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

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 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 H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 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 H2O2 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 H2O2 signal in vivo which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 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.


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 in ammation [3][4][5][6] .Furthermore, an increasing number of studies report the role of low-level ROS as physiologic mediator in normal cellular signaling processes [7][8][9] .Speci cally, 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][12][13] .Nevertheless, limited analytic tools to spatiotemporally monitor speci c oxidants in situ with precision have been a bottleneck to deciphering their speci c 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 speci city 16 .Protein-based peroxide sensors have been engineered to overcome these shortcomings.For example, the roGFP sensor family fuses roGFP, a redox-sensitive green uorescent protein variant, to H 2 O 2 -speci c enzymes like Orp1 (thiol peroxidase), or Tsa2 (typical 2-Cys peroxiredoxin) from yeast to achieve peroxide-speci c roGFP uorescence changes via redox relay 17,18 .The HyPer sensor family is based on the direct fusion of circularly permuted uorescent protein (cpFP) to the regulatory domain of bacterial peroxide sensor protein OxyR for conformational coupling that leads to H 2 O 2 -speci c uorescence change [19][20][21][22][23][24] .Most HyPer sensors use ecOxyR (Escherichia coli OxyR), the most extensively studied OxyR variant, as their sensing domain.However, existing ecOxyRbased 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 exibility in the sensors.Through a series of structure-guided engineering steps, we developed oROS-G (optogenetic hydRogen perOxide Sensor, Green), a green uorescent 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 uorescence 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.Speci cally, 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 e cacy 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 speci city 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 disul de 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.
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 rst characterized the uorescence response of the oROS-G sensor in HEK293 cells in response to exogenously or endogenously sourced H  3A] highlighting the potential robustness of oROS-G expression and functionality in broader biological host systems.In addition, we con rmed 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 con rmed 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 uorescent 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 uorescence response was speci c to peroxide-induced disul de 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-acetylcystine (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  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 speci cally 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 uorescence 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 uorescence 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 uorescent sensor proteins at 405 nm and 488 nm 41,42 .Although this sensor type allows exibility in multi-color optogenetic experiments, illumination at 405 nm could contribute to oxidative stress in mammalian cells. 43We created oROS-Gr, by fusing mCherry to oROS-G, creating an equimolar reference point inert to H 2 O 2 .Flow cytometry analysis con rmed 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.( Menadione treatment has been widely used to model oxidative stress in biological systems [44][45][46][47] .Still, studies to monitor its intracellular effect have been mostly limited to short time windows or noncontinuous 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 dosedependent 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 coe cient 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, con rming improvements in precision [Ratiometric: 0.27 (n = 484), non-Ratiometric: 0.46 (n = 484)].
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 exempli es intricate functional G-protein biased agonists in uence on arrestin-independent inactivation pro le of µ and κ opioid receptors.Brie y, 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 κ-opioid receptor (KOR) has emerged as an promising drug target for pain management with less sideeffects 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 .Nalfura ne 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 Nalfura ne in vivo, con rming 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 uorescence (ex:488/em:510) was monitored by ber photometry in the VTA [Fig.6D].Intraperitoneal administration of 100µg/kg Nalfura ne led to transient increase of H 2 O 2 .Mice pre-treated 30 min prior to a 100µg/kg Nalfura ne injection with a high dose of naloxone (10 mg/kg), su cient to block KOR 68 , showed no signi cant increase in uorescence compared to mice only treated with Nalfura ne [Fig.6E].This con rms that the Nalfura ne induced H 2 O 2 signal in KOR expressing neurons of VTA is opioid receptor speci c.

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 signi cant step in this direction.As a novel green uorescent 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 re nement of the sensor, where we relocated the cpGFP insertion site to maintain exibility of C199-C208 loop of ecOxyR.Drawing inspiration from Akerboom et al. 28 , we incorporated bulky residues adjacent to the cpGFP barrel opening, exempli ed 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 exible 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 e cacy in detecting opioid-induced peroxide increase in vivo, further emphasizing its broad applicability.
In conclusion, the oROS sensors, exempli ed 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 signi cant implications for unraveling the mechanisms underlying various oxidative stress-related diseases.

Declarations
Handling and animal care have been performed according to the Institutional Animal Care and Use Committee of the Institute for Basic Science.
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 tting 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 ampli cation for KLD-based site-directed mutagenesis methods.Primers were ordered from Integrated DNA Technologies (IDT).All gene fragment ampli cations were done using Seither Q5-polymerase (New England Biolabs; M0492L) or Super -II polymerase (Invitrogen; 12368010).Ampli cation of DNA fragments were veri ed 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 (DH5 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 con rm the presence of the intended mutation.Genes encoding the nal 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 veri ed with Sanger sequencing (Genewiz; Seattle, WA) or Whole plasmid nanopore sequencing (Plasmidsarus; Eugene, OR) 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.Brie y, 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% con uent, 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 uorescence response upon H 2 O 2 stimulation.
Neural Differentiation Media was changed twice a week for 21 days at which point the differentiation is considered nished.Neurons were replated at a density of 500,000 cells/cm 2 .

Analysis
Analysis of cell uorescence imaging data was done by FUSE, a custom cloud-based semi-automated time series uorescence data analysis platform written in Python.First, the cell segmentation quality of the selected Cellpose 74 model was manually veri ed.For the segmentation of cells expressing cytosolic uorescent 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, uorescence time-series data is extracted for each region of interest.This allows for unbiased extraction of change in cellular uorescence information for a complete set of experimental samples.Extracted uorescence data is normalized as speci ed in the text using custom python script.
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. 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.
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% iso urane and then decapitated.Generation of stable oROS-Gr expressing HEK293 cells.
HEK293 cells in a T75 ask were transfected (using lipofection, as described above) with oROS-Gr-P2A-Puromycin plasmid.3 Days after the transfection, cells were passaged to 2 T75 asks. 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 24well plates.oROS-Gr stable cells were plated at 75,000/well in 24-well plates.Opioid receptor study AAV: An adenovirus associated double oxed 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 ltered 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 puri ed 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-E a-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.Iso urane 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 ber photometry experiments, mice were injected with 0.5uL AAV1-E a-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 beroptic cannula from Doric Lenses.
Image collection was done using a Bruker Investigator 2-photon microscope, software Prairie View 5.     See image above for gure legend.
Page 28/30 See image above for gure legend.
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 ndings.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 veri ed with one-photon confocal microscopy of post-mortem xed brain slices [Fig.6B].The VTA in ex vivo brain slices showed an acute increase in sensor uorescence 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 rst 5 baseline frames, MOR: 3.35; ci = [1.94,4.82], MOR + NLX: 0.63; ci = [0.42,0.85]) [Fig.6C].

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Figure 5
Figure 5 2 O 2 .Direct application of exogenous H 2 O 2 increases intracellular H 2 O 2 by diffusion across the plasma membrane through speci c aquaporins, which creates an 11,37xt 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 2scavenging effects of molecules.Initially, we expressed oROS-G in the astrocytes and tested H 2 O 2 concentration-dependent functionality [Supp.Figure4C].We observed an increase of 21.27 ± 5.3% and 57.74 ± 9.4% in uorescence with 10 and 100 µM H 2 O 2 , respectively [Supp.Figure4D-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) pathology11,37.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 oROS-G expressed in HEK293 showed a 73% diminished response to exogenous 10 µM peroxide exposure [Supp.Figure4A].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 signi cantly attenuated the response by 72 percent [Supp.Figure4B].(5µM) or putrescine (180 µM).Over a 40-hour continuous recording [Fig.3B],weobserved a signi cant increase in oAβ-induced oROS-G uorescence, 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 presubstrate of MAOB, also signi cantly increased oROS-G sensor uorescence [Fig.3E,F].However, this H 2 O 2 elevation was signi cantly 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 e cacy of potential H 2 O 2 -scavenging compounds.
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-Flash4.0CMOScamera (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) wereintroduced 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 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 ( nal 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 Scienti c, Waltham, MA, USA).Substrates and select inhibitors of the different complexes were added during the measurement to achieve nal 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.