The Reactive Species Interactome in the brain.

SIGNIFICANCE
Redox pioneer Helmut Sies attempted to explain reactive species' challenges faced by organelles, cells, tissues, and organs via three complementary definitions: (1) oxidative stress, i.e. the disturbance in the prooxidant-antioxidant defense balance in favor of the prooxidants, (2) oxidative eustress, the low physiological exposure to prooxidants, and (3) oxidative distress, the supraphysiological exposure to prooxidants. Recent Advances: Identification, concentration and interactions are the most important elements to improve our understanding of reactive species in physiology and pathology. In this context, the reactive species interactome (RSI) is a new multilevel redox regulatory system that identifies reactive species families, reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS), and integrates their interactions with their downstream biological targets.


CRITICAL ISSUES
We propose a united view to fully combine reactive species identification, oxidative eustress and distress, and the RSI system. In this view, we also propose including the forgotten reactive carbonyl species (RCS), an increasingly rediscovered reactive species family related to the other reactive families, and key enzymes within the RSI. We focus on brain physiology and pathology to demonstrate why this united view should be considered.


FUTURE DIRECTIONS
More studies are needed for an improved understanding of the contributions of reactive species through their identification, concentration, and interactions, including in the brain. Appreciating the reactive species interactome in its entirety should unveil new molecular players and mechanisms in physiology and pathology in the brain and elsewhere.


Introduction
"Stress is the spice of life" is a famous quote by Hans Selye (210), who, in 1925, originally, defined stress as a general adaptation syndrome with three stages: alarm reaction, resistance, and exhaustion (208)(209)(210). Subsequently, redox pioneer Helmut Sies helped the concept of stress evolve, especially regarding the prooxidant-antioxidant balance and the concentration-dependent functions of stress molecules. After proving the prooxidant reactive oxygen species (ROS) hydrogen peroxide (H 2 O 2 ) is normally produced in intact eukaryotic cells by studying the steady-state level of the antioxidant defense enzyme catalase, Sies defined oxidative stress in 1985 as "a disturbance in the prooxidantantioxidant balance in favor of the former" (114,216,219,221). This definition was updated in 2007 as "an imbalance between prooxidants and antioxidants in favor of the prooxidants, leading to a disruption of redox signaling and control and/or molecular damage" (222). In 2015, Sies legitimately argued for clearly naming the individual prooxidants instead of applying only the generic labels of oxidative stress and ROS (217).
Finally, in 2017, Sies defined oxidative eustress as "low physiological exposure to prooxidants" with specific targets involved in redox signaling, and oxidative distress as "supraphysiological exposure to prooxidants" with unspecific targets, disrupted redox signaling and damaging molecule modifications (218,220).
From physiology to pathology, likely related to tissue-specific metabolome, excessive stress is indeed a major risk factor in the lungs, liver, kidneys, heart, and brain, the metabolically active organs vital for survival (46,122,142,238). In humans, the brain represents approximately 2% of total body weight but consumes 20% of total basal body oxygen and 20% of the body's energy. It is one of the most metabolically active of all organs (52,196). Importantly, the brain is also highly susceptible to oxidative stress, specifically oxidative distress, for several reasons (52). One of these is due to the multifaceted roles of reactives species themselves, especially superoxide anion ( or Cu + to generate OH . , OHand corresponding reactive Fe 3+ or Cu 2+ in the Fenton reaction (118,225). The Fenton reaction with Fe 2+ occurs mainly in the lysosome, an essential acidic organelle for cellular degradation and recycling of proteins, DNA, RNA, lipids and carbohydrates (64). This lysosomal reation induces lysosomal stress, leakage, and multiple intracellular dysfunctions including bioenergetic metabolism and autophagy alterations (64). Through another reaction, the Haber-Weiss reaction, H 2 O 2 also reacts with O 2 .to generate OH . , OHand O 2 (118,225). Finally, multiple antioxidant defense systems such as glutaredoxin, OXPHOS complex IV cytochrome c oxidase, glutathione (GSH/GSSG), and vitamins contribute to regulating the entire ROS metabolism (53, 223,225). This partial view of ROS metabolism will be completed later with regard to SODs, catalase, XOR, and MPO as the four key enzymes.

Reactive nitrogen species metabolism
Closely related to iron homeostasis, RNS metabolism is the generation of nitric oxide (NO)- mainly expressed in endothelial cells (74,76,120,225). While nNOS and eNOS are calciumdependent enzymes, iNOS is calcium-independent (74,76).
Using O 2 , oxidation of L-arginine by coupled NOS with cofactors including NADPH generates NO, NADP and L-citrulline, which is also an L-arginine precursor through the citrulline-NO cycle (12,74,76,120). Nitric oxide then reacts with O 2 to generate nitrogen dioxide (NO 2 ) and with NO 2 to form dinitrogen trioxide, which reacts with water to produce nitrite (NO 2 -) or dissociates to form NO and NO 2 (74,76,120). Nitrite oxidation with O 2 leads to the generation of nitrate (NO 3 -) (74,76,120). Nitric oxide is also generated by the following process: nitrate reductase (NR) catalyzes the reduction of nitrate into nitrite, and nitrite reductase (NiR) reduces nitrite into NO (120). The RNS metabolism also includes other reactive species such as nitroxyl, nitrous acid, hydroxylamine, and ammonia that are not discussed here (120). This partial view of RNS metabolism will be completed later regarding NO as the key reactive species and NOS as the key enzyme.

Reactive sulfur species metabolism
Reactive sulfur species are derived from hydrogen sulfide (H 2 S) (20, 29, 53, 128, 171, 272) (Fig. 2). From essential amino acid L-methionine recycling and transsulfuration pathway with L-homocysteine and L-cysteine, the RSS-related enzymes cysteine-β-synthase (CBS), cysteine-γ-lyase (CSE here, a.k.a. CGL), cysteine aminotransferase (CAT) and 3mercaptosulfotransferase (3-MST) are the key enzymes involved in H 2 S production (29, 53, 128,157,171,172,233,272). First, L-methionine catabolism leads to L-homocysteine biosynthesis (29, 128,171,171,233,272). Cysteine-γ-lyase then converts L-homocysteine to L-homolanthionine and H 2 S (29, 128,171,171,233,272). The CBS and CSE enzymes convert L-homocysteine and L-cysteine to L-cystathionine and H 2 S, and L-cystathionine is further converted to L-cysteine by CSE (29, 128,171,171,233,272). In addition, L-cysteine is metabolized either by CBS to produce H 2 S, L-serine and L-lanthionine, by CSE to generate H 2 S, ammonium and pyruvate, or by CAT to 3-mercaptopyruvate (29, 128,157,171,171,233,272). The enzyme CAT is also both an aspartate aminotransferase (AST) and a glutamate oxaloacetate transaminase (GOT) that generates α-ketoglutarate and aspartate from oxaloacetate and glutamate in mitochondria (GOT2) catalyzes the reverse reaction in the cytosol (GOT1) (155,157). Cystine, the dimeric form of L-cysteine, is converted by CSE to L-thiocysteine , producing H 2 S through thiol (RSH)-dependent reactions (171,233). The 3-mercaptopyruvate which is also produced by conversion of D-cysteine by D-amino acid oxidase (DAO) and that spontenously generates H 2 S, is the substrate of 3-MST to generate pyruvate, and finally, bound sulfane sulfur (171,233). This sulfur generation results in the release of H 2 S through the thioredoxin (TRX) enzyme and dihydrolipoic acid (DHLA) (171,233). This pathway is standard for H 2 S generation, although TRX and DHLA are part of the unconventional pathway (171). In this pathway, H 2 S is generated from thiosulfate (S 2 O 3 2-) through reductant DHLA or thiosulfate reductase coupled to GSH oxidation or 3-MST (171).
Associated with GSH/GSSG and NADPH/NADH systems, non-enzymatic reactions through thiocysteine persulfides and polysulfides also generate H 2 S (29). Moreover, mitochondrial OXPHOS complex I is a source of H 2 S due to its accessory sulfurtransferase subunit (171).
Finally, not discussed here, volatile organic sulfides such as carbon disulfide, carbonyl sulfide and sulfur dioxide, and acid-labile sulfide in iron centers, including cytochromes and aminothiols such as cysteinylglycine are unconventional sources of H 2 S (171).
Once H 2 S is produced, as its accumulation is toxic, H 2 S catabolism occurs by oxidation to form thiosulfate (29,128,171). This reaction also occurs in mitochondria, where sulfide quinone oxidoreductase oxidizes H 2 S to produce persulfide (29, 128,171). Sulfite and sulfate are also derived from H 2 S and related to thiosulfate (29, 128,171). Thiosulfate is converted into sulfite through thiosulfate sulfurtransferase or rhodanese then into sulfate, and sulfite contributes to H 2 S generation (29, 128,171). Hydrogen sulfide catabolism also occurs by methylation to produce methanethiol, then dimethylsulfide by thiol Smethyltransferase (29). Finally, H 2 S is oxidized into hydrogen thioperoxide, sulfinic acid then sulfonic acid (128). Although many reactive sulfur species are compelling, we will not discuss all the species and pathways. Two species we will discuss are polysulfides (H 2 S n ), including persulfides (H 2 S 2 ). These species are of increasing research interest, and their production and catabolism conventional and unconventional pathways (171). Finally, polysulfides are unstable and their catabolism tend to generate H 2 S (171). This partial view of RSS metabolism will be completed later, considering H 2 S as the notable reactive species.
Notably, RCS, including MGO, are generated as metabolic intermediates during glycolysis and polyol processing (205,211). Methylglyoxal is mainly generated during glycolysis through nonenzymatic catabolism of triose phosphates G3P, dihydroxyacetone-phosphate (DHAP) and the glycation pathway to ultimately produce advanced glycation end products (AGEs) (205,211). The AGEs and lipoxidation end products (ALEs), also induced by RCS are irreversible toxic products (205,211). The end-product fructose from the polyol glucosesorbitol-fructose pathway, is also a precursor of MGO through triose phosphate formation (205,211). Finally, MGO is generated by ketone body catabolism, including oxidation of acetone (205,211). Fortunately, the glyoxalase defense system counteracts the accumulation of MGO by converting MGO into D-lactate (205,211).
Long seen as side products from ROS following lipid peroxidation, careful examination of RCS shows that this family strengthens the connections between ROS, RNS, and RSS families in such a way that the RCS family could be the missing piece of the "stress puzzle" that will change perspectives and contribute to revealing new pathways. This partial view of RCS metabolism has presented the notable reactive species MDA, MGO, and 4-HNE and the key enzymes, GPX as well PRDX.

The metabolism and interactome of notable reactive species and key enzymes
Connections between the notable reactive species, families (including multiple ROS, NO, H 2 S, and MDA, MGO, and 4-HNE) and key enzymes (SOD, catalase, XOR, MPO, NOS, GPX, and PRDX) reinforce the united concept of RSI highlighted in our overview (Fig. 2).
In a recent demonstration of this connectedness, Kenneth Olson and colleagues showed in a concentration-and oxygen-dependent manner that SODs oxidize H 2 S into polysulfides, including persulfide H 2 S 2 , and to a lesser extent H 2 S 3 and H 2 S 5 , which may be an ancient mechanism to detoxify or manage RSS (173 (185). Importantly, GPX and PRDX enzymes regulate one of the most important sources of RCS, lipid hydroperoxides (9,16,137,161,195,269). Specifically, the selenoenzyme GPX4 and PRDX, including PRDX4, which are sensors and regulators of peroxides, neutralize lipid hydroperoxides (9,16,137,161,195,269).
Due to its direct reactions with ROS, RNS, and RSS and key enzymes, the RCS family should be considered as important as ROS, RNS, and RSS families in the reactive species interactome.
Notable reactive species, key enzymes, and the reactive species interactome in the brain As developed in the introduction, we are focused on the brain, which, of all organs, is one of the most metabolically active and sensitive to oxidative distress. From physiology to pathology, from oxidative eustress to distress, considering 1) our strategy of identificationconcentration-interactions, 2) the notable reactive species and key enzymes, and 3) descriptions of individual and puzzling implications of ROS, RNS, RSS, and RCS, we combined key findings to reveal the RSI (including RCS) potential in the brain. We concentrate the brain physiology discussion on adult neurogenesis, cell death and aging, and regard the brain pathology on depression, ischemia, neurodegenerative disorders, and cancers.

Physiology: the brain adult neurogenesis
In mammals, throughout life, neurogenesis, which occurs in several brain regions such as the hippocampal dentate gyrus and lateral ventricles, is defined as "the formation of new neurons from neural stem cells (NSC) and progenitor cells" (19,146). The existence of adult neurogenesis, one that occurs in adulthood has been intensively debated due to discrepancies between different studies using histology and multiple biomarkers (19,146).
Historically, in 1998, adult neurogenesis was observed continuously, at least in the human hippocampus (70). Then, after lengthy debates, in 2019, persistent adult neurogenesis in the hippocampal dentate gyrus was confirmed in healthy adult humans up to 90 years-old, (146,159). As solid evidence is growing in support of human adult neurogenesis (in the hippocampus and elsewhere), the field has moved towards reconciliation of various neurogenesis biomarkers and open questions on the molecular mechanisms regulating neurogenesis including through oxidative stress (146,159,275).

The RSI in brain adult neurogenesis
The neurogenesis field is currently investigating more than twenty different biomarkers (146,275). An increasing number of studies highlight the involvement of several reactive species in brain adult neurogenesis, although the molecular mechanisms between reactive species and neurogenesis biomarkers require further investigation (Fig. 3)  stimulation (271). Superoxide dismutase, catalase and MPO are key enzymes to investigate in adult neurogenesis.
Increasing the complexity, NO generated by neuronal NOS is a negative regulator of NSC and progenitor cell proliferation in healthy brain while its overgeneration after brain damage promotes proliferation and then adult neurogenesis (72,160,177 (191,244,269). The same for PRDXs as PRDX1, PRDX2, and PRDX4 stimulate adult neurogenesis while PRDX6 inhibits adult neurogenesis (268).
In adult neurogenesis, considering interactions between the four reactive species metabolism including through key enzymes, the RSI is fully committed and reveals all its potential when integrated with RCS metabolism (Fig. 2).
The RSI in brain cell death Brain vulnerability increases due to neuroinflammation and decreases in neuroplasticity, dysregulation of neuronal activities, and alteration of bioenergetic metabolism (153). Brain vulnerability is also exacerbated by increased intracellular oxidation, damage, and cell death in neurons, astrocytes, oligodendrocytes and microglial cells, at the intersection of brain physiology and disorders (153). Cell death is an inevitable consequence of cell life, and there are at least twelve types of cell death (80) (Fig.4). In the brain, apoptosis, necrosis including necroptosis, oxytosis, and ferroptosis (two names of the same pathway or two highly similar pathways), autophagy-dependent cell death, and parthanatos poly  Oxidative distress mediated by the RNS family also stimulates the main cell death-types in the brain. Accumulation of NO induces apoptosis through the nitrosylation of key proteins involved in apoptosis signaling, including caspases 3/8/9 and B-cell lymphoma 2 (Bcl-2), and accumulaton of ONOOactivates apoptosis, necroptosis, parthanatos, and autophagydependent cell death in neurons, including through tyrosine nitration of heat shock protein 90 (Hsp90), α-synuclein, cytochrome c, and through DNA damage (188,192). In contrast to RNS, oxidative distress mediated by H 2 S and sulfite is neuroprotective by inhibiting apoptosis and oxytosis. Anti-inflammatory and anti-apoptotic, H 2 S physiologically and pharmacologically reduces cell-death type activation, including after a brain injury, even though very high levels of H 2 S can disrupt mitochondrial function and trigger at least apoptosis in neurons (99, 274). Sulfite also protects neurons against oxytosis, a cell death-type due to H 2 O 2 accumulation, GSH depletion, lipoxygenase activation, and calcium influx (124,135).
Finally, oxidative distress mediated by RCS is also a major cell death-types regulator to consider, at least in the brain. The MGO triggers apoptosis in neurons (55, 61, 86).
Altogether, while oxidative distress mediated by ROS, RNS, and RCS activates several brain cell death-types, i.e. apoptosis, necrosis including necroptosis, autophagy-dependent cell death, and ferroptosis, oxidative distress mediated by RSS inactivates at least apoptosis and oxytosis. Therefore, considering both the RSI in its entirety, including the RCS family and key enzymes at the intersection of the reactive species families, will reveal all RSI potential in brain cell death. This information is important in understanding roles and switching between different cell death-types in physiology, including aging and pathology Is RSI the eleventh hallmark of brain aging ?
Brain aging has a signature of ten hallmarks: neuroinflammation, oxidative damage, adaptive stress response signaling alteration, bioenergetic metabolism dysregulation, mitochondrial dysfunction, aberrant neural network activity, neuronal calcium homeostasis dysregulation, stem cell exhaustion, molecular waste disposal impairment, and DNA repair impairment (153, 264) (Fig. 5). Multiple pathways are involved in brain aging through many different biomarkers not listed here to focus our overview on the reactive species themselves. Impaired adult neurogenesis, overactivation of cell death including apoptosis, and cellular replicative senescence are involved in brain aging (153,264).
It is important to remember that replicative senescence is probably a key driver of aging (14,153,273). Replicative senescence, a powerful anticancer mechanism, is a stable cell cycle arrest associated with telomere shortening, senescence-associated betagalactosidase (SA-β-gal) activity, kinase inhibitor proteins p16INK4 and p21 activation, and a senescence-associated secretory phenotype (SASP) including proteases, growth factors and pro-inflammatory cytokines (133, 264). Senescence is both a physiological and pathological process related to several reactive species and is implicated in aging, including that in post-mitotic neurons and oligodendrocytes, as recent evidence argues that senescence occurs in these cells (14,133,153).
We propose that dysregulated RSI is the 11 th hallmark of brain aging that links cellular replicative senescence, cell death types, adult neurogenesis, and the ten established hallmarks (Fig. 5).
First, at a minimum mitochondrial O 2 accumulation and antioxidant defense decline are the major events that trigger the ten hallmarks of brain aging (153). Oxidative distress mediated by mitochondrial O 2 in the brain, including the frontal cortex, hippocampus, and amygdala, initiates brain aging (228). Interestingly, in mitotic astrocytes, highly glycolytic compared to other glial cells, mitochondrial O 2 does not strongly contribute to cellular senescence while its contribution is significant in other glial cells (228). Overgeneration of H 2 O 2 also leads to senescence of mitotic astrocytes with SA-β-gal activity and probable SASP, and mitotic microglial cells with telomere shortening, SA-β-gal activity and probable SASP (153,256). Compared to astrocytes, post-mitotic neurons, which are much less glycolytic, and so more dependent on mitochondrial function, generate subtantiam 228). Moreover, overgeneration of H 2 O 2 seems to lead to senescence of human neural progenitor cells and post-mitotic neurons with SA-β-gal activity and probable SASP characteristics (153,256,273). All this evidence indicates oxidative distress mediated by H 2 O 2 initiates brain aging. In addition, key enzymes SOD, catalase, XOR, and MPO play key roles in aging, including in the brain as brain aging is associated with progressive decline in SOD and catalase activities. The use of SOD/catalase mimetics may contribute to reversing age-related declines including in cognitive function, and as XOR and MPO activities dangerously increase with aging (50, 87, 243, 250).
Antioxidant defense decline and oxidative distress mediated by ROS are clearly pro-aging, including in the brain.
Reactive nitrogen species metabolism is also actively involved in brain aging, although heterogeneously in nitrosative vulnerability depending on brain cell types (26, 27, 228).
Mitotic glycolytic astrocytes are not vulnerable to NO and ONOOwhile post-mitotic neurons, which are more dependent on mitochondrial respiration, are vulnerable to both NO and ONOO -(26, 27, 228). Nitric oxide, both an anterograde and retrograde neurotransmitter in synapses, and ONOOcontribute to protein nitration, nitrosylation, and consequently brain aging (188). Aging including in brain is associated with nitrative damages, and nitrosylation of amyloid β-peptides, and proteins involved in regulation of immune response, including S100, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (188). Oxidative distress mediated by RNS is pro-aging including in the brain.
Reactive sulfur speices metabolism is also actively involved in brain aging through H 2 S and H 2 S n (polysulfide), which are suggested to act as gliotransmitters (147,149). Oxidative distress mediated by H 2 S and H 2 Sn supports brain function including memory formation during aging by modulating N-methyl-D-Aspartate subtype glutamate receptors (NMDARs) through sulfuration (147,149). The NMDARs are excitatory glutamate receptors involved in neuronal plasticity, and learning and memory. They are dysregulated during brain aging and numerous brain pathologies (147,149). In addition, H 2 S also exerts anti-aging properties by inhibiting ROS and RNS generation, and by activating sirtuin 1 (SIRT1), a major anti-aging and anti-senescence NAD (nicotinamide adenine dinucleotide)-dependent protein deacetylase found in the brain (277). Oxidative distress mediated by RSS is antiaging, including in the brain.
Finally, oxidative distress mediated by 4-HNE and MDA is pro-aging. The 4-HNE and MDA are overgenerated by increased lipid peroxidation and accumulate in tissues and organs with age, including in the brain (115,153). However, with no clear molecular explanation, a progressive decrease in MGO levels associated with an increase in aldolase reductase (AR) expression, which metabolizes MGO, occurs during brain aging, thus suggesting an unsolved key role of MGO in brain aging (127). Moreover, during brain aging, reduced GSH decreases while oxidized GSSG increases, and key enzymes GPXs (including GPX4 activities) as well PRDXs (including PRDX6) decline (15,45,71,145,176,281). Interestingly, GPX4 activity plays a key role at the intersection of apoptosis and ferroptosis, an important topic in aging research, including that exploring the brain (15,96,270). An increase in GPXs and PRDXs expression and activity that improve detoxification of lipid hydroxyperoxides is a promising route of anti-aging therapy exploration to limit detrimental oxidative distress due to 4-HNE and MDA in brain aging.
Considering that antioxidant defense decline and oxidative distress mediated by ROS, RNS, and 4-HNE and MDA are pro-aging while oxidative distress mediated by RSS is anti-aging including in the brain, the entire RSI, including RCS, has to be further investigated. A dysregulated RSI could be the 11 th hallmark of brain aging (Fig. 5).

Conclusion on the RSI in brain physiology
Through adult neurogenesis, cell death and aging in the brain, we showed that the entire RSI should be considered instead of only one reactive species family. Knowing (1) exactly which reactive species is active in, i.e. identification, and (2) the physiological or supraphysiological concentration, i.e. concentration integrating oxidative eustress and distress, will reveal all the RSI potential in physiology, including in the brain.
Reactive species family roles vary in the physiological process, and a family is not solely Oxidative distress mediated by ROS, RNS, and RCS is pro-aging, while that mediated by RSS is anti-aging. Together, the RCS family is coordinated together with the other reactive species families, and therefore only the RSI, including RCS, is fully committed in brain physiology.
Finally, key enzymes SOD, catalase, XOR, MPO, GPXs (including GPX4), and PRDXs require further examination in light of the entire RSI, including RCS in brain physiology. In a physiological process, the global redox effect is created by the combination between (1) effects from different reactives species families in different concentrations, (2) interactions between the different families and (3) activities of the key enzymes that contribute to the RSI dynamics. This axiom is true in physiology and pathologies.

The RSI in mental disorder depression
Oxidative stress, or better, oxidative distress, plays a significant role in mental disorders from addiction to anxiety, depression, bipolar disorder, post-traumatic stress disorder, dementia, and schizophrenia affecting almost 1 billion people worldwide (38, 240).
Depression affects 300 million people worldwide, thus being the leading mental disorder, and by 2030, depression is predicted to be the second leading cause of illness worldwide, following human immunodeficiency virus infection/acquired immunodeficiency syndrome and followed by ischemic heart disease (102,152).
Different complementary approaches such as transcriptomic, proteomic, metabolomic, and epigenetic, depression, including major depression, have been progressively better characterized to improve treatments, although complex molecular mechanisms are involved (230). More than fifteen biomarkers are predictive of this mental disorder, including a reduction in gray matter volume, overgeneration of the stress hormone cortisol, dopamine and noradrenaline reduction, glutamate accumulation, neuroinflammation, tryptophan metabolism alterations, brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF) reduction, and possibly genetic polymorphisms (230) (Fig. 6). The antioxidant defense and reactive species and related targets are also increasingly considered biomarkers for depression, although the molecular mechanisms are still missing (230). The entire RSI, including RCS, should be considered in the study of mental disorders, including depressive disorder, to improve the understanding of the targets and the molecular mechanisms (Fig. 6). First, an imbalance between O 2 and H 2 O 2 due to specific SOD2 genetic polymorphisms increases the risk of depression (54). Overgeneration of ROS, antioxidant defense alterations including SOD dysfunction, and decrease in GSH levels are involved in major depressive disorder through harmful overoxidation of proteins, lipids and DNA (143,199,249). Specific genetic polymorphisms of catalase are also associated with an increased risk of depression (199). A decrease in SOD and catalase activities and increased XOR levels are often found in depressed patients compared to healthy individuals (249). The XOR activity also increases in recurrent depressive episodes in many brain regions, including the hippocampus and thalamus (156). In addition, the accumulation of MPO enzyme in serum is a biomarker of immune dysregulation associated with major depressive disorder (246). Altogether, an imbalance between ROS, oxidative distress mediated by ROS and altered SOD, catalase, XOR, and MPO enzymes exert a prodepressant effect.
Interestingly, it is now known that anti-depressant drugs modulate ROS/RNS generation and antioxidant defense (199,249). Reactive nitrogen metabolism is involved in depression including major depressive disorder. In depression, neuronal NOS enzyme accumulates in several brain regions, including the hippocampus, cortex, and hypothalamus (279). Researchers have proposed that the interaction between nNOS and serotonergic signaling, and the link between nNOS and adult neurogenesis are the key deregulated mechanisms in depression (279). Inhibition of nNOS with NG-nitro-L-arginine methyl ester exhibits antidepressant properties, and several weeks of antidepressant treatment reduces NO levels in depressed patients (199,249,279). Thus, related to nNOS accumulation, oxidative distress mediated by RNS exerts a pro-depressant effect.
Importantly, by increasing synaptic neurotransmission and synaptic plasticity, the H 2 S has an antidepressant-like effect (41). This effect is through induction of adiponectin expression, a hormone involved in glucose and lipid metabolism, that ultimately reduces autophagy and favors synapse formation in the brain, including in the hippocampus (241).
Oxidative distress mediated by RSS exerts an anti-depressant effect. Finally, an increase in lipid peroxidation, the main source of RCS, is associated with  Fig. 6).

The RSI in brain ischemia
When a blood clot blocks or plugs an artery or vein in the brain, an arterial or venous occlusion, this is an ischemic stroke that occurs, creating a medical emergency for which brain aging is the highest non-modifiable risk factor (28, 65, 113,153,202). Every year, stroke, which includes hemorrhagic stroke (20% of the cases) and ischemic stroke (80% of the cases) accounts for approximately 80.1 million cases worldwide and 5.5 million deaths (28,65,113,202). Following a stroke, per hour, 120 million neurons die, 830 billion synapses and 714 km/447 miles of myelinated fibers are lost, so the brain is losing nearly 3.6 years of normal aging (202). To treat arterial or venous occlusion, rapid reperfusion with thrombolysis and endovascular thrombectomy is the main therapy, and while it provides benefits, it also presents adverse effects regarding oxidative distress (28).
Ischemic stroke provokes rapid oxygen and glucose deprivation, inducing excitotoxicity due to glutamate release, mitochondrial dysfunction, oxidative distress, lipid peroxidation, DNA damage, neuroinflammation and neuronal cell death through at least apoptosis and necrosis, including necroptosis (57, 153) (Fig.7). Oxygen deprivation followed by reoxygenation induces an increase in the blood-brain barrier (BBB) permeability partially through activation of the hypoxia-inducible transcriptional factor HIF-1 and an increase in expression of its target genes such as matrix metalloproteinase 2 and VEGF (171,208).  Fig. 7).  be the most powerful therapy to treat ischemia. Regulating the entire RSI will induce key enzyme remodeling, and the best therapy will be one molecule or a combination of molecules directly targeting the heart of the RSI. Therefore, we strongly encourage increased research to better understand the RSI related to the pathophysiology of stroke as the firststep to discover the best therapy.

The RSI in neurodegenerative disorders
Oxidative distress, bioenergetic metabolism, senescence, and cell death are intriguing and complex key players involved in the so-called neurodegenerative disorders, the leading cause of disability worldwide and a major cause of global deaths. These disorders include One significant unknown is whether oxidative distress is a cause or consequence of neurodegeneration (7). A better understanding of the RSI, including RCS and the key enzymes enzymes, will help answer this question and discover new therapies to treat neurodegenerative disorders, including ALS, PD, and AD (Fig.8).
First, related to mitochondrial uncoupling and mitochondrial calcium concentration, oxidative distress mediated by O 2 is associated with AD (262). Oxidative distress mediated by H 2 O 2 occurs during the early stages of protein aggregation in AD and dementia (237). In addition, reduced GSH levels are found in ALS, AD and PD (167). Importantly, the key should be addressed carefully to limit neurotoxic side effects. Once again, the entire RSI, including RCS and the key enzymes, are major players in neurodegeneration and should be considered together in future studies (Fig. 8).

The RSI is a promising target for the treatment of neurodegenerative disorders
Researchers in the neurodegenerative disorder and antioxidant fields need to face the difficult truth that current antioxidant therapies available to treat neurodegenerative disorders have been less than successful. Indeed, all the tested antioxidant drugs that tend to reduce oxidative distress mediated by ROS, increase GSH levels, and increase SOD and catalase activities from in vitro to animal models, ultimately failed at clinical trial levels Complexity is one likely reason why a loss in translation occurs from animal models to human physiology (21). Targeting only one reactive species family is not sufficient. The most powerful therapy for neurodegenerative disorders will target ROS, RNS, RSS, and RCS together and the key enzymes that act upon them. Therefore, we strongly encourage the research community to improve their understanding of the RSI remated to the pathophysiology of neurodegenerative disorders includingALS, PD, and AD, as the first step to discovering the best therapy.

The RSI in brain cancers
Aging is the biggest risk factor for cancer, which is a leading cause of death globally (56).
Cancer is a group of more than 100 malignant diseases throughout the body in which cells grow uncontrollably (56, 125). Glioma accounts for 80% of malignant primary brain cancers (129). They are categorized into three types: oligodendroglioma, ependymoma, and astrocytoma, including the most frequent and the most lethal primary brain cancers, the glioblastoma (129). In addition, it is estimated that malignant secondary brain cancers, called brain metastases, occur in 20% of all the patients with primary cancer with other origins, mainly lung, breast, colorectal, and melanoma cancers (1). In primary brain cancers and metastases, coordination between bioenergetic metabolism and hypoxia plays a critical and harmful role in carcinogenesis, cancer progression, and cancer cell dissemination (22, 98, 179) (Fig. 9).
Interestingly, hypoxia which results from the disequilibrium between oxygen delivery and consumption, is a pervasive stimulus at physiological and pathophysiological levels, in cancer, including brain cancers. Obviously, regarding reactive species, the first concern is always to minimize ROS (Fig. 9).
First, one major concern in cancer, including in the brain, is whether hypoxia modulates In cancer, including that in the brain, RNS metabolism also contributes to regulating the hypoxic status and effects (89). Immune system evasion and anticancer therapy resistance occur in part due to NO overgeneration induced by hypoxia (17,24). In glioma, an increase in iNOS expression is a hallmark of neuroinflammation and of chemoresistance, and iNOS expression and activity inhibitors are promising anti-glioma agents (148). Numerous studies showed that NO regulates glioma growth, cancer angiogenesis, cancer cell invasion, radio-and chemoresistance (5,226,235,242). In addition, ONOOaccumulation in glioblastoma inactivates wild-type p53 function and is associated with an altered inflammatory response in the glioma microenvironment (51). In brain cancer, oxidative distress mediated by RNS is pro-cancer.
Reactive sulfur species metabolism is as important as ROS and RNS in cancer. In brain cancer, associated with hypoxia, oxidative distress mediated by RSS is pro-cancer, although RSS may be promising radiosensitizers depending on their concentration. Hydrogen sulfide has been suggested as a promising radiosensitizer, at least for glioblastoma cells through selective OXPHOS complex III impairment and mitochondrial respiration reduction, an increase in H 2 O 2 levels, and an increase in DNA damage (265). However, H 2 S also favors C6 glioma cell proliferation and survival through increased cyclooxygenase-2 expression and apoptosis reduction (278). The debate surrounding the anti-cancer and pro-cancer roles of H 2 S in brain cancers is made more complicated by the puzzling increase in expression of the H 2 S producing enzymes CBS and CSE in astrocytomas and brain metastases (132).
Hypoxia increases this complexity, as hypoxia induces H 2 S generation, and H 2 S regulates HIF-1α subunit expression and stabilization (257).
Finally, RCS metabolism also plays a key role in cancer, including in the brain.
Methylglyoxal induces cell cycle arrest, inhibits cell proliferation, and initiates apoptosis in glioblastoma cells (186). Interestingly, in glioblastoma, 4-HNE and MDA levels decrease, related to high aldehyde dehydrogenase 1A3 levels, which detoxify cancer cells from aldehydes (190,261). In addition, in glioma, key enzyme GPX activity decreases, while a high level of GPX2 is a poor prognosis biomarker in glioblastoma (95,193). Finally, an increase in PRDX4 activity in glioblastoma promotes cancer proliferation and anticancer therapy resistance (184). Thus, targeting GPX and PRDX as well as inducing oxidative distress mediated by RCS could be promising anti-cancer therapies, including against glioblastoma.
Once again, in brain cancer, the entire RSI is the ideal target through the coordination of the different reactive species and key enzymes (Fig. 9). In the brain, associated with hypoxia, oxidative distress mediated by ROS, RNS, and RSS is pro-cancer, while inducing oxidative distress mediated by RCS is a promising anti-cancer therapy. Additionally, altered SOD, catalase, XOR, MPO, GPX, and PRDX enzymes are promising targets in anti-cancer therapy, including brain cancer.

Antioxidants and cancer: bad and good news
Anti-ROS antioxidants such as N-acetylcysteine (NAC) and vitamin E, at high concentrations, accelerate lung cancer by disrupting the ROS-p53 axis, as p53 is a tumor suppressor gene involved in stress response following oxidative stress, hypoxia and increases HIF-1α subunit stability, increases ROS generation, and dysregulates H 2 S generation (105,106,110,123,144,260,267). Thus, gain-of-function mutated IDH1 and IDH2 in cancers (including primary brain cancers) that account for more than 70-80% of lower-grade glioma and the majority of secondary glioblastoma, induce epigenetic reprogramming, bioenergetic metabolism dysregulation (at least both ROS and RSS dysregulation) and finally cancer invasion (105,106,110,123,144,260,267). Reactive species interactome should be considered to ultimately design more efficient and powerful anti-cancer therapies in all cancers. Combining RSI modulation therapy with radiotherapy, chemotherapy, nanoparticle therapy and/or immunotherapy may provide especially promising pathways.

Conclusion on the RSI in brain pathology
Through depression, ischemia, neurodegenerative disorders, and cancers in the brain, we  Importantly, in addition to the reactive species, the key enzymes SOD, catalase, XOR, MPO, GPX, and PRDX should be considered in all these brain pathologies.
In a pathological process, what creates the global redox effect is the combination between (1) effects from different reactive species families in different concentrations, (2) interactions between the different families, and (3) activities of key enzymes that contribute to the RSI dynamics. The RSI, including RCS and key enzymes, is fully committed in brain pathologies.

Conclusions
Oxidative eustress and distress must be seriously considered in relation to reactive species concentrations. From the most studied ROS, it appears that the reactive species interactome (RSI) in its entirety has to be seriously considered related to reactive species identification and interactions. We highlighted that the RSI, meaning ROS, RNS, RSS, RCS and key associated enzymes, is fully committed in brain physiology and pathology (Fig. 10).
Depending on organ oxygenation, physiology, pathology, and key enzyme dynamics, the complex coordination of the four reactive species families related to their concentrations creates the global redox effect. The ROS, RNS, RSS, and RCS form a link between physiology and pathologies, between the antioxidant defense and the bioenergetic metabolism, between cell death and cell survival. From adult neurogenesis to cell death, aging, depression, ischemia, neurodegeneration, and finally, cancers, the brain is a vital organ in which the RSI combined in eutress or distress is playing all its potential for mechanistic and therapeutic clues.

Authorship confirmation statement
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Funding statement
The forum review article was supported by the Region Normandie, the Inkermann fund hosted at the Fondation de France, the CNRS and Université de Caen-Normandie (UNICAEN).

4-HNE 4-hydroxy-trans-2-nonenal
Aβ               considered, including SOD, catalase, MPO, XOR, GPXs and PRDXs. Targeting these enzymes is also a promising anti-cancer therapy, including in brain cancer. Circled reactive species are the known species involved in brain cancer. HIF-1, hypoxia-inducible factor 1; GPX, glutathione peroxidase; MPO, myeloperoxidase; NRF2, nuclear transcription erythroid-2related factor 2; NOS, nitric oxide synthase; PRDX, peroxiredoxin; SOD, superoxide dismutase; XOR, xanthine oxidoreductase.  NOS, GPXs, PRDXs, and XOR, play essential roles in physiology and pathology, and have to be also investigated. The brain is a vital organ in which the RSI combined in eustress or distress is playing all its potential for mechanistic and therapeutic clues. Graphical Abstract