate. Two days after transfection, cells were harvested and subjected to three freeze–thaw cycles. The supernatant was transferred to a Beckman tube containing a 40% sucrose cushion and spun at 27,000 rpm overnight at 4°C. Pellets were resuspended in CsCl at a density of 1.37 g/ml and spun at 65000 rpm 4 hours at 4°C. 1 ml CsCl fractions were run on an agarose gel, and genome-containing fractions were selected and spun at 50000 rpm overnight at 4°C. The 1 ml fractions were collected again, and genome containing fractions were dialyzed overnight. The filtered solution was transferred to a Beckman tube containing a 40% sucrose cushion and spun at 27,000 rpm overnight at 4°C. The pellet (containing purified AAV) was resuspended in 150 μl 1× HBSS. Virus was aliquoted and stored at -80 ° C until use. Animals and surgeries: Test naive C57BL/6 male mice were ear punched at least 21 days after birth and genotyped using Transnetyx genotyping services. PCR screening was performed for the presence of Cre recombinase. For brain slice studies, mice between 5-7 weeks of age were injected with 0.5uL AAV1-Efla-FLEX-oROS-mCherry (CITE) construct containing oROS-Gr into a MOR CRE positive mouse bilaterally into the VTA using coordinates: ML: +/- 0.5, AP: -3.28, DN: -4.5 zeroed at bregma. Isoflurane was used for anesthesia and carprofen for pain relief. Mice were mounted on a stereotaxic alignment system and injections were made using a Hamilton 2.0uL model 7002 KH syringe. Similarly, for fiber photometry experiments, mice were injected with 0.5uL AAV1-Efla-FLEX-oROS-mCherry unilaterally at a 15-degree angle, using the coordinates ML: -1.71, AP: -3.28, DN: -4.67 then implanted with a 400/430 µm diameter Mono fiberoptic cannula from Doric Lenses. 2-photon imaging of µ-opioid receptor expressing neurons in VTA: Two-four weeks after viral injection, the brain was dissected and 200um horizontal slices were prepared using a vibratome. Slices were incubated in NMDG (92mM NMDG, 2.5mM KCl, 1.25mM NaH2PO4, 30mM NaHCO3, 20mM HEPES, 25mM Glucose, 2mM Thiourea, 5mM Na-ascorbate, 3mM Na-pyruvate, pH to 7.4, 0.5mM CaCl•4H2O, 10mM MgSO4•7H2O). Recordings were made in a HEPES solution (92mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 30mM NaHCO3, 20mM HEPES, 25mM Glucose, 2mM Thiourea, 5mM Na-Ascorbate, 3mM Na-Pyruvate). Image collection was done using a Bruker Investigator 2-photon microscope, software Prairie View 5.5, simultaneously collecting both the mCherry (1040 nm fixed) and GFP (920 nm tunable) signals with a Nikon 16X water immersion objective, as well as a z-stack spanning 60um across an hour time course. Baseline recordings were made in ACSF (124mM NaCl, 3mM KCl, 2mM MgSO4, 1.25mM NaH2PO4, 2.5mM CaCl2, 26mM NaHCO3, 10mM Glucose) at 32C, before treatment. For confocal images, animals were perfused intracardially with phosphate-buffered saline (PBS) and 10% formalin. Brains were stored in 10% formalin for up to 24 hours then switched to a 20% sucrose solution at 4C until sectioning. Coronal slices of the VTA were collected at 40um each and mounted using VECTASHIELD HardSet mounting Medium with DAPI. Confocal images were taken with the Leica SP8x Confocal microscope located in the Keck Center at UW. Fiber photometry of kappa-opioid expressing neurons in the VTA : A real-time signal processor (RZ5P; Tucker-Davis Technologies) connected to Synapse Software (Fiber Photometry) to set frequency of light stimulation and to record input from photodetectors. The RZ5P was connected to a light emitting diode (LED) driver (Doric Lenses) that controlled the power of a 465 nm and 560 nm Doric LED. A low autofluorescence patch cord (400/430) was attached to the LED, to a fluorescent MiniCube (Doric Lenses) with dichroic mirrors. Connected optical patch cords to the MiniCube with pigtailed rotary joining (FRJ; Doric Lenses) allowed free animal movement during data collection. Patch Cords were bleached with light prior to photometry sessions to minimize autofluorescence. Power of the LED at the fiber tip was set to ~30 uW and was tested prior to the start of each session. Signals were collected at a sampling frequency of 1017 Hz. Each of the sessions was downsampled by a factor of 100 and normalized to a 15-minute baseline period in the beginning of the recording. Data were then smoothed using a moving average filter (100s window) to remove high frequency noise and detrended to remove linear drift. The isosbestic channel (405 nm) was fitted to the 470 nm channel using a least-squares method and subtracted to remove motion artifacts. Each session started with a 15 min baseline recording period prior to pharmacological experiments to calculate fluorescent change from baseline (ΔF/Fo; Change in fluorescence from baseline fluorescence/baseline fluorescence). References Falkowski, P. G. et al. The rise of oxygen over the past 205 million years and the evolution of large placental mammals. Science 309 , 2202–2204 (2005). Koch, L. G. & Britton, S. L. Aerobic metabolism underlies complexity and capacity. J. Physiol. 586 , 83–95 (2008). Westerblad, H. & Allen, D. G. Emerging roles of ROS/RNS in muscle function and fatigue. Antioxid. Redox Signal. 15 , 2487–2499 (2011). Barnham, K. J., Masters, C. L. & Bush, A. I. Neurodegenerative diseases and oxidative stress. Nat. Rev. Drug Discov. 3 , 205–214 (2004). Lee, Y. M., He, W. & Liou, Y.-C. The redox language in neurodegenerative diseases: oxidative post-translational modifications by hydrogen peroxide. Cell Death Dis. 12 , 58 (2021). Forrester, S. J., Kikuchi, D. S., Hernandes, M. S., Xu, Q. & Griendling, K. K. Reactive Oxygen Species in Metabolic and Inflammatory Signaling. Circ. Res. 122 , 877–902 (2018). D’Autréaux, B. & Toledano, M. B. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat. Rev. Mol. Cell Biol. 8 , 813–824 (2007). Schieber, M. & Chandel, N. S. ROS function in redox signaling and oxidative stress. Curr. Biol. 24 , R453-62 (2014). Finkel, T. Signal transduction by reactive oxygen species. J. Cell Biol. 194 , 7–15 (2011). Sies, H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biol 11 , 613–619 (2017). Chun, H. et al. Severe reactive astrocytes precipitate pathological hallmarks of Alzheimer’s disease via H2O2- production. Nat. Neurosci. 23 , 1555–1566 (2020). Chen, Q. M., Tu, V. C., Wu, Y. & Bahl, J. J. Hydrogen peroxide dose dependent induction of cell death or hypertrophy in cardiomyocytes. Arch. Biochem. Biophys. 373 , 242–248 (2000). Afanas’ev, I. New nucleophilic mechanisms of ros-dependent epigenetic modifications: comparison of aging and cancer. Aging Dis. 5 , 52–62 (2014). Sies, H. & Jones, D. P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat. Rev. Mol. Cell Biol. 21 , 363–383 (2020). Sies, H. et al. Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. Nat. Rev. Mol. Cell Biol. 23 , 499–515 (2022). Murphy, M. P. et al. Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab 4 , 651–662 (2022). Gutscher, M. et al. Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases. J. Biol. Chem. 284 , 31532–31540 (2009). Morgan, B. et al. Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes. Nat. Chem. Biol. 12 , 437–443 (2016). Belousov, V. V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3 , 281–286 (2006). Markvicheva, K. N. et al. A genetically encoded sensor for H2O2 with expanded dynamic range. Bioorg. Med. Chem. 19 , 1079–1084 (2011). Bilan, D. S. et al. HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime imaging. ACS Chem. Biol. 8 , 535–542 (2013). Ermakova, Y. G. et al. Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide. Nat. Commun. 5 , 5222 (2014). Subach, O. M. et al. Slowly Reducible Genetically Encoded Green Fluorescent Indicator for In Vivo and Ex Vivo Visualization of Hydrogen Peroxide. Int. J. Mol. Sci. 20 , (2019). Pak, V. V. et al. Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function. Cell Metab. 31 , 642-653.e6 (2020). Pang, Y. et al. SHRIMP: Genetically Encoded mScarlet-derived Red Fluorescent Hydrogen Peroxide Sensor with High Brightness and Minimal Photoactivation. bioRxiv 2023.08.09.552302 (2023) doi:10.1101/2023.08.09.552302. Lee, C. et al. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nat. Struct. Mol. Biol. 11 , 1179–1185 (2004). Jo, I. et al. Structural details of the OxyR peroxide-sensing mechanism. Proc. Natl. Acad. Sci. U. S. A. 112 , 6443–6448 (2015). Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32 , 13819–13840 (2012). Bienert, G. P. & Chaumont, F. Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide. Biochim. Biophys. Acta 1840 , 1596–1604 (2014). Montiel, V. et al. Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide. Sci. Transl. Med. 12 , (2020). Wragg, D., Leoni, S. & Casini, A. Aquaporin-driven hydrogen peroxide transport: a case of molecular mimicry? RSC Chem Biol 1 , 390–394 (2020). Lim, J. B., Langford, T. F., Huang, B. K., Deen, W. M. & Sikes, H. D. A reaction-diffusion model of cytosolic hydrogen peroxide. Free Radic. Biol. Med. 90 , 85–90 (2016). Jan, Y.-H. et al. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication. Toxicol. Appl. Pharmacol. 288 , 114–120 (2015). Bienert, G. P. et al. Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. J. Biol. Chem. 282 , 1183–1192 (2007). Pedre, B., Barayeu, U., Ezeriņa, D. & Dick, T. P. The mechanism of action of N-acetylcysteine (NAC): The emerging role of H2S and sulfane sulfur species. Pharmacol. Ther. 228 , 107916 (2021). Ezeriņa, D., Takano, Y., Hanaoka, K., Urano, Y. & Dick, T. P. N-Acetyl Cysteine Functions as a Fast-Acting Antioxidant by Triggering Intracellular H2S and Sulfane Sulfur Production. Cell Chem Biol 25 , 447-459.e4 (2018). Chun, H., Lim, J., Park, K. D. & Lee, C. J. Inhibition of monoamine oxidase B prevents reactive astrogliosis and scar formation in stab wound injury model. Glia 70 , 354–367 (2022). Lee, S. et al. Channel-mediated tonic GABA release from glia. Science 330 , 790–796 (2010). Jo, S. et al. GABA from reactive astrocytes impairs memory in mouse models of Alzheimer’s disease. Nat. Med. 20 , 886–896 (2014). Jankowsky, J. L. et al. Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Hum. Mol. Genet. 13 , 159–170 (2004). Kritsiligkou, P., Shen, T. K. & Dick, T. P. A comparison of Prx- and OxyR-based H2O2 probes expressed in S. cerevisiae. J. Biol. Chem. 297 , 100866 (2021). Smolyarova, D. D., Podgorny, O. V., Bilan, D. S. & Belousov, V. V. A guide to genetically encoded tools for the study of H2 O2. FEBS J. 289 , 5382–5395 (2022). Ramakrishnan, P., Maclean, M., MacGregor, S. J., Anderson, J. G. & Grant, M. H. Cytotoxic responses to 405nm light exposure in mammalian and bacterial cells: Involvement of reactive oxygen species. Toxicol. In Vitro 33 , 54–62 (2016). Birnbaum, J. H. et al. Oxidative stress and altered mitochondrial protein expression in the absence of amyloid-β and tau pathology in iPSC-derived neurons from sporadic Alzheimer’s disease patients. Stem Cell Res. 27 , 121–130 (2018). Loor, G. et al. Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis. Free Radic. Biol. Med. 49 , 1925–1936 (2010). Criddle, D. N. et al. Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells. J. Biol. Chem. 281 , 40485–40492 (2006). Shneyvays, V., Leshem, D., Shmist, Y., Zinman, T. & Shainberg, A. Effects of menadione and its derivative on cultured cardiomyocytes with mitochondrial disorders. J. Mol. Cell. Cardiol. 39 , 149–158 (2005). Ishii, T., Warabi, E. & Mann, G. E. Mechanisms underlying Nrf2 nuclear translocation by non-lethal levels of hydrogen peroxide: p38 MAPK-dependent neutral sphingomyelinase2 membrane trafficking and ceramide/PKCζ/CK2 signaling. Free Radic. Biol. Med. 191 , 191–202 (2022). Espinosa-Diez, C. et al. Antioxidant responses and cellular adjustments to oxidative stress. Redox Biol 6 , 183–197 (2015). Klima, J. C. et al. Incorporation of sensing modalities into de novo designed fluorescence-activating proteins. Nat. Commun. 12 , 856 (2021). Goodman, J. B. et al. Redox-Resistant SERCA [Sarco(endo)plasmic Reticulum Calcium ATPase] Attenuates Oxidant-Stimulated Mitochondrial Calcium and Apoptosis in Cardiac Myocytes and Pressure Overload-Induced Myocardial Failure in Mice. Circulation 142 , 2459–2469 (2020). Qin, F. et al. Cytosolic H2O2 mediates hypertrophy, apoptosis, and decreased SERCA activity in mice with chronic hemodynamic overload. Am. J. Physiol. Heart Circ. Physiol. 306 , H1453-63 (2014). Gonnot, F. et al. SERCA2 phosphorylation at serine 663 is a key regulator of Ca2+ homeostasis in heart diseases. Nat. Commun. 14 , 3346 (2023). Akaike, T. et al. A Sarcoplasmic Reticulum Localized Protein Phosphatase Regulates Phospholamban Phosphorylation and Promotes Ischemia Reperfusion Injury in the Heart. JACC: Basic to Translational Science 2 , 160–180 (2017). Wong, H.-S., Dighe, P. A., Mezera, V., Monternier, P.-A. & Brand, M. D. Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions. J. Biol. Chem. 292 , 16804–16809 (2017). Huang, J.-H., Co, H. K., Lee, Y.-C., Wu, C.-C. & Chen, S.-H. Multistability maintains redox homeostasis in human cells. Mol. Syst. Biol. 17 , e10480 (2021). Forman, H. J. & Zhang, H. Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nat. Rev. Drug Discov. 20 , 689–709 (2021). Zhang, Y. et al. A comparative genomics study of carbohydrate/glucose metabolic genes: from fish to mammals. BMC Genomics 19 , 246 (2018). Audzeyenka, I. et al. Hyperglycemia alters mitochondrial respiration efficiency and mitophagy in human podocytes. Exp. Cell Res. 407 , 112758 (2021). Schattauer, S. S. et al. Peroxiredoxin 6 mediates Gαi protein-coupled receptor inactivation by cJun kinase. Nat. Commun. 8 , 743 (2017). Schattauer, S. S. et al. Reactive oxygen species (ROS) generation is stimulated by κ opioid receptor activation through phosphorylated c-Jun N-terminal kinase and inhibited by p38 mitogen-activated protein kinase (MAPK) activation. J. Biol. Chem. 294 , 16884–16896 (2019). Stanicka, J., Russell, E. G., Woolley, J. F. & Cotter, T. G. NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells. J. Biol. Chem. 290 , 9348–9361 (2015). Brewer, T. F., Garcia, F. J., Onak, C. S., Carroll, K. S. & Chang, C. J. Chemical approaches to discovery and study of sources and targets of hydrogen peroxide redox signaling through NADPH oxidase proteins. Annu. Rev. Biochem. 84 , 765–790 (2015). Muñoz, M. et al. Hydrogen peroxide derived from NADPH oxidase 4- and 2 contributes to the endothelium-dependent vasodilatation of intrarenal arteries. Redox Biol 19 , 92–104 (2018). Johnson, F. & Giulivi, C. Superoxide dismutases and their impact upon human health. Mol. Aspects Med. 26 , 340–352 (2005). El Daibani, A. et al. Molecular mechanism of biased signaling at the kappa opioid receptor. Nat. Commun. 14 , 1338 (2023). Schattauer, S. S., Kuhar, J. R., Song, A. & Chavkin, C. Nalfurafine is a G-protein biased agonist having significantly greater bias at the human than rodent form of the kappa opioid receptor. Cell. Signal. 32 , 59–65 (2017). Douglas, A. J. et al. Effects of the kappa-opioid agonist U50,488 on parturition in rats. Br. J. Pharmacol. 109 , 251–258 (1993). Aslund, F., Zheng, M., Beckwith, J. & Storz, G. Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status. Proc. Natl. Acad. Sci. U. S. A. 96 , 6161–6165 (1999). García-Nafría, J., Watson, J. F. & Greger, I. H. IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly. Sci. Rep. 6 , 27459 (2016). Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat. Protoc. 8 , 162–175 (2013). Bremner, S. B. et al. Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues. J. Tissue Eng. 13 , 20417314221119628 (2022). Tohyama, S. et al. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 12 , 127–137 (2013). Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods 18 , 100–106 (2021). Pachitariu, M. & Stringer, C. Cellpose 2.0: how to train your own model. Nat. Methods 19 , 1634–1641 (2022). Woo, D. H. et al. TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation. Cell 151 , 25–40 (2012). Additional Declarations There is NO Competing Interest. Supplementary Files LeeetaloROSGsupp.pdf Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Justin Lee","email":"","orcid":"https://orcid.org/0000-0002-3555-0980","institution":"IBS (Institute for Basic Science)","correspondingAuthor":false,"prefix":"","firstName":"C.","middleName":"Justin","lastName":"Lee","suffix":""},{"id":278759447,"identity":"0ee322ae-db74-4557-b30c-99b35c9a0a06","order_by":17,"name":"Charles Chavkin","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Charles","middleName":"","lastName":"Chavkin","suffix":""}],"badges":[],"createdAt":"2024-03-08 19:50:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4048855/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4048855/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53388093,"identity":"43553b88-d010-4a17-835f-986afd31345a","added_by":"auto","created_at":"2024-03-25 11:45:57","extension":"png","order_by":1,"title":"Figure 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11:53:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2946281,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4048855/v1/bdaaa0ef-7657-4187-8a50-8b1997944a33.pdf"},{"id":53388089,"identity":"3c90ec88-e764-4d1f-80a9-b680f04fca50","added_by":"auto","created_at":"2024-03-25 11:45:56","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2010575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"LeeetaloROSGsupp.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4048855/v1/eab282983e2596e24dc88a2b.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Ultra-fast genetically encoded sensor for precise real-time monitoring of physiological and pathophysiological peroxide dynamics","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndogenous Reactive Oxygen Species (ROS) are indispensable components of aerobic metabolism, which hallmarks the rise of complex life\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Due to their damaging impact on biological macromolecules at high concentrations, redox homeostasis is tightly regulated in most aerobic systems, and high-level accumulation of ROS is often viewed as a pathogenic marker in degenerative diseases (e.g. Alzheimer\u0026rsquo;s disease, Duchenne Muscular Dystrophy), tumorigenesis, and inflammation\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Furthermore, an increasing number of studies report the role of low-level ROS as physiologic mediator in normal cellular signaling processes\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Specifically, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is a key redox signaling molecule, owing to its relative stability and ability to modify cysteine residues in proteins, enabling selective downstream signaling\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. On the other hand, excessive H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is a common pathological marker affecting phenotypic and disease progression in various cell types\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Nevertheless, limited analytic tools to spatiotemporally monitor specific oxidants \u003cem\u003ein situ\u003c/em\u003e with precision have been a bottleneck to deciphering their specific role in physiology and the cause and effect of their imbalance\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Thus, methods to interrogate the role of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e would be broadly applicable to the study of redox biology\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMost synthetic ROS-sensitive dyes are unsuited for these considerations because of their short working time window, low sensitivity, and low specificity\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Protein-based peroxide sensors have been engineered to overcome these shortcomings. For example, the roGFP sensor family fuses roGFP, a redox-sensitive green fluorescent protein variant, to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-specific enzymes like Orp1 (thiol peroxidase), or Tsa2 (typical 2-Cys peroxiredoxin) from yeast to achieve peroxide-specific roGFP fluorescence changes via redox relay\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The HyPer sensor family is based on the direct fusion of circularly permuted fluorescent protein (cpFP) to the regulatory domain of bacterial peroxide sensor protein OxyR for conformational coupling that leads to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-specific fluorescence change\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Most HyPer sensors use ecOxyR (\u003cem\u003eEscherichia coli\u003c/em\u003e OxyR), the most extensively studied OxyR variant, as their sensing domain. However, existing ecOxyR-based peroxide sensors exhibit low sensitivity and slow oxidation kinetics (seconds under saturation conditions)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, while studies reported peroxide-dependent oxidation of ecOxyR at a sub-second scale\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We hypothesized that the discrepancy stems from the disruption of structural flexibility in the sensors. Through a series of structure-guided engineering steps, we developed oROS-G (optogenetic hydRogen perOxide Sensor, Green), a green fluorescent protein (GFP, excitation: 488 nm, emission: 515 nm) and an ecOxyR-based peroxide sensor that exhibits exceptional sensitivity and kinetics enabling the visualization of peroxide diffusion. We also engineered oROS-Gr, a ratiometric variant of oROS-G by fusing it with mCherry, which allows measurement of the precise sensor oxidation state by normalizing sensor fluorescence intensity for the expression level. Here, we present diverse use cases of oROS sensors to monitor both steady-state and transient H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in various model systems. Specifically, we showed how oROS can detect varying H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in astrocytes in the context of Alzheimer\u0026rsquo;s disease models and assessed the efficacy of a drug in reducing aberrant peroxide levels. Also, we investigated how different glucose levels can result in different intracellular oxidative environments in conjunction with mitochondrial respiratory depression. Lastly, we monitored opioid dependent acute H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e generation in mouse brain both \u003cem\u003eex vivo\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, demonstrating potential utility of oROS sensors as a functional downstream reporter for G-protein biased opioid receptor activation.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e \u003cb\u003eStructure-guided engineering strategies for ecOxyR-based H\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003esensor with improved sensitivity and kinetics.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOxyR is a bacterial H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sensor protein that regulates the transcription of antioxidative genes in response to low-level cellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The specificity of OxyR for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stems from its unique H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e binding pocket\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that binding H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e leads to an intermediate state that facilitates the disulfide bridging of two conserved cysteine residues (C199-C208), which triggers the transition into the oxidized conformational state of OxyR. Due to its unique characteristic as an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sensor with low scavenging capacity\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, OxyR is an attractive scaffold for building a protein-based H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reporter. Nevertheless, the slow kinetics and low sensitivity of existing ecOxyR sensors\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e deviate from the reported ecOxyR kinetics, prompting us to revisit the sensor design \u003cb\u003e[Fig.\u0026nbsp;1A, Supp. Figure\u0026nbsp;1A].\u003c/b\u003e OxyR-based peroxide sensors have circular permuted fluorescent proteins (cpFP) within the loop between residues C199 and C208. However, the crystal structure of oxidized ecOxyR [PDB:1I6A] predicted an evident peak of B-factor \u003cb\u003e[Fig.\u0026nbsp;1B]\u003c/b\u003e indicating this loop region is more flexible than its surroundings. We hypothesized that inserting the bulky cpFP there (e.g. in HyPer sensors) could diminish sensing performance by possibly increasing the conformational entropy of the intermediate state that brings C199 and C208 into proximity\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. We performed pairwise residue distance analysis between oxidized and reduced ecOxyR structures and found that the region between residues 209\u0026ndash;220 goes through noticeable peroxide-dependent conformational change \u003cb\u003e[Supp. Figure\u0026nbsp;1B].\u003c/b\u003e Therefore, we tested alternative cpGFP insertion within this region. The functional screening for oROS sensors was performed in Human Embryonic Kidney (HEK293) cells to ensure compatibility with other mammalian host systems. cpGFP insertion between residue 211 and 212 elicited a robust response (97.55% increase in ∆F/Fo; confidence interval 95% (ci) = [96.6, 98.52]) to 300 \u0026micro;M extracellular peroxide, which has been reported to induce full oxidation of OxyR-based sensors\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e \u003cb\u003e[Fig.\u0026nbsp;1B, C].\u003c/b\u003e The 211\u0026ndash;212 variant responded immediately (in 25\u0026ndash;75% sensor saturation response kinetics, 1.06s; ci = [1.05, 1.07]) which was not observed in other ecOxyR-based sensors\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Moreover, the variant showed improved response amplitudes (20.41% in ∆F/Fo; ci = [19.62, 21.17]) to low peroxide levels (10\u0026micro;M) compared to HyperRed (2.8% in ∆F/Fo; ci = [2.61, 3.0]), which incorporates the red fluorescent protein cpmApple between OxyR positions 205 and 206 \u003cb\u003e[Fig.\u0026nbsp;1D].\u003c/b\u003e Next, based on the guiding principles learned from engineering of the calcium indicator GCaMP5\u003csup\u003e28\u003c/sup\u003e, we introduced large and apolar amino acid tyrosine at the residue sites putatively proximal to the cpGFP predicted opening to reduce solvent access. We found the E215Y mutation increased response amplitude (∆F/Fo) by 2.1-fold at full oxidation (ci = [1.99, 2.26]) and we named this variant oROS-G \u003cb\u003e[Fig.\u0026nbsp;1E, Supp. Figure\u0026nbsp;1C]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization of ultrasensitive and fast peroxide sensor, oROS-G.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe first characterized the fluorescence response of the oROS-G sensor in HEK293 cells in response to exogenously or endogenously sourced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Direct application of exogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e increases intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by diffusion across the plasma membrane through specific aquaporins, which creates an extracellular-to-intracellular gradient of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e29\u0026ndash;31\u003c/sup\u003e. Under these conditions, the intracellular concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is reported to be about 2 or 3 magnitudes lower than that of extracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e22,32\u003c/sup\u003e. On the other hand, the pharmacological agent menadione produces H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e intracellularly through various redox cycling mechanisms\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The signal amplitude of oROS-G (192.34% in ∆F/Fo; ci = [190.45, 194.23]) at saturation (300 uM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was \u0026asymp;\u0026thinsp;2-fold greater than that of HyPerRed (97.74% in ∆F/Fo; ci = [96.52, 99.06]), Improved sensitivity of oROS-G yielded a\u0026thinsp;\u0026asymp;\u0026thinsp;7.08 times larger response at low-level peroxide stimulation. (oROS-G: 116.22% in ∆F/Fo; ci = [110.85, 121.73] vs HyPerRed: 16.45% in ∆F/Fo; ci = [15.98, 16.95]) [\u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e] oROS-G also exhibited significant improvement in on-kinetics compared to HyPerRed with \u0026asymp;\u0026thinsp;38 times faster 25\u0026ndash;75% ∆F/Fo kinetics \u003cb\u003e[Fig.\u0026nbsp;2B].\u003c/b\u003e Intriguingly, the fast oxidation kinetics of the oROS-G sensor captured the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e diffusion across the imaging field of view from the media mixing, in contrast to HyPerRed which exhibited uniform population response \u003cb\u003e[Supp. Figure\u0026nbsp;2A, B].\u003c/b\u003e Further analysis revealed the speed of peroxide diffusion during media mixing to be \u0026asymp;\u0026thinsp;824\u0026micro;m/s \u003cb\u003e[Supp. Figure\u0026nbsp;2C, D].\u003c/b\u003e The speed of peroxide travel slows down to \u0026asymp;\u0026thinsp;100\u0026micro;m/s after passing the cell plasma membrane during intracellular diffusion. This potentially represents peroxide travel becoming rate limited by aquaporin-driven passive transmembrane diffusion\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e \u003cb\u003e[Supp. Figure\u0026nbsp;2E, F].\u003c/b\u003e Taken together, visualization of bolus H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e introduction using oROS-G was only rate-limited by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e travel speed and transmembrane transport rate, allowing real-time observation of intracellular peroxide diffusion in mammalian cells. Thus, oROS-G can be a vital tool for expanding our understanding of the dynamic topological and temporal landscape of peroxide in biological systems.\u003c/p\u003e \u003cp\u003eIn HEK293 cells, oROS-G also acutely responded to 10\u0026micro;M and 50\u0026micro;M menadione in a dose-dependent manner (∆F/Fo, 10\u0026micro;M: 89.56%; ci = [81.79, 97.57], 50\u0026micro;M: 173.68%; ci = [166.81, 180.35]) \u003cb\u003e[Fig.\u0026nbsp;2C].\u003c/b\u003e The result was consistent in human primary astrocytes, \u003cb\u003e[Supp. Figure\u0026nbsp;3A]\u003c/b\u003e highlighting the potential robustness of oROS-G expression and functionality in broader biological host systems. In addition, we confirmed the robust expression and function of oROS-G in rat cortical neurons and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) \u003cb\u003e[Fig.\u0026nbsp;2D].\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext, we confirmed that oROS-G is a fully reversible sensor by directly reducing it using 10 mM Dithiothreitol (DTT) \u003cb\u003e[Fig.\u0026nbsp;2E]\u003c/b\u003e or media washout \u003cb\u003e[Supp. Figure\u0026nbsp;2B]\u003c/b\u003e. Here, we noticed that the endogenous reduction kinetics of the sensor in mammalian cells was faster than other OxyR-based sensors\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For example, both HyPerRed and HyPer7 took 20\u0026thinsp;~\u0026thinsp;30 minutes for them to return to baseline after the sensor saturation. HyPer7 is the newest green iteration of the HyPer sensor family that was engineered by swapping the sensing domain with a different OxyR domain from \u003cem\u003eNeisseria meningitidis\u003c/em\u003e (nmOxyR)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e with fluorescent reporter insertion contained to the C-C loop region. oROS-G reached\u0026thinsp;\u0026asymp;\u0026thinsp;90% reduction from its maximum saturation in 4.17 minutes, whereas HyPer7 only achieved about\u0026thinsp;\u0026asymp;\u0026thinsp;20% reduction from its full saturation in the same duration, consistent with the previous report. (HyPer7: 0.81; ci = [0.8, 0.82], oROSG: 0.12; ci = [0.1, 0.15]) oROS-G showed 2.63 times faster decay kinetics than HyPer7 based on approximation with reduction time to 85% of saturation, making oROS-G a more compelling candidate for measurement of peroxide transient rise and decay of intracellular peroxide species \u003cb\u003e[Fig.\u0026nbsp;2F, Supp. Figure\u0026nbsp;3C]\u003c/b\u003e. Lastly, we created a C199S mutant of oROS-G to show that the fluorescence response was specific to peroxide-induced disulfide bridging of C199-C208, which is consistent with other OxyR-based peroxide sensors \u003cb\u003e[Supp. Figure\u0026nbsp;3D]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMonitoring the effect of antioxidants on intracellular peroxide level in Alzheimer\u0026rsquo;s model.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we explored using oROS-G in the context of antioxidants that target intracellular peroxides. N-acetyl-cystine (NAC) is a cysteine prodrug widely used as a classical \u0026ldquo;antioxidant\u0026rdquo;. Although its detailed mechanism of action has not been established, recent studies highlight its antioxidative role via the production of low-level sulfane sulfur species\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Using oROS-G, we measured the effect of NAC-dependent catabolism on cellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in real-time. With a 1-hour preincubation of 1 mM NAC, oROS-G expressed in HEK293 showed a 73% diminished response to exogenous 10 \u0026micro;M peroxide exposure \u003cb\u003e[Supp. Figure\u0026nbsp;4A]\u003c/b\u003e. Similarly, we incubated oROS-G expressing HEK293 cells to either NAC (10 mM) or Vehicle (DMSO) for 20 minutes before 10 \u0026micro;M menadione exposure. NAC significantly attenuated the response by 72 percent \u003cb\u003e[Supp. Figure\u0026nbsp;4B]\u003c/b\u003e. We next examined the ability of the oROS-G sensor in primary cultured astrocytes to quantitatively detect endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels to assess the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-scavenging effects of molecules. Initially, we expressed oROS-G in the astrocytes and tested H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration-dependent functionality \u003cb\u003e[Supp. Figure\u0026nbsp;4C]\u003c/b\u003e. We observed an increase of 21.27\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3% and 57.74\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4% in fluorescence with 10 and 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, respectively \u003cb\u003e[Supp. Figure\u0026nbsp;4D-F]\u003c/b\u003e, indicating a functional response of the oROS-G sensor in primary cultured astrocytes. Previously, we have shown that aberrant H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production by reactive astrocytes, a pathological form of astrocytes, is a contributing factor to Alzheimer\u0026rsquo;s disease (AD) pathology\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In detail, upregulated monoamine oxidase B (MAOB) in reactive astrocytes produces H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by breaking down oligomerized amyloid beta (oAβ)-metabolites, such as putrescine, leading to pathological H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e generation\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e \u003cb\u003e[Fig.\u0026nbsp;3A].\u003c/b\u003e Therefore, we tested whether we could monitor aberrant endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production in oROS-G transfected astrocytes treated with oAβ (5 \u0026micro;M) or putrescine (180 \u0026micro;M). Over a 40-hour continuous recording \u003cb\u003e[Fig.\u0026nbsp;3B]\u003c/b\u003e, we observed a significant increase in oAβ-induced oROS-G fluorescence, indicating a notable rise in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. \u003cb\u003e[Fig.\u0026nbsp;3C, D].\u003c/b\u003e Conversely, the application of KDS2010 (1 \u0026micro;M), a selective MAOB inhibitor, and sodium pyruvate (1 mM), a potential H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenger, showed a smaller increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels. Additionally, incubation with putrescine, a pre-substrate of MAOB, also significantly increased oROS-G sensor fluorescence \u003cb\u003e[Fig.\u0026nbsp;3E, F]\u003c/b\u003e. However, this H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e elevation was significantly reduced by KDS2010 and partially reduced by sodium pyruvate. Taken together, these results suggest that the oROS-G sensor in primary cultured astrocytes is a reliable tool for monitoring endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production under AD-like conditions and evaluating the efficacy of potential H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-scavenging compounds.\u003c/p\u003e \u003cp\u003eThen we asked whether we could measure the endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in mouse brains. To test this idea, we bilaterally injected the AAV5-GFAP104-oROS-G virus into the CA1 hippocampus of APP/PS1 mice\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, a well-known AD model, to overexpress oROS-G sensor specifically in the astrocytes \u003cb\u003e[Fig.\u0026nbsp;3G].\u003c/b\u003e Two weeks post-injection, we prepared brain slices and tested the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration-dependent functionality of the sensor. Again, to test the functionality of the sensor, we applied 10 and 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e through bath application. We found an increase of 13.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% and 39.46\u0026thinsp;\u0026plusmn;\u0026thinsp;3.12% in fluorescence with 10 and 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, respectively \u003cb\u003e[Fig.\u0026nbsp;3H, I].\u003c/b\u003e Like \u003cem\u003ein vitro\u003c/em\u003e, the oROS-G sensor functions effectively in astrocytes \u003cem\u003eex vivo\u003c/em\u003e. Next, we examined the capability to measure elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in astrocytes of APP/PS1 mice. We hypothesized that treatment with DTT would unmask the portion activated by astrocytic H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Following DTT (10 mM) administration, we observed a reduction in fluorescence below the baseline levels. Notably, we demonstrated that APP/PS1 mice exhibited a greater reduction compared to wild-type, suggesting a potential method for measuring endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels \u003cb\u003e[Fig.\u0026nbsp;3J-L].\u003c/b\u003e Taken together, these results demonstrate that the oROS-G sensor functions effectively \u003cem\u003eex vivo\u003c/em\u003e, presenting a potential method for measuring endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels and investigating the antioxidant capacity of various molecules.\u003c/p\u003e \u003cp\u003e \u003cb\u003eoROS-Gr for long-term and non-continuous monitoring of intracellular H\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eMost ratiometric sensors designed for peroxide response are based on dual-excitation of the green fluorescent sensor proteins at 405 nm and 488 nm\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Although this sensor type allows flexibility in multi-color optogenetic experiments, illumination at 405 nm could contribute to oxidative stress in mammalian cells.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e We created oROS-Gr, by fusing mCherry to oROS-G, creating an equimolar reference point inert to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Flow cytometry analysis confirmed a strong linear correlation between green (Em. 510 nm) and red (Em. 605 nm) emission intensity of oROS-Gr expressed in HEK293 cells (n\u0026thinsp;=\u0026thinsp;16,326) \u003cb\u003e[Fig.\u0026nbsp;4A]\u003c/b\u003e. Thus, oROS-Gr can be used for long-term and non-continuous monitoring by calculating the green-to-red light emission ratio independent of sensor expression levels. Upon exogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation, the oROS-Gr ratio (Em. 510/605) showed a dose-dependent response in HEK293 cells. (1 \u0026micro;M: 0.06 (n\u0026thinsp;=\u0026thinsp;327); ci = [0.06, 0.06], 10 \u0026micro;M: 0.11 (n\u0026thinsp;=\u0026thinsp;306); ci = [0.1, 0.11], 25 \u0026micro;M: 0.14 (n\u0026thinsp;=\u0026thinsp;405); ci = [0.14, 0.14], 50 \u0026micro;M: 0.15 (n\u0026thinsp;=\u0026thinsp;469); ci = [0.15, 0.15]) \u003cb\u003e[Fig.\u0026nbsp;4B]\u003c/b\u003e In addition, the oROS-Gr green-to-red ratio predictably followed a sequence of exogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation (100 uM) and DTT (10 mM) reduction \u003cb\u003e[Fig.\u0026nbsp;4C, Supp. Figure\u0026nbsp;5]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eMenadione treatment has been widely used to model oxidative stress in biological systems\u003csup\u003e\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Still, studies to monitor its intracellular effect have been mostly limited to short time windows or non-continuous snapshots at varying time points. These studies did not provide insights into the real-time impact on redox homeostasis over a longer period. Here, we used to continuously measure (sampling every 5 minutes) the effects of menadione on cellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels over a ten-hour time window using stable oROS-Gr expressing HEK293 cells. Initially, menadione at 0, 1, 10, and 50 uM induced acute dose-dependent elevation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. However, within 30 minutes, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels at 10\u0026micro;M were higher than those at 50\u0026micro;M, which returned to a dose-dependent trend within four hours. \u003cb\u003e[Fig.\u0026nbsp;4D]\u003c/b\u003e Further analysis of intracellular redox landscape analysis and functional role of putative cellular antioxidative elements\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e is required to understand this phenomenon fully. Nevertheless, the temporally perplexing effects of menadione on intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e level gives us a cautionary insight to avoid the assumption that an oxidative agent such as menadione has always a direct dose-dependent effect on the intracellular peroxide level. We also tested if using oROS-Gr can improve the readout precision over oROS-G. We compared the coefficient of variation (CoV) of the ratiometric data (Em. 510/605) against data acquired in single wavelength mode (Em. 510) during long-term menadione exposure \u003cb\u003e[Fig.\u0026nbsp;4E].\u003c/b\u003e The ratiometric readout showed about 2-fold lower CoV compared to the single wavelength mode, confirming improvements in precision [Ratiometric: 0.27 (n\u0026thinsp;=\u0026thinsp;484), non-Ratiometric: 0.46 (n\u0026thinsp;=\u0026thinsp;484)].\u003c/p\u003e \u003cp\u003eWe confirmed the robust expression and functionality of oROS-Gr in various human stem cell-derived cells. For example, we measured peroxide levels in hiPSC-derived cortical neurons in response to 24-hour 10 \u0026micro;M and 50 \u0026micro;M Menadione incubation to be 1.77-fold and 2-fold of oROS-Gr ratio observed at vehicle negative control, respectively (Vehicle: 1.0; ci = [0.82, 1.21], 10 \u0026micro;M: 1.77; ci = [1.62, 1.88], 50 \u0026micro;M: 2.01; ci = [1.97, 2.03]) \u003cb\u003e[Fig.\u0026nbsp;4F]\u003c/b\u003e. Next, we used the sarcoendoplasmic reticulum calcium ATPase (SERCA) blocker cyclopiazonic acid (CPA, 10 \u0026micro;M) to elevate Ca\u003csup\u003e2+\u003c/sup\u003e in the cytosol of hiPSC-cardiomyocytes (CM)\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. When hiPSC-CMs expressed oROS-Gr, we measured increased cytosolic peroxide levels within 2 hours of CPA incubation \u003cb\u003e[Supp. Figure\u0026nbsp;4].\u003c/b\u003e As previously reported, this confirms a tight coupling between intracellular Ca\u003csup\u003e2+\u003c/sup\u003e and ROS levels\u003csup\u003e\u003cspan additionalcitationids=\"CR52 CR53\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGlucose-dependent basal oxidation level in mammalian cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSuperoxide and peroxide are continuously generated as byproducts through electron transfers during aerobic metabolism\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In this context, glucose, as one of the primary substrates of aerobic metabolic pathways, plays a crucial role in modulating cellular metabolic activity\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Intriguingly, low as well as high glucose levels were reported to result in depressed respiratory activity in cultured human podocytes\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The study also showed that the reduction of metabolic rates in high-glucose conditions can be reversed by incubation with the antioxidant NAC, indicating that respiratory suppression is correlated with oxidative stress. Thus, we hypothesized high glucose (HG\u0026thinsp;=\u0026thinsp;25 mM) but also low glucose (LG\u0026thinsp;=\u0026thinsp;1 mM) media would result in higher basal peroxide levels than medium glucose (MG\u0026thinsp;=\u0026thinsp;10 mM). We incubated HEK293 cells for 48 hours in HG, NG, and LG media and compared the ratiometric oROS-Gr signals. Here, low and high glucose conditions caused higher peroxide levels than MG (MG: 0.38; ci = [0.378, 0.382], LG: 0.402; ci = [0.4, 0.404], HG: 0.392; ci = [0.389, 0.394]). \u003cb\u003e[Fig.\u0026nbsp;5A].\u003c/b\u003e We directly measured metabolic activities and found that basal and maximum respiratory rates were also the lowest under low and high glucose conditions \u003cb\u003e[Fig.\u0026nbsp;5B, Supp. Figure\u0026nbsp;7A, B]\u003c/b\u003e, indicating an inverse correlation with increased peroxide levels. Indeed, cells that were pre-incubated with 1 mM of the antioxidant NAC under HG conditions brought the oROS-Gr level 84% closer to MG conditions, indicating modest suppression of oxidative stress \u003cb\u003e[Supp. Figure\u0026nbsp;7C].\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eG-protein biased agonists elicit H\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003egeneration in κ and \u0026micro; opioid receptor-expressing neurons in the Ventral Tegmental Area\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWe previously reported that peroxide generated by a NADPH oxidase (NOX) mechanism regulated opioid receptor signaling\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, which exemplifies intricate functional G-protein biased agonists influence on arrestin-independent inactivation profile of \u0026micro; and κ opioid receptors. Briefly, G-protein biased opioid receptor activation triggers cJUN N-terminal kinase (JNK) phosphorylation. Phosphorylated JNK then activates peroxiredoxin 6 (PRDX6), producing superoxide (SO) from NOX. SO can quickly oxidize the Gαi protein complex to inactivate the opioid receptors. This event can be captured using H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as a marker of opioid receptor activation because superoxide is readily transformed into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by superoxide dismutase\u003csup\u003e\u003cspan additionalcitationids=\"CR63 CR64\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e \u003cb\u003e[Fig.\u0026nbsp;6A]\u003c/b\u003e. With impressive sensitivity and robust expressibility of oROS-Gr, we sought to monitor transient H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e generation in the animal brain in response to G-protein biased agonists.\u003c/p\u003e \u003cp\u003eAs a proof of concept, we showed that morphine, a potent G-protein biased agonist of \u0026micro;-opioid receptors (MOR), triggers transient peroxide generation in MOR-expressing neurons in the ventral tegmental area (VTA) of MOR-Cre transgenic mice, which is consistent with our previous findings. The oROS signals were measured using 2-photon microscopy on \u003cem\u003eex vivo\u003c/em\u003e live brain slices after viral delivery of the AAV1-DIO-oROS-Gr into the VTA of MOR-Cre transgenic mice. Expression of oROS-Gr in the VTA was verified with one-photon confocal microscopy of post-mortem fixed brain slices \u003cb\u003e[Fig.\u0026nbsp;6B]\u003c/b\u003e. The VTA in \u003cem\u003eex vivo\u003c/em\u003e brain slices showed an acute increase in sensor fluorescence during bath application of 1\u0026micro;M morphine over 30 minutes of monitoring. This increase was blocked by the opioid receptor antagonist 1\u0026micro;M Naloxone (Normalized ∆F/Fo to first 5 baseline frames, MOR: 3.35; ci = [1.94, 4.82], MOR\u0026thinsp;+\u0026thinsp;NLX: 0.63; ci = [0.42, 0.85]) \u003cb\u003e[Fig.\u0026nbsp;6C]\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eκ-opioid receptor (KOR) has emerged as an promising drug target for pain management with less side-effects\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. We previously showed behavioral and pharmacological evidence on how the oxidative pressure of JNK-PRDX6-PLA2-NOX cascade from KOR results in acute analgesic tolerance as shown in the warm water tail withdrawal assay\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Nalfurafine is a functionally selective G-protein biased κ-opioid agonist shown to have therapeutic potential as a non-dysphoric antipruritic analgesic\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Here, we explored the use of the oROS sensor to directly monitor acute H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e response to Nalfurafine \u003cem\u003ein vivo\u003c/em\u003e, confirming activation of JNK-PRDX6-PLA2-NOX in KOR positive neurons in the VTA. KOR-Cre transgenic mice were injected with AAV1-DIO-oROS-Gr, and the sensor fluorescence (ex:488/em:510) was monitored by fiber photometry in the VTA \u003cb\u003e[Fig.\u0026nbsp;6D]\u003c/b\u003e. Intraperitoneal administration of 100\u0026micro;g/kg Nalfurafine led to transient increase of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Mice pre-treated 30 min prior to a 100\u0026micro;g/kg Nalfurafine injection with a high dose of naloxone (10 mg/kg), sufficient to block KOR\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, showed no significant increase in fluorescence compared to mice only treated with Nalfurafine \u003cb\u003e[Fig.\u0026nbsp;6E]\u003c/b\u003e. This confirms that the Nalfurafine induced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e signal in KOR expressing neurons of VTA is opioid receptor specific.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo further improve our understanding of redox biology, we need the ability to monitor oxidative agents in diverse, multi-faceted contexts. Our development of the oROS sensor framework represents a significant step in this direction. As a novel green fluorescent sensor, oROS-G demonstrates unparalleled sensitivity and response kinetics for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e monitoring, surpassing the capabilities of previous ecOxyR-based sensors. This enhancement is largely attributed to our structural refinement of the sensor, where we relocated the cpGFP insertion site to maintain flexibility of C199-C208 loop of ecOxyR. Drawing inspiration from Akerboom et al.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, we incorporated bulky residues adjacent to the cpGFP barrel opening, exemplified by the E215Y mutation in oROS-G. Our study has revealed a novel insertion site within ecOxyR, paving the way for the creation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e sensors that are both fast and sensitive. Our hypothesis suggests that the flexible region in the ligand sensing domain is intrinsically linked to sensor function. These principles could lay the groundwork for future optogenetic sensors tailored to detecting other analytes.\u003c/p\u003e \u003cp\u003eInterestingly, the diversity of OxyR variants in nature, each characterized by a conserved peroxide oxidation mechanism, opens avenues for exploring a range of sensor functionalities. Notably, OxyRs from different bacterial strains exhibit distinct reduction mechanisms; ecOxyR predominantly follows a Grx (glutaredoxin)-dependent reduction pathway, where Grx proteins facilitate the reduction of oxidized proteins\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. In contrast, other variants like nmOxyR (\u003cem\u003eNeisseria meningitidis\u003c/em\u003e) might employ a Trx (thioredoxin)-dependent reduction mechanism, involving the Trx system known for mitigating cellular oxidative stress (e.g. HyPer7)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This variation necessitates further exploration of these domains for sensors in mammalian systems, where they could serve as complementary tools for dissecting peroxide biology in various redox environments.\u003c/p\u003e \u003cp\u003eOur study also demonstrated the practical versatility of oROS sensors in a range of experimental setups. With oROS-G, we successfully monitored H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in astrocytes, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e, shedding light on cellular redox states. Moreover, the ratiometric oROS-Gr sensor enabled us to observe the effects of glucose on cytoplasmic peroxide levels, which correlated with known patterns of mitochondrial oxidative stress. Future studies should aim to clarify the sources of peroxide accumulation, considering factors like NADPH oxidase activity and mitochondrial respiration. Additionally, our work highlights the oROS sensor's efficacy in detecting opioid-induced peroxide increase \u003cem\u003ein vivo\u003c/em\u003e, further emphasizing its broad applicability.\u003c/p\u003e \u003cp\u003eIn conclusion, the oROS sensors, exemplified by oROS-G and oROS-Gr, offer a new paradigm for studying peroxide biology. Their application across various model systems has the potential to revolutionize our approach to understanding and monitoring complex redox processes, with significant implications for unraveling the mechanisms underlying various oxidative stress-related diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.D.L was supported by 1F31DA056121-01A1 and an ISCRM Fellowship. A.B was supported by the Brain Research Foundation, UW Royalty Research Fund, UW ISCRM IPA, NIGMS R01 GM139850-01, P30 DA048736, NIMH RF1MH130391, NINDS U01NS128537, NIDA R21DA051193 and the McKnight Foundation\u0026rsquo;s Technologies in Neuroscience Award. K. E. was supported by T32AG066574. The research received additional support from the Lynn and Mike Garvey Imaging Core, the UW NAPE Center, and ISCRM Shared Equipment. We want to thank Dr. Randy Moon for his support. Also, this work was supported by the Institute for Basic Science, Center for Cognition and Sociality (IBS-R001-D2) to C.J.L. We are also grateful to the IBS virus facility for providing a virus packaging service for in vivo experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData\u003c/strong\u003e \u003cstrong\u003eAvailability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSource data will be available via figshare shortly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSource code will be available at https://github.com/justindaholee/oROS-G_manuscript shortly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures performed at the University of Washington have been approved by the University of Washington\u0026rsquo;s Animal Use Committee (protocol #4422-01) and follow the National Institute of Health and the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. Handling and animal care have been performed according to the Institutional Animal Care and Use Committee of the Institute for Basic Science.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterial requests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003ePlasmid name:\u003c/u\u003e Addgene #\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003epC3.1_CMV_oROS-G:\u003c/u\u003e 216111, \u003cu\u003epC3.1_CMV_oROS-G_LF:\u003c/u\u003e 216112, \u003cu\u003epCAG_oROS-Gr:\u003c/u\u003e 216113, \u003cu\u003epCAG_oROS-Gr-LF:\u003c/u\u003e 216114, \u003cu\u003eAAV2_CAG_oROS-G:\u003c/u\u003e 216115, \u003cu\u003eAAV2_CAG_oROS-G_LF:\u003c/u\u003e 216116\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProtein structure analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein structure analysis and plotting were performed using Chimera-X-1.2.1.\u0026nbsp;Oxidized [PDB:1I6A] and reduced [PDB:1I69] crystal structures of ecOxyR were imported from the Protein Data Bank (PDB). Pairwise residue distance between reduced and oxidized ecOxyR structure was achieved by aligning both structures using a matchmaker algorithm that superimposes protein structures by creating a pairwise sequence alignment and then fitting the aligned residue pairs to derive pairwise residue distances.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMolecular Biology\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eoROS-HT variants were cloned based on the pC1 plasmid backbone from pC1-HyPer-Red (Addgene ID: 48249). Primers for point mutations or fragment assembly required to generate the oROS-HT screening variants were designed for In Vitro Assembly cloning (IVA) technique\u003csup\u003e70\u003c/sup\u003e, gibson assembly (New England Biolabs; E2611L) or blunt-end amplification for KLD-based site-directed mutagenesis methods. Primers were ordered from Integrated DNA Technologies (IDT). All gene fragment amplifications were done using Seither Q5-polymerase (New England Biolabs; M0492L) or Superfi-II polymerase (Invitrogen; 12368010). Amplification of DNA fragments were verified with agarose gel electrophoresis. 30 minutes of DpnI enzyme treatment were done on every PCR product to remove the plasmid template from PCR samples. For IVA cloning circularization or assembly of the PCR products was achieved by transforming linear DNA products into competent E.Coli cells (DH5ɑ or TOP10) and grown on agar plates that contain\u0026nbsp;either ampicillin or kanamycin selection antibiotic (50 \u0026micro;g/mL).\u0026nbsp;For gibson assembly and KLD cloning,\u0026nbsp;circularized DNA was transformed as above. Upon colony formation, single colonies were picked and grown in 5mL cultures containing LB Broth (Fisher BioReagents; BP9723-2) and selection antibiotic (ampicillin/kanamycin; 50 \u0026micro;g/mL) overnight (37\u0026deg;C, 230 RPM). DNA was isolated using Machery Nagel DNA prep kits (Machery Nagel; 740490.250). Sanger sequencing (Genewiz; Seattle, WA) or Whole plasmid nanopore sequencing (Plasmidsarus; Eugene, OR) of the isolated plasmid DNA was used to confirm the presence of the intended mutation. Genes encoding the final variants were cloned into a CAG-driven backbone, pCAG-Archon1-KGC-EGFP-ER2-WPRE (Addgene; #108423), using the methods above. England Biolabs; E2621L). All subsequences were verified with Sanger sequencing (Genewiz; Seattle, WA) or Whole plasmid nanopore sequencing (Plasmidsarus; Eugene, OR)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eChemicals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e working solutions were freshly prepared before every experiment from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution 30 % (w/w) in H\u003csub\u003e2\u003c/sub\u003eO (Sigma-Aldrich, H1009). Stock solution of Menadione (VENDOR, CAT) was prepared in 100% DMSO at 50mM. Stock solution of Cyclopiazonic Acid (Tocris, 1235) was prepared in 100% DMSO at 20mM. Chemicals specific to other method sections can be found in their respective sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell culture and transfection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman Embryonic Kidney (HEK293; ATCC Ref: CRL-1573)\u0026nbsp;cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium + GlutaMAX (Gibco; 10569-010) supplemented with 10% fetal bovine serum (Biowest; S1620). When cultures reached 85% confluency, the cultures were seeded at 150,000/75,000 cells per well in 24/48-well plates, respectively. 24 hours after cell seeding, the cells were transfected using Lipofectamine3000 (Invitrogen; L3000015) at 1000/500 ng of DNA per well of a 24/48-well plate, according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePrimary rat neuron isolation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary cortical neurons were prepared as previously described \u003csup\u003e47,48\u003c/sup\u003e. Briefly, 24-well tissue culture plates were coated with Matrigel (mixed 1:20 in cold-PBS, Corning; 356231) solution and incubated at 4\u0026deg;C overnight prior to use. Sterile dissection tools were used to isolate cortical brain tissue from P0 rat pups (male and female). Tissue was minced until 1mm pieces remained, then lysed in equilibrated (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e) enzyme (20 U/mL Papain (Worthington Biochemical Corp; LK003176) in 5mL of EBSS (Sigma; E3024)) solution for 30 minutes at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator. Lysed cells were centrifuged at 200xg for 5 minutes at room temperature, and the supernatant was removed before cells were resuspended in 3 mLs of EBSS (Sigma; E3024). Cells were triturated 24x with a pulled Pasteur pipette in EBSS until homogenous. EBSS was added until the sample volume reached 10 mLs prior to spinning at 0.7 rcf for 5 minutes at room temperature. Supernatant was removed, and enzymatic dissociation was stopped by resuspending cells in 5 mLs EBSS (Sigma; E3024) + final concentration of 10 mM HEPES Buffer (Fisher; BP299-100) + trypsin inhibitor soybean (1 mg/ml in EBSS at a final concentration of 0.2%; Sigma, T9253) + 60 \u0026micro;l of fetal bovine serum (Biowest; S1620) + 30 \u0026micro;l 100 U/mL DNase1 (Sigma;11284932001). Cells were washed 2x by spinning at 0.7 rcf for 5 minutes at room temperature and removing supernatant + resuspending in 10 mLs of Neuronal Basal Media (Invitrogen; 10888022) supplemented with B27 (Invitrogen; 17504044) and glutamine (Invitrogen; 35050061) (NBA++). After final wash spin and supernatant removal, cells were resuspended in 10 mLs of NBA++ prior to counting. Just before neurons were plated, matrigel was aspirated from the wells. Neurons were plated on the prepared culture plates at desired seeding density. Twenty-four hours after plating, 1\u0026micro;M AraC (Sigma; C6645) was added to the NBA++ growth media to prevent the growth of glial cells.Plates were incubated at 37\u0026deg;C and 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand maintained by exchanging half of the media volume for each well with fresh, warmed Neuronal Basal Media (Invitrogen; 10888022) supplemented with B27 (Invitrogen; 17504044) and glutamine (Invitrogen; 35050061) every three days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHuman primary astrocytes, and stem cell derived cardiomyocytes and neurons\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAstrocytes: Human primary cortical astrocytes were purchased from ScienCell Research Laboratories (Carlsbad, CA) and were stored, thawed and sub-cultured based on the manufacturer\u0026rsquo;s protocol. Briefly, the astrocytes were cultured for 72 h in a base medium with an astrocyte growth supplement and fetal bovine serum provided by the same manufacturer. Cultures were maintained in a 37\u0026deg;C/5% CO\u003csub\u003e2\u003c/sub\u003e incubator throughout the culture period, and the astrocytes with low passage numbers (p0-p3) were used to guarantee consistent phenotype expression. When the culture became 70% confluent, the cells were dissociated with TrypLE (Thermo Fisher), followed by passaging on the PDL-coated 24 cover glasses for oROS-G1 transfection. The transfected cells were then cultured for an additional 96 h before H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment (10 \u0026micro;M, 100 \u0026micro;M) for recording the fluorescence response upon H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCardiomyocytes: Undifferentiated IMR90 (WiCell) hiPSCs were maintained on Matrigel (Corning) coated tissue culture plates in mTeSR1 (Stemcell Technologies). Cardiomyocyte directed differentiation was performed using a modified small molecule Wnt-modulating protocol using Chiron 99021 and IWP-4 as previously described.\u003csup\u003e71,72\u003c/sup\u003e. Lactate enrichment was performed following differentiation to purify hiPSC-CMs.\u003csup\u003e73\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eCortical neurons: Neurons were generated from the previously characterized wild type CV background human induced pluripotent stem cell line (Young et al. 2015). Neural progenitor cells (NPCs) from this cell line were differentiated from hiPSCs using dual-SMAD inhibition and NPCs were differentiated to neurons as previously described (Knupp et al., 2020; Shin et al., 2023). Briefly, for cortical neuron differentiation from NPCs, NPCs were expanded into 10 cm plates in Basal Neural Maintenance Media (BNMM) (1:1 DMEM/F12 (#11039047 Life Technologies) + glutamine media/neurobasal media (#21103049, GIBCO), 0.5% N2 supplement (# 17502-048; Thermo Fisher Scientific,) 1% B27 supplement (# 17504-044; Thermo Fisher Scientific), 0.5% GlutaMax (# 35050061; Thermo Fisher Scientific), 0.5% insulin-transferrin-selenium (#41400045; Thermo Fisher Scientific), 0.5% NEAA (# 11140050; Thermo Fisher Scientific), 0.2% \u0026beta;-mercaptoethanol (#21985023, Life Technologies) \u0026nbsp;+ 20 ng/mL FGF (R\u0026amp;D Systems, Minneapolis, MN). Once the NPCs reached 100% confluence, they were switched to Neural Differentiation Media (BNMM +0.2 mg/mL brain-derived neurotrophic factor (CC# 450\u0026ndash;02; PeproTech) + 0.2 mg/mL glial-cell-derived neurotrophic factor (CC# 450\u0026ndash;10; PeproTech) + 0.5 M dbcAMP (CC# D0260; Sigma Aldrich). Neural Differentiation Media was changed twice a week for 21 days at which point the differentiation is considered finished. Neurons were replated at a density of 500,000 cells/cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImaging\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImaging experiments described in this study were performed as follows unless specifically noted. Epifluorescence imaging experiments were performed on a Leica DMI8 microscope (Semrock bandpass filter:\u0026nbsp;GFP ex/em: FF01-474-27/FF01-520-35, RFP ex/em:FF01-578-21/FF01-600-37) \u0026nbsp;controlled by MetaMorph Imaging software, using a sCMOS camera (Photometrics Prime95B) and 20x magnification lens (Leica HCX PL FLUOTAR L 20x/0.40 NA CORR) or 10\u0026times; objective (Leica HCX PL FLUOTAR L 10x/0.32 NA).\u0026nbsp;Confocal imaging experiments were performed on a Leica SP8 confocal microscope from the Lynn and Mike Garvey Imaging Core at the Institute of Stem Cell and Regenerative Medicine. Cells were imaged in live cell imaging solution with 10mM glucose (LCIS+, Gibco, A14291DJ).\u0026nbsp;Image analysis methods described below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnalysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of cell fluorescence imaging data was done by FUSE, a custom cloud-based semi-automated time series fluorescence data analysis platform written in Python. First, the cell segmentation quality of the selected Cellpose\u003csup\u003e74\u003c/sup\u003e model was manually verified. For the segmentation of cells expressing cytosolic fluorescent indicators, model \u0026lsquo;cyto\u0026rsquo; was selected as our base model. If the selected Cellpose model was low-performing, we further trained the Cellpose model using the Cellpose 2.0 human-in-the-loop system\u003csup\u003e75\u003c/sup\u003e. Using an \u0026ldquo;optimized\u0026rdquo; segmentation model, fluorescence time-series data is extracted for each region of interest. This allows for unbiased\u0026nbsp;extraction of change in cellular fluorescence information for a complete set of experimental samples. Extracted fluorescence data is normalized as specified in the text using custom python script.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAstrocyte study\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePrimary mouse astrocyte culture:\u0026nbsp;\u003c/em\u003ePrimary mouse cultured astrocytes were prepared from P1-P3 C57BL/6J mouse pups as previously described.\u003csup\u003e76\u003c/sup\u003e Briefly, 60-mm culture dishes were coated with 0.1 mg/ml poly-D-lysine (PDL, Sigma; P6407) solution prior to use. The hippocampal tissue was isolated, and dissociated into single cell suspension by trituration in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium supplemented with 4.5 g/L glucose, L-glutamine, sodium pyruvate (DMEM, Corning; 10-013-CV) + 10% heat-inactivated horse serum (Gibco; 26050-088) + 10% heat-inactivated fetal bovine serum (Dawin bio; A0100-010) + 1000 unit/ml penicillin-streptomycin (Gibco; 15140122). Dissociated cells were plated onto the PDL coated dishes. Cultures were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. On the third day, cells were vigorously washed with repeated pipetting using medium to get rid of debris and other floating cell types.\u003cbr\u003e\u0026nbsp;On the 10th day of culture, cultured primary astrocytes were electrophoretically transfected with oROS-G plasmid with a voltage protocol (1200 V, 20 pulse width, 2 pulses) using the Microporator (Invitrogen Neon Transfection System; MPK5000S) and replated onto coverglass coated with PDL (Sigma; P6407) or \u0026micro;-Plate 96 Well Black (ibid; 89626).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImaging of cultured primary mouse astrocytes:\u0026nbsp;\u003c/em\u003eOn the 14th day of culture, the oROS-G transfected cultured primary astrocytes were transferred to a recording chamber which were mounted on an inverted Nikon Ti2-U microscope and continuously perfused with an external solution contained (in mM): 150 NaCl, 10 HEPES, 5.5 glucose, 3 KCl, 2MgCl2, 2 CaCl2, and pH adjusted to pH 7.3. Intensity images of 525 nm wavelength were taken at 485 nm excitation wavelengths using ORCA-Flash4.0 CMOS camera (Hamamatsu; C13440). \u0026nbsp;Imaging workbench (INDEC Biosystem) and ImageJ (NIH) were utilized for image acquisition and ROI analysis of cultured astrocytes. To examine H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-dose dependent responses of oROS-G transfected cultured astrocytes, concentration of 10 and 100 \u0026micro;M of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(Sigma; 88597) were introduced by bath application. The peak response of the sensor was normalized to its baseline (\u0026Delta;F/Fo), which was measured 90 seconds before introducing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. For confocal live-cell imaging and monitoring antioxidant drugs, confocal imaging was performed by using Nikon A1R confocal microscope mounted onto a Nikon Eclipse Ti body with 20x objective lens. A Live-cell imaging chamber and incubation system were used for maintaining environmental conditions at 10% CO2 and 37\u0026deg;C during 40-hour continuous recording. Images were acquired by using NIS-element AR (Nikon). For image analysis, NIS-element (Nikon) and ImageJ (NIH) were used.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimals:\u0026nbsp;\u003c/em\u003eAll APP/PS1 mice were group-housed in a temperature- and humidity-controlled environment with a 12 h light/dark cycle and had free access to food and water.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVirus injection:\u0026nbsp;\u003c/em\u003eThe AAV5-GFAP104-oROS-G viral vector was cloned and AAV containing GFAP-104-oROS-G was packaged by the IBS virus facility (Daejeon, Korea). Mice were deeply anesthetized via vaporized 1% isoflurane and immobilized in a stereotaxic (RWD Life Science). Following an incision on the midline of the scalp, bilateral craniotomies were performed above the hippocampus CA1 (anterior/posterior, -2 mm; medial/lateral, \u0026plusmn;1.6 mm; dorsal/ventral, -1.45 mm from the bregma) using a microdrill. The virus was bilaterally microinjected (0.1 \u0026mu;l/min for 10 min; total 0.8 \u0026mu;l) using a syringe pump (KD Scientific).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eoROS-G imaging of GFAP-positive astrocytes in the brain slices:\u003c/em\u003e A total of 2 weeks after the virus injection into the hippocampus, animals were anesthetized with 1% isoflurane and then decapitated. The brains were submerged in chilled cutting solution that contained (in mM): 250 Sucrose, 26 NaHCO3, 10 D(+)-glucose, 4 MgCl2, 0.1 CaCl2, 2.5 KCl, \u0026nbsp;2 Sodium Pyruvate, 1.25 NaH2PO4, 0.5 ascorbic acid, and pH adjusted to pH 7.4. Coronal slices (300 \u0026mu;m thick) were prepared with a vibrating-knife microtome D.S.K LinearSlicer pro 7 (Dosaka EM Co. Ltd). For stabilization, brain slices were incubated at room temperature for at least 1 h before imaging. For imaging, the slices were transferred to a recording chamber which were mounted on an upright Zeiss Examiner D1 microscope and continuously perfused with an artificial cerebrospinal fluid (aCSF) solution that contained (in mM): 130 NaCl, 24 NaHCO3, 1.25 NaH2PO4, 3.5 KCl, 1.5 MgCl2, 1.5 CaCl2, D(+)-glucose, and pH adjusted to pH 7.4. All solutions were equilibrated with 95% O2 and 5% CO2. Imaging was acquired at 0.25 frame per second with 60X water-immersion objective lens, a ORCA-Flash4.0 CMOS camera (Hamamatsu; C13440), and a LED (CoolLED) filtered with 485-nm fluorescence was applied. Imaging workbench (INDEC Biosystem) and ImageJ (NIH) were utilized for image acquisition and ROI analysis. To examine H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-dose dependent responses of sensor-expressing astrocytes, concentration of 10 and 100 \u0026micro;M of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewere introduced by bath application. The peak response of the sensor was normalized to its baseline (\u0026Delta;F/Fo), wich was measured 90 seconds before introducing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. To measure endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in astrocytes of APP/PS1 mice and their littermates, we used 10 mM DTT (Thermo; R0861). This method reduced the oROS-G sensor bound to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, resulting in fluorescence below the baseline levels. These reduced fluorescence responses were normalized to its baseline (\u0026Delta;F/Fo), suggesting the basal endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGeneration of stable oROS-Gr expressing HEK293 cells.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293 cells in a T75 flask were transfected (using lipofection, as described above) with oROS-Gr-P2A-Puromycin plasmid. 3 Days after the transfection, cells were passaged to 2 T75 flasks. 2 Days after, puromycin-based selection was performed for a week using complete DMEM media (as previously described) supplemented with puromycin (1\u0026micro;g/mL). Cells after selection were expanded for 3 passages. Enrichment of cell populations with robust oROS-Gr expression was achieved with BD FACSAria II Cell Sorter at Flow and Imaging Core Lab of University of Washington South Lake Union Campus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGlucose experiment and Seahorse Assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eoROS-Gr stable cells cultured in complete DMEM with 10mM glucose were plated at 75,000/well in 24-well plates. oROS-Gr stable cells were plated at 75,000/well in 24-well plates. 1 day post seeding, FBS in the DMEM media was brought down to 2% from 10%. 2 day post seeding cells were in serum-free DMEM with various levels of glucose. Mannose was supplemented as needed to keep osmotic pressure of each media consistent (final total sugar content: 25mM). oROS-Gr ratio (GFP/RFP) were imaged in LCIS media with varying glucose and mannose level. For Seahorse assay, oROS-Gr stable cells mentioned above were plated in a Matrigel-coated 96 well Seahorse plate at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well for an equivalent procedure as above. The MitoStress protocol in the Seahorse XF96 Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA) was performed two weeks later. An hour before the assay, the culture media was replaced with media (Agilent Seahorse XF base medium, 103334-100 Agilent Technologies, Santa Clara, CA, USA) supplemented with 25 mM glucose and 1 mM Sodium pyruvate (11360070 Gibco/Thermo Scientific, Waltham, MA, USA). Substrates and select inhibitors of the different complexes were added during the measurement to achieve final concentrations of oligomycin (2.5 \u0026mu;M), FCCP (1 \u0026mu;M), rotenone (2.5 \u0026mu;M) and antimycin (2.5 \u0026mu;M). The oxygen consumption rate (OCR) values were then normalized with readings from Hoechst staining (HO33342 Sigma-Aldrich, St. Louis, MO, USA), which corresponded to the number of cells in the well.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOpioid receptor study\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAAV\u003c/em\u003e: An adenovirus associated double floxed inverted (AAV1-DIO) virus was generated containing the oROS-Gr by cloning oROS-Gr into pAAV1-Ef1a-DIO using Nhe1 and Asc1 restriction sites. AAV1 were prepared by the NAPE Molecular Genetics Resources Core as described previously (Gore, et al, 2013). HEK293T cells were transfected with 25 \u0026mu;g AAV1 vector plasmid and 50 \u0026mu;g packaging vector (pDG1) per 15 cm plate. Two days after transfection, cells were harvested and subjected to three freeze\u0026ndash;thaw cycles. The supernatant was transferred to a Beckman tube containing a 40% sucrose cushion and spun at 27,000 rpm overnight at 4\u0026deg;C. Pellets were resuspended in CsCl at a density of 1.37 g/ml and spun at 65000 rpm 4 hours at 4\u0026deg;C. 1 ml CsCl fractions were run on an agarose gel, and genome-containing fractions were selected and spun at 50000 rpm overnight at 4\u0026deg;C. The 1 ml fractions were collected again, and genome containing fractions were dialyzed overnight. The filtered solution was transferred to a Beckman tube containing a 40% sucrose cushion and spun at 27,000 rpm overnight at 4\u0026deg;C. The pellet (containing purified AAV) was resuspended in 150 \u0026mu;l 1\u0026times; HBSS. Virus was aliquoted and stored at -80 \u0026deg; C until use.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimals and surgeries:\u003c/em\u003e Test naive C57BL/6 male mice were ear punched at least 21 days after birth and genotyped using Transnetyx genotyping services. PCR screening was performed for the presence of Cre recombinase. For brain slice studies, mice between 5-7 weeks of age were injected with 0.5uL AAV1-Efla-FLEX-oROS-mCherry (CITE) construct containing oROS-Gr into a MOR CRE positive mouse bilaterally into the VTA using coordinates: ML: +/- 0.5, AP: -3.28, DN: -4.5 zeroed at bregma. Isoflurane was used for anesthesia and carprofen for pain relief. Mice were mounted on a stereotaxic alignment system and injections were made using a Hamilton 2.0uL model 7002 KH syringe. Similarly, for fiber photometry experiments, mice were injected with 0.5uL AAV1-Efla-FLEX-oROS-mCherry unilaterally at a 15-degree angle, using the coordinates ML: -1.71, AP: -3.28, DN: -4.67 then implanted with a 400/430 \u0026micro;m diameter Mono fiberoptic cannula from Doric Lenses.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2-photon imaging of \u0026micro;-opioid receptor expressing neurons in VTA:\u0026nbsp;\u003c/em\u003eTwo-four weeks after viral injection, the brain was dissected and 200um horizontal slices were prepared using a vibratome. Slices were incubated in NMDG (92mM NMDG, 2.5mM KCl, 1.25mM NaH2PO4, 30mM NaHCO3, 20mM HEPES, 25mM Glucose, 2mM Thiourea, 5mM Na-ascorbate, 3mM Na-pyruvate, pH to 7.4, 0.5mM CaCl\u0026bull;4H2O, 10mM MgSO4\u0026bull;7H2O). Recordings were made in a HEPES solution (92mM NaCl, 2.5mM KCl, 1.25mM NaH2PO4, 30mM NaHCO3, 20mM HEPES, 25mM Glucose, 2mM Thiourea, 5mM Na-Ascorbate, 3mM Na-Pyruvate). Image collection was done using a Bruker Investigator 2-photon microscope, software Prairie View 5.5, simultaneously collecting both the mCherry (1040 nm fixed) and GFP (920 nm tunable) signals with a Nikon 16X water immersion objective, as well as a z-stack spanning 60um across an hour time course. Baseline recordings were made in ACSF (124mM NaCl, 3mM KCl, 2mM MgSO4, 1.25mM NaH2PO4, 2.5mM CaCl2, 26mM NaHCO3, 10mM Glucose) at 32C, before treatment. For confocal images, animals were perfused intracardially with phosphate-buffered saline (PBS) and 10% formalin. Brains were stored in 10% formalin for up to 24 hours then switched to a 20% sucrose solution at 4C until sectioning. Coronal slices of the VTA were collected at 40um each and mounted using VECTASHIELD HardSet mounting Medium with DAPI. Confocal images were taken with the Leica SP8x Confocal microscope located in the Keck Center at UW.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFiber photometry of kappa-opioid expressing neurons in the VTA\u003c/em\u003e: A real-time signal processor (RZ5P; Tucker-Davis Technologies) connected to Synapse Software (Fiber Photometry) to set frequency of light stimulation and to record input from photodetectors. The RZ5P was connected to a light emitting diode (LED) driver (Doric Lenses) that controlled the power of a 465 nm and 560 nm Doric LED. A low autofluorescence patch cord (400/430) was attached to the LED, to a fluorescent MiniCube (Doric Lenses) with dichroic mirrors. Connected optical patch cords to the MiniCube with pigtailed rotary joining (FRJ; Doric Lenses) allowed free animal movement during data collection. Patch Cords were bleached with light prior to photometry sessions to minimize autofluorescence. Power of the LED at the fiber tip was set to ~30 uW and was tested prior to the start of each session. Signals were collected at a sampling frequency of 1017 Hz. Each of the sessions was downsampled by a factor of 100 and normalized to a 15-minute baseline period in the beginning of the recording. Data were then smoothed using a moving average filter (100s window) to remove high frequency noise and detrended to remove linear drift. The isosbestic channel (405 nm) was fitted to the 470 nm channel using a least-squares method and subtracted to remove motion artifacts. Each session started with a 15 min baseline recording period prior to pharmacological experiments to calculate fluorescent change from baseline (\u0026Delta;F/Fo; Change in fluorescence from baseline fluorescence/baseline fluorescence). \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFalkowski, P. G. \u003cem\u003eet al.\u003c/em\u003e The rise of oxygen over the past 205 million years and the evolution of large placental mammals. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e309\u003c/strong\u003e, 2202\u0026ndash;2204 (2005).\u003c/li\u003e\n\u003cli\u003eKoch, L. G. \u0026amp; Britton, S. L. Aerobic metabolism underlies complexity and capacity. \u003cem\u003eJ. Physiol.\u003c/em\u003e \u003cstrong\u003e586\u003c/strong\u003e, 83\u0026ndash;95 (2008).\u003c/li\u003e\n\u003cli\u003eWesterblad, H. \u0026amp; Allen, D. G. Emerging roles of ROS/RNS in muscle function and fatigue. \u003cem\u003eAntioxid. Redox Signal.\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 2487\u0026ndash;2499 (2011).\u003c/li\u003e\n\u003cli\u003eBarnham, K. J., Masters, C. L. \u0026amp; Bush, A. I. Neurodegenerative diseases and oxidative stress. \u003cem\u003eNat. Rev. Drug Discov.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 205\u0026ndash;214 (2004).\u003c/li\u003e\n\u003cli\u003eLee, Y. M., He, W. \u0026amp; Liou, Y.-C. The redox language in neurodegenerative diseases: oxidative post-translational modifications by hydrogen peroxide. \u003cem\u003eCell Death Dis.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 58 (2021).\u003c/li\u003e\n\u003cli\u003eForrester, S. J., Kikuchi, D. S., Hernandes, M. S., Xu, Q. \u0026amp; Griendling, K. K. Reactive Oxygen Species in Metabolic and Inflammatory Signaling. \u003cem\u003eCirc. Res.\u003c/em\u003e \u003cstrong\u003e122\u003c/strong\u003e, 877\u0026ndash;902 (2018).\u003c/li\u003e\n\u003cli\u003eD\u0026rsquo;Autr\u0026eacute;aux, B. \u0026amp; Toledano, M. B. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. \u003cem\u003eNat. Rev. Mol. Cell Biol.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 813\u0026ndash;824 (2007).\u003c/li\u003e\n\u003cli\u003eSchieber, M. \u0026amp; Chandel, N. S. ROS function in redox signaling and oxidative stress. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, R453-62 (2014).\u003c/li\u003e\n\u003cli\u003eFinkel, T. Signal transduction by reactive oxygen species. \u003cem\u003eJ. Cell Biol.\u003c/em\u003e \u003cstrong\u003e194\u003c/strong\u003e, 7\u0026ndash;15 (2011).\u003c/li\u003e\n\u003cli\u003eSies, H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. \u003cem\u003eRedox Biol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 613\u0026ndash;619 (2017).\u003c/li\u003e\n\u003cli\u003eChun, H. \u003cem\u003eet al.\u003c/em\u003e Severe reactive astrocytes precipitate pathological hallmarks of Alzheimer\u0026rsquo;s disease via H2O2- production. \u003cem\u003eNat. Neurosci.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1555\u0026ndash;1566 (2020).\u003c/li\u003e\n\u003cli\u003eChen, Q. M., Tu, V. C., Wu, Y. \u0026amp; Bahl, J. J. Hydrogen peroxide dose dependent induction of cell death or hypertrophy in cardiomyocytes. \u003cem\u003eArch. Biochem. Biophys.\u003c/em\u003e \u003cstrong\u003e373\u003c/strong\u003e, 242\u0026ndash;248 (2000).\u003c/li\u003e\n\u003cli\u003eAfanas\u0026rsquo;ev, I. New nucleophilic mechanisms of ros-dependent epigenetic modifications: comparison of aging and cancer. \u003cem\u003eAging Dis.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 52\u0026ndash;62 (2014).\u003c/li\u003e\n\u003cli\u003eSies, H. \u0026amp; Jones, D. P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. \u003cem\u003eNat. Rev. Mol. Cell Biol.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 363\u0026ndash;383 (2020).\u003c/li\u003e\n\u003cli\u003eSies, H. \u003cem\u003eet al.\u003c/em\u003e Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. \u003cem\u003eNat. Rev. Mol. Cell Biol.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 499\u0026ndash;515 (2022).\u003c/li\u003e\n\u003cli\u003eMurphy, M. P. \u003cem\u003eet al.\u003c/em\u003e Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. \u003cem\u003eNat Metab\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 651\u0026ndash;662 (2022).\u003c/li\u003e\n\u003cli\u003eGutscher, M. \u003cem\u003eet al.\u003c/em\u003e Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e284\u003c/strong\u003e, 31532\u0026ndash;31540 (2009).\u003c/li\u003e\n\u003cli\u003eMorgan, B. \u003cem\u003eet al.\u003c/em\u003e Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes. \u003cem\u003eNat. Chem. Biol.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 437\u0026ndash;443 (2016).\u003c/li\u003e\n\u003cli\u003eBelousov, V. V. \u003cem\u003eet al.\u003c/em\u003e Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. \u003cem\u003eNat. Methods\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 281\u0026ndash;286 (2006).\u003c/li\u003e\n\u003cli\u003eMarkvicheva, K. N. \u003cem\u003eet al.\u003c/em\u003e A genetically encoded sensor for H2O2 with expanded dynamic range. \u003cem\u003eBioorg. Med. Chem.\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 1079\u0026ndash;1084 (2011).\u003c/li\u003e\n\u003cli\u003eBilan, D. S. \u003cem\u003eet al.\u003c/em\u003e HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime imaging. \u003cem\u003eACS Chem. Biol.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 535\u0026ndash;542 (2013).\u003c/li\u003e\n\u003cli\u003eErmakova, Y. G. \u003cem\u003eet al.\u003c/em\u003e Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 5222 (2014).\u003c/li\u003e\n\u003cli\u003eSubach, O. M. \u003cem\u003eet al.\u003c/em\u003e Slowly Reducible Genetically Encoded Green Fluorescent Indicator for In Vivo and Ex Vivo Visualization of Hydrogen Peroxide. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003ePak, V. V. \u003cem\u003eet al.\u003c/em\u003e Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function. \u003cem\u003eCell Metab.\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 642-653.e6 (2020).\u003c/li\u003e\n\u003cli\u003ePang, Y. \u003cem\u003eet al.\u003c/em\u003e SHRIMP: Genetically Encoded mScarlet-derived Red Fluorescent Hydrogen Peroxide Sensor with High Brightness and Minimal Photoactivation. \u003cem\u003ebioRxiv\u003c/em\u003e 2023.08.09.552302 (2023) doi:10.1101/2023.08.09.552302.\u003c/li\u003e\n\u003cli\u003eLee, C. \u003cem\u003eet al.\u003c/em\u003e Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. \u003cem\u003eNat. Struct. Mol. Biol.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1179\u0026ndash;1185 (2004).\u003c/li\u003e\n\u003cli\u003eJo, I. \u003cem\u003eet al.\u003c/em\u003e Structural details of the OxyR peroxide-sensing mechanism. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e, 6443\u0026ndash;6448 (2015).\u003c/li\u003e\n\u003cli\u003eAkerboom, J. \u003cem\u003eet al.\u003c/em\u003e Optimization of a GCaMP calcium indicator for neural activity imaging. \u003cem\u003eJ. Neurosci.\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 13819\u0026ndash;13840 (2012).\u003c/li\u003e\n\u003cli\u003eBienert, G. P. \u0026amp; Chaumont, F. Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide. \u003cem\u003eBiochim. Biophys. Acta\u003c/em\u003e \u003cstrong\u003e1840\u003c/strong\u003e, 1596\u0026ndash;1604 (2014).\u003c/li\u003e\n\u003cli\u003eMontiel, V. \u003cem\u003eet al.\u003c/em\u003e Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide. \u003cem\u003eSci. Transl. Med.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, (2020).\u003c/li\u003e\n\u003cli\u003eWragg, D., Leoni, S. \u0026amp; Casini, A. Aquaporin-driven hydrogen peroxide transport: a case of molecular mimicry? \u003cem\u003eRSC Chem Biol\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 390\u0026ndash;394 (2020).\u003c/li\u003e\n\u003cli\u003eLim, J. B., Langford, T. F., Huang, B. K., Deen, W. M. \u0026amp; Sikes, H. D. A reaction-diffusion model of cytosolic hydrogen peroxide. \u003cem\u003eFree Radic. Biol. Med.\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e, 85\u0026ndash;90 (2016).\u003c/li\u003e\n\u003cli\u003eJan, Y.-H. \u003cem\u003eet al.\u003c/em\u003e Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication. \u003cem\u003eToxicol. Appl. Pharmacol.\u003c/em\u003e \u003cstrong\u003e288\u003c/strong\u003e, 114\u0026ndash;120 (2015).\u003c/li\u003e\n\u003cli\u003eBienert, G. P. \u003cem\u003eet al.\u003c/em\u003e Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e282\u003c/strong\u003e, 1183\u0026ndash;1192 (2007).\u003c/li\u003e\n\u003cli\u003ePedre, B., Barayeu, U., Ezeriņa, D. \u0026amp; Dick, T. P. The mechanism of action of N-acetylcysteine (NAC): The emerging role of H2S and sulfane sulfur species. \u003cem\u003ePharmacol. Ther.\u003c/em\u003e \u003cstrong\u003e228\u003c/strong\u003e, 107916 (2021).\u003c/li\u003e\n\u003cli\u003eEzeriņa, D., Takano, Y., Hanaoka, K., Urano, Y. \u0026amp; Dick, T. P. N-Acetyl Cysteine Functions as a Fast-Acting Antioxidant by Triggering Intracellular H2S and Sulfane Sulfur Production. \u003cem\u003eCell Chem Biol\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 447-459.e4 (2018).\u003c/li\u003e\n\u003cli\u003eChun, H., Lim, J., Park, K. D. \u0026amp; Lee, C. J. Inhibition of monoamine oxidase B prevents reactive astrogliosis and scar formation in stab wound injury model. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, 354\u0026ndash;367 (2022).\u003c/li\u003e\n\u003cli\u003eLee, S. \u003cem\u003eet al.\u003c/em\u003e Channel-mediated tonic GABA release from glia. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e330\u003c/strong\u003e, 790\u0026ndash;796 (2010).\u003c/li\u003e\n\u003cli\u003eJo, S. \u003cem\u003eet al.\u003c/em\u003e GABA from reactive astrocytes impairs memory in mouse models of Alzheimer\u0026rsquo;s disease. \u003cem\u003eNat. Med.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 886\u0026ndash;896 (2014).\u003c/li\u003e\n\u003cli\u003eJankowsky, J. L. \u003cem\u003eet al.\u003c/em\u003e Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. \u003cem\u003eHum. Mol. Genet.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 159\u0026ndash;170 (2004).\u003c/li\u003e\n\u003cli\u003eKritsiligkou, P., Shen, T. K. \u0026amp; Dick, T. P. A comparison of Prx- and OxyR-based H2O2 probes expressed in S. cerevisiae. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e297\u003c/strong\u003e, 100866 (2021).\u003c/li\u003e\n\u003cli\u003eSmolyarova, D. D., Podgorny, O. V., Bilan, D. S. \u0026amp; Belousov, V. V. A guide to genetically encoded tools for the study of H2 O2. \u003cem\u003eFEBS J.\u003c/em\u003e \u003cstrong\u003e289\u003c/strong\u003e, 5382\u0026ndash;5395 (2022).\u003c/li\u003e\n\u003cli\u003eRamakrishnan, P., Maclean, M., MacGregor, S. J., Anderson, J. G. \u0026amp; Grant, M. H. Cytotoxic responses to 405nm light exposure in mammalian and bacterial cells: Involvement of reactive oxygen species. \u003cem\u003eToxicol. In Vitro\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 54\u0026ndash;62 (2016).\u003c/li\u003e\n\u003cli\u003eBirnbaum, J. H. \u003cem\u003eet al.\u003c/em\u003e Oxidative stress and altered mitochondrial protein expression in the absence of amyloid-\u0026beta; and tau pathology in iPSC-derived neurons from sporadic Alzheimer\u0026rsquo;s disease patients. \u003cem\u003eStem Cell Res.\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 121\u0026ndash;130 (2018).\u003c/li\u003e\n\u003cli\u003eLoor, G. \u003cem\u003eet al.\u003c/em\u003e Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis. \u003cem\u003eFree Radic. Biol. Med.\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 1925\u0026ndash;1936 (2010).\u003c/li\u003e\n\u003cli\u003eCriddle, D. N. \u003cem\u003eet al.\u003c/em\u003e Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e281\u003c/strong\u003e, 40485\u0026ndash;40492 (2006).\u003c/li\u003e\n\u003cli\u003eShneyvays, V., Leshem, D., Shmist, Y., Zinman, T. \u0026amp; Shainberg, A. Effects of menadione and its derivative on cultured cardiomyocytes with mitochondrial disorders. \u003cem\u003eJ. Mol. Cell. Cardiol.\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 149\u0026ndash;158 (2005).\u003c/li\u003e\n\u003cli\u003eIshii, T., Warabi, E. \u0026amp; Mann, G. E. Mechanisms underlying Nrf2 nuclear translocation by non-lethal levels of hydrogen peroxide: p38 MAPK-dependent neutral sphingomyelinase2 membrane trafficking and ceramide/PKC\u0026zeta;/CK2 signaling. \u003cem\u003eFree Radic. Biol. Med.\u003c/em\u003e \u003cstrong\u003e191\u003c/strong\u003e, 191\u0026ndash;202 (2022).\u003c/li\u003e\n\u003cli\u003eEspinosa-Diez, C. \u003cem\u003eet al.\u003c/em\u003e Antioxidant responses and cellular adjustments to oxidative stress. \u003cem\u003eRedox Biol\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 183\u0026ndash;197 (2015).\u003c/li\u003e\n\u003cli\u003eKlima, J. C. \u003cem\u003eet al.\u003c/em\u003e Incorporation of sensing modalities into de novo designed fluorescence-activating proteins. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 856 (2021).\u003c/li\u003e\n\u003cli\u003eGoodman, J. B. \u003cem\u003eet al.\u003c/em\u003e Redox-Resistant SERCA [Sarco(endo)plasmic Reticulum Calcium ATPase] Attenuates Oxidant-Stimulated Mitochondrial Calcium and Apoptosis in Cardiac Myocytes and Pressure Overload-Induced Myocardial Failure in Mice. \u003cem\u003eCirculation\u003c/em\u003e \u003cstrong\u003e142\u003c/strong\u003e, 2459\u0026ndash;2469 (2020).\u003c/li\u003e\n\u003cli\u003eQin, F. \u003cem\u003eet al.\u003c/em\u003e Cytosolic H2O2 mediates hypertrophy, apoptosis, and decreased SERCA activity in mice with chronic hemodynamic overload. \u003cem\u003eAm. J. Physiol. Heart Circ. Physiol.\u003c/em\u003e \u003cstrong\u003e306\u003c/strong\u003e, H1453-63 (2014).\u003c/li\u003e\n\u003cli\u003eGonnot, F. \u003cem\u003eet al.\u003c/em\u003e SERCA2 phosphorylation at serine 663 is a key regulator of Ca2+ homeostasis in heart diseases. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 3346 (2023).\u003c/li\u003e\n\u003cli\u003eAkaike, T. \u003cem\u003eet al.\u003c/em\u003e A Sarcoplasmic Reticulum Localized Protein Phosphatase Regulates Phospholamban Phosphorylation and Promotes Ischemia Reperfusion Injury in the Heart. \u003cem\u003eJACC: Basic to Translational Science\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 160\u0026ndash;180 (2017).\u003c/li\u003e\n\u003cli\u003eWong, H.-S., Dighe, P. A., Mezera, V., Monternier, P.-A. \u0026amp; Brand, M. D. Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e292\u003c/strong\u003e, 16804\u0026ndash;16809 (2017).\u003c/li\u003e\n\u003cli\u003eHuang, J.-H., Co, H. K., Lee, Y.-C., Wu, C.-C. \u0026amp; Chen, S.-H. Multistability maintains redox homeostasis in human cells. \u003cem\u003eMol. Syst. Biol.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e10480 (2021).\u003c/li\u003e\n\u003cli\u003eForman, H. J. \u0026amp; Zhang, H. Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. \u003cem\u003eNat. Rev. Drug Discov.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 689\u0026ndash;709 (2021).\u003c/li\u003e\n\u003cli\u003eZhang, Y. \u003cem\u003eet al.\u003c/em\u003e A comparative genomics study of carbohydrate/glucose metabolic genes: from fish to mammals. \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 246 (2018).\u003c/li\u003e\n\u003cli\u003eAudzeyenka, I. \u003cem\u003eet al.\u003c/em\u003e Hyperglycemia alters mitochondrial respiration efficiency and mitophagy in human podocytes. \u003cem\u003eExp. Cell Res.\u003c/em\u003e \u003cstrong\u003e407\u003c/strong\u003e, 112758 (2021).\u003c/li\u003e\n\u003cli\u003eSchattauer, S. S. \u003cem\u003eet al.\u003c/em\u003e Peroxiredoxin 6 mediates G\u0026alpha;i protein-coupled receptor inactivation by cJun kinase. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 743 (2017).\u003c/li\u003e\n\u003cli\u003eSchattauer, S. S. \u003cem\u003eet al.\u003c/em\u003e Reactive oxygen species (ROS) generation is stimulated by \u0026kappa; opioid receptor activation through phosphorylated c-Jun N-terminal kinase and inhibited by p38 mitogen-activated protein kinase (MAPK) activation. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e294\u003c/strong\u003e, 16884\u0026ndash;16896 (2019).\u003c/li\u003e\n\u003cli\u003eStanicka, J., Russell, E. G., Woolley, J. F. \u0026amp; Cotter, T. G. NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e290\u003c/strong\u003e, 9348\u0026ndash;9361 (2015).\u003c/li\u003e\n\u003cli\u003eBrewer, T. F., Garcia, F. J., Onak, C. S., Carroll, K. S. \u0026amp; Chang, C. J. Chemical approaches to discovery and study of sources and targets of hydrogen peroxide redox signaling through NADPH oxidase proteins. \u003cem\u003eAnnu. Rev. Biochem.\u003c/em\u003e \u003cstrong\u003e84\u003c/strong\u003e, 765\u0026ndash;790 (2015).\u003c/li\u003e\n\u003cli\u003eMu\u0026ntilde;oz, M. \u003cem\u003eet al.\u003c/em\u003e Hydrogen peroxide derived from NADPH oxidase 4- and 2 contributes to the endothelium-dependent vasodilatation of intrarenal arteries. \u003cem\u003eRedox Biol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 92\u0026ndash;104 (2018).\u003c/li\u003e\n\u003cli\u003eJohnson, F. \u0026amp; Giulivi, C. Superoxide dismutases and their impact upon human health. \u003cem\u003eMol. Aspects Med.\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 340\u0026ndash;352 (2005).\u003c/li\u003e\n\u003cli\u003eEl Daibani, A. \u003cem\u003eet al.\u003c/em\u003e Molecular mechanism of biased signaling at the kappa opioid receptor. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1338 (2023).\u003c/li\u003e\n\u003cli\u003eSchattauer, S. S., Kuhar, J. R., Song, A. \u0026amp; Chavkin, C. Nalfurafine is a G-protein biased agonist having significantly greater bias at the human than rodent form of the kappa opioid receptor. \u003cem\u003eCell. Signal.\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 59\u0026ndash;65 (2017).\u003c/li\u003e\n\u003cli\u003eDouglas, A. J. \u003cem\u003eet al.\u003c/em\u003e Effects of the kappa-opioid agonist U50,488 on parturition in rats. \u003cem\u003eBr. J. Pharmacol.\u003c/em\u003e \u003cstrong\u003e109\u003c/strong\u003e, 251\u0026ndash;258 (1993).\u003c/li\u003e\n\u003cli\u003eAslund, F., Zheng, M., Beckwith, J. \u0026amp; Storz, G. Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e, 6161\u0026ndash;6165 (1999).\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Nafr\u0026iacute;a, J., Watson, J. F. \u0026amp; Greger, I. H. IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 27459 (2016).\u003c/li\u003e\n\u003cli\u003eLian, X. \u003cem\u003eet al.\u003c/em\u003e Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/\u0026beta;-catenin signaling under fully defined conditions. \u003cem\u003eNat. Protoc.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 162\u0026ndash;175 (2013).\u003c/li\u003e\n\u003cli\u003eBremner, S. B. \u003cem\u003eet al.\u003c/em\u003e Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues. \u003cem\u003eJ. Tissue Eng.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 20417314221119628 (2022).\u003c/li\u003e\n\u003cli\u003eTohyama, S. \u003cem\u003eet al.\u003c/em\u003e Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. \u003cem\u003eCell Stem Cell\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 127\u0026ndash;137 (2013).\u003c/li\u003e\n\u003cli\u003eStringer, C., Wang, T., Michaelos, M. \u0026amp; Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. \u003cem\u003eNat. Methods\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 100\u0026ndash;106 (2021).\u003c/li\u003e\n\u003cli\u003ePachitariu, M. \u0026amp; Stringer, C. Cellpose 2.0: how to train your own model. \u003cem\u003eNat. Methods\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 1634\u0026ndash;1641 (2022).\u003c/li\u003e\n\u003cli\u003eWoo, D. H. \u003cem\u003eet al.\u003c/em\u003e TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e151\u003c/strong\u003e, 25\u0026ndash;40 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4048855/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4048855/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHydrogen Peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity and response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e dynamics. We successfully tracked real-time transient and steady-state H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse brain \u003cem\u003eex vivo\u003c/em\u003e and\u003cem\u003e in vivo\u003c/em\u003e. These applications demonstrate oROS's capabilities to monitor H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as a secondary response to pharmacologically induced oxidative stress and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aβ-putriscine-MAOB axis, highlighting the sensor’s relevance in validating neurodegenerative disease models. Lastly, we demonstrated acute opioid-induced generation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e signal \u003cem\u003ein vivo\u003c/em\u003e which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool, offering a window into the dynamic landscape of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for understanding diseases associated with oxidative stress, such as cancer, neurodegenerative, and cardiovascular diseases.\u003c/p\u003e","manuscriptTitle":"Ultra-fast genetically encoded sensor for precise real-time monitoring of physiological and pathophysiological peroxide dynamics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-25 11:45:51","doi":"10.21203/rs.3.rs-4048855/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ae12b4ec-e780-4b94-aded-f6299e833f2b","owner":[],"postedDate":"March 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":29359031,"name":"Biological sciences/Biological techniques/Molecular engineering/Protein design"},{"id":29359032,"name":"Biological sciences/Biological techniques/Imaging/Fluorescence imaging"}],"tags":[],"updatedAt":"2024-03-25T11:45:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-25 11:45:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4048855","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4048855","identity":"rs-4048855","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}