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Primary cultures of bovine chromaffin cells are considered a good model to evaluate potential neuroprotective compounds for two major reasons: (i) they share many common features to neurons as they synthesize, store, and release neurotransmitters; they are excitable cells that express voltage-dependent calcium, potassium, and sodium channels; they express different neuronal receptor subtypes; and (ii) they can be easily cultured in high quantities from adult animals; as adult para-neurons, they can be used to reproduce different neurodegenerative-like cytotoxicity models. In this chapter, we describe protocols to mimic calcium overload (veratridine and thapsigargin) and oxidative stress (rotenone plus oligomycin-A and 6-hydroxydopamine) to evaluate potential neuroprotective compounds.

Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jpain.2022.07.014., Neuropathic pain (NP) is a challenging condition to treat, as the need for new drugs to treat NP is an unmet goal. We investigated the analgesic potential of a new sulfated disaccharide compound, named BIS014. Oral administration (p.o.) of this compound induced ameliorative effects in formalin-induced nociception and capsaicin-induced secondary mechanical hypersensitivity in mice, but also after partial sciatic nerve transection (spared nerve injury), chemotherapy (paclitaxel)-induced NP, and diabetic neuropathy induced by streptozotocin. Importantly, BIS014, at doses active on neuropathic hypersensitivity (60 mg/kg/p.o.), did not alter exploratory activity or motor coordination (in the rotarod test), unlike a standard dose of gabapentin (40 mg/kg/p.o.) which although inducing antiallodynic effects on the NP models, it also markedly decreased exploration and motor coordination. In docking and molecular dynamic simulation studies, BIS014 interacted with TRPV1, a receptor involved in pain transmission where it behaved as a partial agonist. Additionally, similar to capsaicin, BIS014 increased cytosolic Ca2+ concentration ([Ca2+]c) in neuroblastoma cells expressing TRPV1 receptors; these elevations were blocked by ruthenium red. BIS014 did not block capsaicin-elicited [Ca2+]c transients, but inhibited the increase in the firing rate of action potentials in bradykinin-sensitized dorsal root ganglion neurons stimulated with capsaicin. Perspective: We report that the oral administration of a new sulfated disaccharide compound, named BIS014, decreases neuropathic pain from diverse etiology in mice. Unlike the comparator gabapentin, BIS014 does not induce sedation. Thus, BIS014 has the potential to become a new efficacious non-sedative oral medication for the treatment of neuropathic pain., Laboratorios Bioibérica (Barcelona), Universidad Autónoma de Madrid (UAM)

By replacing a phenolic ring of (E)-resveratrol with an 1,3,4-oxadiazol-2(3H)-one heterocycle, new resveratrol-based multi- target-directed ligands (MTDLs) were obtained. They were evaluated in several assays related to oxidative stress and inflammation (monoamine oxidases, nuclear erythroid 2-related factor, quinone reductase-2, and oxygen radical trapping) and then in experiments of increasing complexity (neurogenic properties and neuroprotection vs okadaic acid). 5-[(E)-2-(4-Methoxyphenyl)ethenyl]-3-(prop-2-yn-1- yl)-1,3,4-oxadiazol-2(3H)-one (4e) showed a well-balanced MTDL profile: cellular activation of the NRF2-ARE pathway (CD = 9.83 μM), selective inhibition of both hMAO-B and QR2 (IC50s = 8.05 and 0.57 μM), and the best ability to promote hippocampal neurogenesis. It showed a good drug-like profile (positive in vitro central nervous system permeability, good physiological solubility, no glutathione conjugation, and lack of PAINS or Lipinski alerts) and exerted neuroprotective and antioxidant actions in both acute and chronic Alzheimer models using hippocampal tissues. Thus, 4e is an interesting MTDL that could stimulate defensive and regenerative pathways and block early events in neurodegenerative cascades., The authors gratefully acknowledge the following financial supports: the Spanish Ministry of Science, Innovation and Universities; Spanish Research Agency; European Regional Development Funds (grants RTI2018-093955-B-C21, SAF2015-64948-C2-1-R, and PID2021-122650OB-I00 to M.I.R.-F.; RTI2018-095793-B-I00 to M.G.L.); Spanish Na- tional Research Council (CSIC grants, PIE-202080E118 to M.I.R.-F. and PIE-202080I026 to R.L.); Health Institute Carlos III (grant PI17/01700 to R.L.); and General Council for Research and Innovation of the Community of Madrid and European Structural Funds (grant B2017/BMD-3827- NRF24ADCM). They thank the Ministry of Education of Spain for the following fellowships: FPU16/01704 and mobility grant FPUEST17/00233 (to C.H.-A.), FPU15/ 03269 (to C.F.-M.), FPU18/00630 (to E.d.S.), and FPU13/ 03737 (to P.M.). R.L. and M.G.L. also thank “Fundación Teófilo Hernando” for its continued support. The Spanish Medicinal Chemistry Society (SEQT) awarded the “Lilly Prize for Young Researchers” to this work, presented by C.H.-A. at the XIX SEQT Awards.

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1,2,3,4, a phenomenon that occurs in hypoxia4,5,6,7,8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism., This research has been financed by Spanish Government grants (ISCIII and AEI agencies, partially funded by the European Union FEDER/ERDF) CSD2007-00020 (RosasNet, Consolider-Ingenio 2010 programme to A.M.-R. and J.A.E.); CP07/00143, PS09/00101, PI12/00875, PI15/00107 and RTI2018-094203-B-I00 (to A.M.-R.); CP12/03304 and PI15/01100 (to L.M.); CP14/00008, CPII19/00005 and PI16/00735 (to J.E.); SAF2016-77222-R (to A. Cogolludo); PI17/01286 (to P.N.); SAF2015-65633-R, RTI2018-099357-B-I00 and CB16/10/00282 (to J.A.E.); RTI2018-095793-B-I00 (to M.G.L.); and SAF2017-84494-2-R (to J.R.-C.), by the European Union (ITN GA317433 to J.A.E. and MC-CIG GA304217 to R.A.-P.), by grants from the Comunidad de Madrid B2017/BMD-3727 (to A. Cogolludo) and B2017/BMD-3827 (to M.G.L.), by a grant from the Fundación Domingo Martínez (to M.G.L. and A.M.-R.), by the Human Frontier Science Program grant HFSP-RGP0016/2018 (to J.A.E.), by grants from the Fundación BBVA (to R.A.-P. and J.R.-C.), by the UCM-Banco Santander grant PR75/18-21561 (to A.M.-d.-P.), by the Programa Red Guipuzcoana de Ciencia, Tecnología e Información 2018-CIEN-000058-01 (to J.R.-C.) and from the Basque Government under the ELKARTEK Program (grant no. KK-2019/bmG19 to J.R.-C.), by the Swiss National Science Foundation (SNF) grant 310030_124970/1 (to A.B.), by a travel grant from the IIS-IP (to P.H.-A.) and by the COST actions TD0901 (HypoxiaNet) and BM1203 (EU-ROS). The CNIC is supported by the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (Spanish Government award SEV-2015-0505). CIC biomaGUNE is supported by the María de Maeztu Units of Excellence Program from the Spanish Government (MDM-2017-0720). P.H.-A. was a recipient of a predoctoral FPU fellowship from the Spanish Government. E.N. is a recipient of a predoctoral FPI fellowship from the Universidad Autónoma de Madrid (UAM). A.M.-R., L.M. and J.E. are supported by the I3SNS or ‘Miguel Servet’ programmes (ISCIII, Spanish Government; partially funded by the FEDER/ERDF).

The transcription factor nuclear factor erythroid 2‐related factor 2 (NRF2) is considered the master regulator of the phase II antioxidant response. It controls a plethora of cytoprotective genes related to oxidative stress, inflammation, and protein homeostasis, among other processes. Activation of these pathways has been described in numerous pathologies., Ministerio de Ciencia e Innovación, Grant/Award Numbers: PID2019‐110061RB‐ I00, PID2021‐122650OB‐I00, PID2021‐ 123481OB‐I00, PID2021‐125986OB‐I00; Instituto de Salud Carlos III, Grant/Award Number: PI17/01700; Comunidad de Madrid, Grant/Award Number: B2017/BMD‐3827‐ NRF24ADCM

Disrupted homeostasis of the microtubule binding protein tau is a shared feature of a set of neurodegenerative disorders known as tauopathies. Acetylation of soluble tau is an early pathological event in neurodegeneration. In this work, we find that a large fraction of neuronal tau is degraded by chaperone-mediated autophagy (CMA) whereas, upon acetylation, tau is preferentially degraded by macroautophagy and endosomal microautophagy. Rerouting of acetylated tau to these other autophagic pathways originates, in part, from the inhibitory effect that acetylated tau exerts on CMA and results in its extracellular release. In fact, experimental blockage of CMA enhances cell-to-cell propagation of pathogenic tau in a mouse model of tauopathy. Furthermore, analysis of lysosomes isolated from brains of patients with tauopathies demonstrates similar molecular mechanisms leading to CMA dysfunction. This study reveals that CMA failure in tauopathy brains alters tau homeostasis and could contribute to aggravate disease progression., The tau protein has been implicated in neurodegenerative disorders and can propagate from cell to cell. Here, the authors show that tau acetylation reduces its degradation by chaperone-mediated autophagy, causing re-routing to other autophagic pathways and increasing extracellular tau release.

The P2X7 receptor (P2X7R) is a ligand-gated ion channel that is being recognized as a major player in neuropsychiatric disorders such as Major Depressive Disorder (MDD). P2X7R activation is triggered by high extracellular ATP concentrations, leading to channel opening and inducing an increase in cytosolic calcium concentration ([Ca2+]c), that activates the inflammatory pathway. Those receptors are expressed not only in CNS cells but also in peripheral blood cells, where they are activated in response to inflammatory molecules such as bacterial lipopolysaccharide (LPS). LPS induced-tissue damage promotes an elevation of extracellular ATP, triggering the NRLP3-inflammasome assembly and activation that, sequentially, induces caspase-1 cleavage and IL-1β processing and secretion. In this context, we attempt to understand the role of P2X7R in [Ca2+]c homeostasis regulation, inflammasome expression and its pharmacological modulation in MDD. For this purpose, monocytes were isolated from peripheral blood of MDD patients and [Ca2+]c was monitored with the intracellular probe Fura-2. Our results point out to P2X7R as the responsible of the Ca2+ imbalance, as well as TNF-α-dependent activation of caspase-1 in MDD patients. In addition, P2X7R blockade with its specific antagonist, JNJ-47965567, reduces the Ca2+ entry upon Bz-ATP exposure. Altogether, our results point that MDD patients have both, Ca2+ homeostasis alteration and an inflammatory status, which promote an independent-inflammasome activation of caspase-1. Therefore, we propose the pharmacological modulation of P2X7R as a therapeutic approach against MDD symptoms.

Tauopathies are a group of neurodegenerative diseases characterized by the alteration/aggregation of TAU protein, for which there is still no effective treatment. Therefore, new pharmacological targets are being sought, such as elements of the endocannabinoid system (ECS). We analysed the occurrence of changes in the ECS in tauopathies and their implication in the pathogenesis. By integrating gene expression analysis, immunofluorescence, genetic and adeno-associated virus expressing TAU mouse models, we found a TAU-dependent increase in CB receptor expression in hippocampal neurons, that occurs as an early event in the pathology and was maintained until late stages. These changes were accompanied by alterations in the endocannabinoid metabolism. Remarkably, CB ablation in mice protects from neurodegeneration induced by hTAU overexpression, corroborated at the level of cognitive behaviour, synaptic plasticity, and aggregates of insoluble TAU. At the level of neuroinflammation, the absence of CB did not produce significant changes in concordance with a possible neuronal location rather than its classic glial expression in these models. These findings were corroborated in post-mortem samples of patients with Alzheimer’s disease, the most common tauopathy. Our results show that neurons with accumulated TAU induce the expression of the CB receptor, which enhances neurodegeneration. These results are important for our understanding of disease mechanisms, providing a novel therapeutic strategy to be investigated in tauopathies., Spanish Ministry of Economy and Competitiveness Grants refs. SAF2016-76520-R and PID2019-105600RB-I00 to ILB; Ref. RTI2018-095793-B-I00 to MGL. General Council for Research and Innovation of the Community of Madrid and European Structural Funds Ref. S2017/BMD-3813-ELA_Madrid to ILB and B2017/BMD-3827 – NRF24ADCM to MGL. Fundacion Tatiana Perez de Guzman el Bueno P-043-FTPGB 2020 to ILB

Heme oxygenase-1 (HO-1) is an inducible enzyme known for its anti-inflammatory, antioxidant and neuroprotective effects. However, increased expression of HO-1 during aging and age-related neurodegenerative diseases have been associated to neurotoxic ferric iron deposits. Being microglia responsible for the brain's innate immune response, the aim of this study was to understand the role of microglial HO-1 under inflammatory conditions in aged mice. For this purpose, aged wild type (WT) and LysMCreHmox1△△ (HMOX1M-KO) mice that lack HO-1 in microglial cells, were used. Aged WT mice showed higher basal expression levels of microglial HO-1 in the brain than adult mice. This increase was even higher when exposed to an inflammatory stimulus (LPS via i.p.) and was accompanied by alterations in different iron-related metabolism proteins, resulting in an increase of iron deposits, oxidative stress, ferroptosis and cognitive decline. Furthermore, microglia exhibited a primed phenotype and increased levels of inflammatory markers such as iNOS, p65, IL-1β, TNF-α, Caspase-1 and NLRP3. Interestingly, all these alterations were prevented in aged HMOX1M-KO and WT mice treated with the HO-1 inhibitor ZnPPIX. In order to determine the effects of microglial HO-1-dependent iron overload, aged WT mice were treated with the iron chelator deferoxamine (DFX). DFX caused major improvements in iron, inflammatory and behavioral alterations found in aged mice exposed to LPS. In conclusion, this study highlights how microglial HO-1 overexpression contributes to neurotoxic iron accumulation providing deleterious effects in aged mice exposed to an inflammatory insult., Graphical abstract Image 1, Highlights • Microglial HO-1 increases with aging and under an acute inflammatory stimulus. • LPS-dependent microglial HO-1 upregulation during aging leads to iron overload. • Microglial HO-1-dependent iron accumulation leads to ferroptosis. • HO-1-dependent iron alterations lead to neuroinflammation. • HO-1 inhibitors/iron chelators reduce iron accumulation and neuroinflammation.

Approximately 44 million people worldwide live with Alzheimer’s disease (AD) or a related form of dementia. Aggregates of the microtubule-associated protein tau are a common marker of these neurodegenerative diseases collectively termed as tauopathies. However, all therapeutic attempts based on tau have failed, suggesting that tau may only indicate a higher-level causal mechanism. For example, increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation. Here we show that type 4 NADPH oxidase (NOX), the most abundant isoform of the only dedicated reactive oxygen producing enzyme family, is upregulated in dementia and AD patients and in a humanized mouse model of tauopathy. Both global knockout and neuronal knockdown of theNox4gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP). Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevented cognitive decline, suggesting a direct and causal role for neuronal NOX4. Thus, NOX4 is a previously unrecognized causal, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease.Graphical abstract

Alzheimer&rsquo, s disease is a chronic and irreversible pathological process that has become the most prevalent neurodegenerative disease. Currently, it is considered a multifactorial disease where oxidative stress and chronic neuroinflammation play a crucial role in its onset and development. Its characteristic neuronal loss has been related to the formation of neurofibrillary tangles mainly composed by hyperphosphorylated tau protein. Hyperphosphorylation of tau protein is related to the over-activity of GSK-3&beta, a kinase that participates in several pathological mechanisms including neuroinflammation. Neuronal loss is also related to cytosolic Ca2+ homeostasis dysregulation that triggers apoptosis and free radicals production, contributing to oxidative damage and, finally, neuronal death. Under these premises, we have obtained a new family of 4,7-dihydro-2H-pyrazolo[3&ndash, b]pyridines as multitarget directed ligands showing potent antioxidant properties and able to scavenge both oxygen and nitrogen radical species, and also, with anti-inflammatory properties. Further characterization has demonstrated their capacity to inhibit GSK-3&beta, and to block L-type voltage dependent calcium channels. Novel derivatives have also demonstrated an interesting neuroprotective profile on in vitro models of neurodegeneration. Finally, compound 4g revokes cellular death induced by tau hyperphosphorylation in hippocampal slices by blocking reactive oxygen species (ROS) production. In conclusion, the multitarget profile exhibited by these compounds is a novel therapeutic strategy of potential interest in the search of novel treatments for Alzheimer&rsquo, s disease.

7 p.-2 fig., Leucine-rich repeat kinase 2 (LRRK2) is a protein kinase whose activity plays an important role in neurodegenerative diseases. Although mutations in LRRK2 gene are the most common cause of monogenic Parkinson's disease, it has been reported that LRRK2 may promote Tau phosphorylation, increasing its aggregation. Thus, the modulation of LRRK2 activity by small molecules able to inhibit this kinase activity could be an innovative therapeutic strategy for different tauopathies. We examined the therapeutic effects of a new benzothiazole-based LRRK2 inhibitor, known as JZ1.40, in a mouse model of tauopathy. Mice were injected in the right hippocampus with an adeno-associated vector expressing human-TAUP301L and treated daily with JZ1.40 (10 mg/kg, i.p) or vehicle for three weeks. JZ1.40 reaches the brain and modulates RAB10 and Tau phosphorylation at the epitopes modified by LRRK2. Moreover, JZ1.40 treatment ameliorates the cognitive impairment induced by TAUP301L overexpression, which correlates with prevention of granular cell layer degeneration by improving synaptic plasticity. These data show that JZ1.40 is neuroprotective in vivo, which is translated into cognition enhancement., This work was supported by MINECO (Competitiveness Grants SAF2016-76520-R to ILB, SAF2016-76693-R to AM, and RTI2018-095793-B-I00 to MGL), MECD (FPU grant FPU13-003262 to JZD), SCS is beneficiary of the call for predoctoral contracts for the training of research personnel 2018 (FPI-UAM) and Fundación Tatiana Pérez de Guzmán el Bueno to EL.

New multi-target indole and naphthalene derivatives containing the oxadiazolone scaffold as a bioisostere of the melatonin acetamido group have been developed. The novel compounds were characterized at melatonin receptors MT1R and MT2R, quinone reductase 2 (QR2), lipoxygenase-5 (LOX-5), and monoamine oxidases (MAO-A and MAO-B), and also as radical scavengers. We found that selectivity within the oxadiazolone series can be modulated by modifying the side chain functionality and coplanarity with the indole or naphthalene ring. In phenotypic assays, several oxadiazolone-based derivatives induced signalling mediated by the transcription factor NRF2 and promoted the maturation of neural stem-cells into a neuronal phenotype. Activation of NRF2 could be due to the binding of indole derivatives to KEAP1, as deduced from surface plasmon resonance (SPR) experiments. Molecular modelling studies using the crystal structures of QR2 and the KEAP1 Kelch-domain, as well as the recently described X-ray free-electron laser (XFEL) structures of chimeric MT1R and MT2R, provided a rationale for the experimental data and afforded valuable insights for future drug design endeavours, The authors gratefully acknowledge the following financial supports: Spanish Ministry of Science, Innovation and Universities; Spanish Research Agency; and European Regional Development Funds (grants RTI2018-093955-B-C21 and SAF2015-64948-C2-1-R to M.I.R.-F.; RTI2018-095793-B-I00 to M.G.L., SAF2015-64629-C2- 2-R to F.G.), General Council for Research and Innovation of the Community of Madrid and European Structural Funds (grant B2017/BMD-3827 e NRF24ADCM), Health Institute Carlos III (Miguel Servet II ProgramCP16/00014 and grant PI17/01700 to R.L.). CH-A and P.M. thank their PhD fellowships from Spanish Ministry of Education (MEC, PhD grant FPU16/01704 and mobility grant FPUEST17/00233 to CH-A and FPU13/03737 to P.M.).

Chronic neuroinflammation is a hallmark of the onset and progression of brain proteinopathies such as Alzheimer disease (AD) and it is suspected to participate in the neurodegenerative process. Transcription factor NRF2, a master regulator of redox homeostasis, controls acute inflammation but its relevance in low-grade chronic inflammation of AD is inconclusive due to lack of good mouse models. We have addressed this question in a transgenic mouse that combines amyloidopathy and tauopathy with either wild type (AT-NRF2-WT) or NRF2-deficiency (AT-NRF2-KO). AT-NRF2-WT mice died prematurely, at around 14 months of age, due to motor deficits and a terminal spinal deformity but AT-NRF2-KO mice died roughly 2 months earlier. NRF2-deficiency correlated with exacerbated astrogliosis and microgliosis, as determined by an increase in GFAP, IBA1 and CD11b levels. The immunomodulatory molecule dimethyl fumarate (DMF), a drug already used for the treatment of multiple sclerosis whose main target is accepted to be NRF2, was tested in this preclinical model. Daily oral gavage of DMF during six weeks reduced glial and inflammatory markers and improved cognition and motor complications in the AT-NRF2-WT mice compared with the vehicle-treated animals. This study demonstrates the relevance of the inflammatory response in experimental AD, tightly regulated by NRF2 activity, and provides a new strategy to fight AD., This work was funded by SAF2016-76520-R of the Spanish Ministry of Economy and Competitiveness, a Pathfinder grant of the Centres of Excellence in Neurodegeneration (COEN) of the EU-Joint Program for Neurodegenerative diseases, and European Regional Development Fund, Competitiveness Operational Program 2014–2020, through the grant P_37_732/2016 REDBRAIN and a collaborative CIBERNED project PI2017/04-3. M.P. is recipient of a FPU fellowship of Autonomous University of Madrid.

Human erythropoietin is mainly recognized for its hematopoietic function; however, by binding to its receptor (EpoR), it can activate different signaling pathways as STAT, PI3K, MAPK and RAS to increase cellular differentiation or provide neuroprotective effects, among others. A recombinant human erythropoietin variant with low glycosylation and without hematopoietic effect (EpoL) was purified from skimmed goat milk. Recombinant human erythropoietin (Epo) was obtained from CHO cell line and used as control to compare EpoL effects. Neuroprotection studies were performed in PC12 cells and rat hippocampal slices. Cells were pretreated during 1Â h with EpoL or Epo and exposed to oxidative agents (H2O2 or FCCP); cell viability was assayed at the end of the experiment by the MTT method. Hippocampal slices were exposed to 15Â min of oxygen and glucose deprivation (OGD) and the neuroprotective drugs EpoL or Epo were incubated for 2Â h post-OGD in re-oxygenated medium. Cell cultures stressed with oxidative agents, and pretreated with EpoL, showed neuroprotective effects of 30% at a concentration 10 times lower than that of Epo. Moreover, similar differences were observed in OGD ex vivo assays. Neuroprotection elicited by EpoL was lost when an antibody against EpoR was present, indicating that its effect is EpoR-dependent. In conclusion, our results suggest that EpoL has a more potent neuroprotective profile than Epo against oxidative stress, mediated by activation of EpoR, thus EpoL represents an important target to develop a potential biopharmaceutical to treat different central nervous system pathologies related to oxidative stress such as stroke or neurodegenerative diseases. Keywords: Erythropoietin, Erythropoietin receptor, Neuroprotection, Oxidative stress

Background and aims Saffron is an antioxidant herbal derivative; however, its efficacy as a nutritional cardioprotective agent has not been fully elucidated. We investigated the cardioprotective properties of a standardized saffron aqueous extract (SFE) against ischemia/reperfusion (I/R) injury in Wild-Type (WT) and ApoE (−/−) mice and the underlying molecular mechanisms. Methods and results WT and ApoE (−/−) mice were subjected to 30 min I and 2 h R, with the following per os interventions for 4 weeks: 1) WT Control Group, receiving Water for Injection (WFI); 2) WT Crocus Group, receiving SFE at a dose of 60 mg/kg/day; 3) WT Crocus + Wort group, receiving SFE as described above and wortmannin at a dose of 60 μg/kg bolus 15 min before R; 4) ApoE (−/−) Control Group, receiving WFI; 5) ApoE (−/−) Crocus Group, receiving SFE at a dose of 60 mg/kg/day and 6) ApoE (−/−) Crocus + Wort: receiving SFE as described above and wortmannin at a dose of 60 μg/kg bolus, 15 min before R. Ischemic area/area at risk (I/R%) ratio was measured. Blood samples and ischemic myocardial tissue were collected at the 10th min of reperfusion for assessment of troponin I, malondialdehyde (MDA), nitrotyrosine (NT), p-eNOS, eNOS, p-Akt, Akt, p-p42/p-p44, p-GSK3β, GSK3β, IL-6, Nrf2, HO-1 and MnSOD expression. The effect of SFE on Nrf2 expression was also evaluated in vitro . SFE reduced infarct size in WT (16.15 ± 3.7% vs 41.57 ± 2.48%, ***p (−/−) mice (16.14 ± 1.47% vs 45.57 ± 1.73%, ***p (−/−) mice). Mice receiving SFE showed increased levels of eNOS, p-Akt, p-ERK1/2, p-44/p-42 and p-GSK3β-Ser9 and reduced expression of IL-6 and iNOS; furthermore, SFE reduced the levels of MDA and NT. SFE induced Nrf2 expression and its downstream targets, HO-1 and MnSOD in the myocardium of the treated animals, and induced Nrf2 expression in vitro in a dose-dependent manner. Conclusions SFE limits myocardial infarction in Wild-Type and ApoE (−/−) mice in a multifaceted manner including activation of Akt/eNOS/ERK1/2/GSK3-β and through Nrf2 pathway, bestowing antioxidant protection against I/R.

In this review, we show how chromaffin cells have contributed to evaluate neuroprotective compounds with diverse mechanisms of action. Chromaffin cells are considered paraneurons, as they share many common features with neurons: (i) they synthesize, store, and release neurotransmitters upon stimulation and (ii) they express voltage-dependent calcium, sodium, and potassium channels, in addition to a wide variety of receptors. All these characteristics, together with the fact that primary cultures from bovine adrenal glands or chromaffin cells from the tumor pheochromocytoma cell line PC12 are easy to culture, make them an ideal model to study neurotoxic mechanisms and neuroprotective drugs. In the first part of this review, we will analyze the different cytotoxicity models related to calcium dyshomeostasis and neurodegenerative disorders like Alzheimer's or Parkinson's. Along the second part of the review, we describe how different classes of drugs have been evaluated in chromaffin cells to determine their neuroprotective profile in different neurodegenerative-related models.

Folic acid (folate) is a vitamin of the B-complex group crucial for neurological function. Considering that excitotoxicity and cell death induced by glutamate are involved in many disorders, the potential protective effect of folic acid on glutamate-induced cell damage in rat hippocampal slices and the possible intracellular signaling pathway involved in such effect were investigated. The treatment of hippocampal slices with folic acid (100 μM) significantly abrogated glutamate (1 mM)-induced reduction of cell viability measured by MTT reduction assay and inhibited glutamate-induced D-[3H]-aspartate release. To investigate the putative intracellular signaling pathways implicated in the protective effect of folic acid, we used a PI3K inhibitor, LY294002, which abolished the protective effects of folic acid against glutamate-induced cell damage and D-[3H] aspartate release. Moreover, hippocampal slices incubated with folic acid alone for 30 min presented increased phosphorylation of GSK-3β at Ser9, indicating an inhibition of the activity of this enzyme. Furthermore, folic acid in the presence of glutamate insult in hippocampal slices maintained for an additional period of 6 h in fresh culture medium without glutamate and/or folic acid induced phosphorylation of GSK-3β and β-catenin expression. In addition, glutamate-treated hippocampal slices showed increased iNOS expression that was reversed by folic acid. In conclusion, the results of this study show that the protective effect of folic acid against glutamate-induced excitotoxicity may involve the modulation of PI3K/GSK-3β/β-catenin pathway and iNOS inhibition.

Neuronal hippocampal death can be induced by exacerbated levels of cortisol, a condition usually observed in patients with Major depressive disorder (MDD). Previous in vitro and in vivo studies showed that ursolic acid (UA) elicits antidepressant and neuroprotective properties. However, the protective effects of UA against glucocorticoid-induced cytotoxicity have never been addressed. Using an in vitro model of hippocampal cellular death induced by elevated levels of corticosterone, we investigated if UA prevents corticosterone-induced cytotoxicity in HT22 mouse hippocampal derived cells. Concentrations lower than 25 µM UA did not alter cell viability. Co-incubation with UA for 48 h was able to protect HT22 cells from the reduction on cell viability and from the increase in apoptotic cells induced by corticosterone. Inhibition of protein kinase A (PKA), protein kinase C (PKC) and, Ca2+/calmodulin-dependent protein kinase II (CaMKII), but not phosphoinositide 3-kinase(PI3K), by using the pharmacological the inhibitors: H-89, chelerythrine, KN-62, and LY294002, respectively totally abolished the cytoprotective effects of UA. Finally, UA abrogated the reduction in phospho-extracellular signal–regulated kinases 1 and 2 (ERK1/2) but not in phospho-c-Jun kinases induced by corticosterone. These results indicate that the protective effect of UA against the cytotoxicity induced by corticosterone in HT22 cells may involve PKA, PKC, CaMKII, and ERK1/2 activation. The cytoprotective potential of UA against corticosterone-induced cytotoxicity and its ability to modulate intracellular signaling pathways involved in cell proliferation and survival suggest that UA may be a relevant strategy to manage stress-related disorders such as MDD.

Significance Current one drug–one target–one disease approaches in drug discovery have become increasingly inefficient. Network pharmacology defines disease mechanisms as networks best targeted by multiple, synergistic drugs. Using the high unmet medical need indication stroke, we here develop an integrative in silico approach based on a primary target, NADPH oxidase type 4, to identify a mechanistically related cotarget, NO synthase, for network pharmacology. Indeed, we validate both in vivo and in vitro, including humans, that both NOX4 and NOS inhibition is highly synergistic, leading to a significant reduction of infarct volume, direct neuroprotection, and blood–brain-barrier stabilization. This systems medicine approach provides a ground plan to decrease current failure in the field by being implemented in other complex indications., Drug discovery faces an efficacy crisis to which ineffective mainly single-target and symptom-based rather than mechanistic approaches have contributed. We here explore a mechanism-based disease definition for network pharmacology. Beginning with a primary causal target, we extend this to a second using guilt-by-association analysis. We then validate our prediction and explore synergy using both cellular in vitro and mouse in vivo models. As a disease model we chose ischemic stroke, one of the highest unmet medical need indications in medicine, and reactive oxygen species forming NADPH oxidase type 4 (Nox4) as a primary causal therapeutic target. For network analysis, we use classical protein–protein interactions but also metabolite-dependent interactions. Based on this protein–metabolite network, we conduct a gene ontology-based semantic similarity ranking to find suitable synergistic cotargets for network pharmacology. We identify the nitric oxide synthase (Nos1 to 3) gene family as the closest target to Nox4. Indeed, when combining a NOS and a NOX inhibitor at subthreshold concentrations, we observe pharmacological synergy as evidenced by reduced cell death, reduced infarct size, stabilized blood–brain barrier, reduced reoxygenation-induced leakage, and preserved neuromotor function, all in a supraadditive manner. Thus, protein–metabolite network analysis, for example guilt by association, can predict and pair synergistic mechanistic disease targets for systems medicine-driven network pharmacology. Such approaches may in the future reduce the risk of failure in single-target and symptom-based drug discovery and therapy.

Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic option. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier. Its mechanism, however, is unknown. Here, we identified type 5 NADPH oxidase (NOX5), a calciumactivated, ROS-forming enzyme, as the missing link. Using a humanized knockin (KI) mouse model and in vitro organotypic cultures, we found that reoxygenation or calcium overload increased brain ROS levels in a NOX5-dependent manner. In vivo, postischemic ROS formation, infarct volume, and functional outcomes were worsened in NOX5-KI mice. Of clinical and therapeutic relevance, in a human blood-barrier model, pharmacological NOX inhibition also prevented acute reoxygenationinduced leakage. Our data support further evaluation of poststroke recanalization in the presence of NOX inhibition for limiting stroke-induced damage.

Acetylcholine α7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain as well as in neuropsychiatric, neurodegenerative, and inflammatory processes. Positive allosteric modulators (PAMs) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here, we report the preparation and biological activity of a fluoro-containing compound, 1-(2′,5′-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the α7 receptors and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in Alzheimer's disease (AD)-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aβ, with cell death almost completely prevented at 10 and 30 μM, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the complete Freund's adjuvant (CFA)-induced paw inflammation model after intraperitoneal administration., This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (MINECO-FEDER) grant number SAF2015-66275-C2-R and RTI2018-097189-C2 to RGM and AFM, and RTI2018-095793-B-I00 to MGL, the Comunidad de Madrid/European Union Ref S2017/BMD-3827 to MGL and the CSIC, grant number 201980E030 to RGM.

Erythropoietin is a glycoproteic hormone that regulates hematopoiesis by acting on its specific receptor (EpoR). The expression of EpoR in the central nervous system (CNS) suggests a role for this hormone in the brain. Recently, we developed a new Epo variant without hematopoietic activity called EpoL, which showed marked neuroprotective effects against oxidative stress in brain ischemia related models. In this study, we have evaluated the neuroprotective effects of EpoL against oxidative stress induced by chronic treatment with Aβ. Our results show that EpoL was neuroprotective against Aβ-induced toxicity by a mechanism that implicates EpoR, reduction in reactive oxygen species, and reduction in astrogliosis. Furthermore, EpoL treatment improved calcium handling and SV2 levels. Interestingly, the neuroprotective effect of EpoL against oxidative stress induced by chronic Aβ treatment was achieved at a concentration 10 times lower than that of Epo. In conclusion, EpoL, a new variant of Epo without hematopoietic activity, is of potential interest for the treatment of diseases related to oxidative stress in the CNS such as Alzheimer disease., Grant Innova-Corfo “13IDL2-18688”; Conicyt Grant “Beca Doctorado Nacional no. 21130386”; Fondecyt Grant “1161078”

This review describes recent developments in the search for effective therapeutic agents that target redox homeostasis in neurodegenerative disease. The disruption to thiol redox homeostasis in Alz ...

Agmatine, an endogenous neuromodulator, is a potential candidate to constitute an adjuvant/monotherapy for the management of depression. A recent study by our group demonstrated that agmatine induces Nrf2 and protects against corticosterone effects in a hippocampal neuronal cell line. The present study is an extension of this previous study by assessing the antidepressant-like effect of agmatine in an animal model of depression induced by corticosterone in mice. Swiss mice were treated simultaneously with agmatine or imipramine at a dose of 0.1 mg/kg/day (p.o.) and corticosterone for 21 days and the daily administrations of experimental drugs were given immediately prior to corticosterone (20 mg/kg/day, p.o.) administrations. Wild-type C57BL/6 mice (Nrf2 (+/+)) and Nrf2 KO (Nrf2 (−/−)) were treated during 21 days with agmatine (0.1 mg/kg/day, p.o.) or vehicle. Twenty-four hours after the last treatments, the behavioral tests and biochemical assays were performed. Agmatine treatment for 21 days was able to abolish the corticosterone-induced depressive-like behavior and the alterations in the immunocontent of mature BDNF and synaptotagmin I, and in the serotonin and glutamate levels. Agmatine also abolished the corticosterone-induced changes in the morphology of astrocytes and microglia in CA1 region of hippocampus. In addition, agmatine treatment in control mice increased noradrenaline, serotonin, and dopamine levels, CREB phosphorylation, mature BDNF and synaptotagmin I immunocontents, and reduced pro-BDNF immunocontent in the hippocampus. Agmatine’s ability to produce an antidepressant-like effect was abolished in Nrf2 (−/−) mice. The present results reinforce the participation of Nrf2 in the antidepressant-like effect produced by agmatine and expand literature data concerning its mechanisms of action., This work was supported by CNPq, CAPES/PDSE, CAPES/PROCAD, Núcleo de Excelência em Neurociências Aplicadas de Santa Catarina (NENASC) Project/ PRONEX Program CNPq/FAPESC (Brazil) to ALSR. The 8th Convocatoria de proyectos de Cooperación Interuniversitaria UAM-Santander con America Latina, Spanish Ministry of Economy and Competence Ref. SAF2012-32223 to MGL and ALSR. This work was also supported by FIS CA12/00122 to ARN and FPU12/02220 to AW.

Neuroinflammation and oxidative stress are being recognized as characteristic hallmarks in many neurodegenerative diseases, especially those that portray proteinopathy, such as Alzheimer&rsquo, s disease (AD). Heme-oxygenase 1 (HO-1) is an inducible enzyme with antioxidant and anti-inflammatory properties, while microglia are the immune cells in the central nervous system. To elucidate the brain expression profile of microglial HO-1 in aging and AD-progression, we have used the 5xFAD (five familial AD mutations) mouse model of AD and their littermates at different ages (four, eight, 12, and 18 months). Total brain expression of HO-1 was increased with aging and such increase was even higher in 5xFAD animals. In co-localization studies, HO-1 expression was mainly found in microglia vs. other brain cells. The percentage of microglial cells expressing HO-1 and the amount of HO-1 expressed within microglia increased progressively with aging. Furthermore, this upregulation was increased by 2&ndash, 3-fold in the elder 5xFAD mice. In addition, microglia overexpressing HO-1 was predominately found surrounding beta-amyloid plaques. These results were corroborated using postmortem brain samples from AD patients, where microglial HO-1 was found up-regulated in comparison to brain samples from aged matched non-demented patients. This study demonstrates that microglial HO-1 expression increases with aging and especially with AD progression, highlighting HO-1 as a potential biomarker or therapeutic target for AD.

Glycine-proline-glutamate (GPE) is a cleaved tripeptide of IGF-I that can be processed to cycloprolylglycine (cPG) in the brain. IGF-I protects the hippocampal somatostatinergic system from β-amyloid (Aβ) insult and although neither IGF-I-derived peptides bind to IGF-I receptors, they exert protective actions in several neurological disorders. As their effects on the hippocampal somatostatinergic system remain unknown, the objective of this study was to evaluate if cPG and/or GPE prevent the deleterious effects of Aβ25-35 infusion on this system and whether changes in intracellular-related signaling and interleukin (IL) content are involved in their protective effect. We also determined the effect of cPG or GPE co-administration with Aβ25-35 on IL secretion in glial cultures and the influence of these ILs on signaling activation and somatostatin synthesis in neuronal cultures. cPG or GPE co-administration reduced Aβ-induced cell death and pro-inflammatory ILs, increased IL-4 and partially avoided the reduction of components of the somatostatinergic system affected by Aβ25-35. GPE increased activation of Akt and CREB and reduced GSK3β activation and astrogliosis, whereas cPG increased phosphorylation of extracellular signal-regulated kinases. Both peptides converged in the activation of mTOR and S6 kinase. Co-administration of these peptides with Aβ25-35 to glial cultures increased IL-4 and reduced IL-1β; this release of IL-4 could be responsible for activation of Akt and increased somatostatin in neuronal cultures. Our findings suggest that cPG and GPE exert protective effects against Aβ on the somatostatinergic system by a reduction of the inflammatory environment that may activate different pro-survival pathways in these neurons.

All metazoans depend on O2delivery and consumption by the mitochondrial oxidative phosphorylation (OXPHOS) system to produce energy. A decrease in O2availability (hypoxia) leads to profound metabolic rewiring. In addition, OXPHOS uses O2to produce reactive oxygen species (ROS) that can drive cell adaptations through redox signalling, but also trigger cell damage1–4, and both phenomena occur in hypoxia4–8. However, the precise mechanism by which acute hypoxia triggers mitochondrial ROS production is still unknown. Ca2+is one of the best known examples of an ion acting as a second messenger9, yet the role ascribed to Na+is to serve as a mere mediator of membrane potential and collaborating in ion transport10. Here we show that Na+acts as a second messenger regulating OXPHOS function and ROS production by modulating fluidity of the inner mitochondrial membrane (IMM). We found that a conformational shift in mitochondrial complex I during acute hypoxia11drives the acidification of the matrix and solubilization of calcium phosphate precipitates. The concomitant increase in matrix free-Ca2+activates the mitochondrial Na+/Ca2+exchanger (NCLX), which imports Na+into the matrix. Na+interacts with phospholipids reducing IMM fluidity and mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III, generating a redox signal. Inhibition of mitochondrial Na+import through NCLX is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+import into the mitochondrial matrix controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences in cellular metabolism.

Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic options. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain-barrier. Its mechanism, however, is unknown. Here we identify type 5 NADPH oxidase (NOX5), a calcium-activated, reactive oxygen species (ROS)-forming enzyme as missing link. Using a humanised knock-in mouse model and in vitro in organotypic cultures, we find re-oxygenation or calcium overload to increase brain ROS levels in a NOX5-dependent manner. In vivo, post-ischemic ROS formation, infarct volume and functional outcomes were worsened in NOX5 knock-in mice. Of clinical and therapeutic relevance, in a human blood-barrier model pharmacological NOX inhibition also prevented acute re-oxygenation induced leakage. Our data therefore identify NOX5 as sufficient to induce acute post-reperfusion calcium-dependent blood-brain-barrier breakdown. We suggest urgent clinical validation by conducting protective post-stroke re-canalisation in the presence of a NOX inhibitor.

Systems medicine has a mechanism-based rather than a symptom- or organ-based approach to disease and identifies therapeutic targets in a nonhypothesis-driven manner. In this work, we apply this to transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) by cross-validating its position in a protein-protein interaction network (the NRF2 interactome) functionally linked to cytoprotection in low-grade stress, chronic inflammation, metabolic alterations, and reactive oxygen species formation. Multiscale network analysis of these molecular profiles suggests alterations of NRF2 expression and activity as a common mechanism in a subnetwork of diseases (the NRF2 diseasome). This network joins apparently heterogeneous phenotypes such as autoimmune, respiratory, digestive, cardiovascular, metabolic, and neurodegenerative diseases, along with cancer. Importantly, this approach matches and confirms in silico several applications for NRF2-modulating drugs validated in vivo at different phases of clinical development. Pharmacologically, their profile is as diverse as electrophilic dimethyl fumarate, synthetic triterpenoids like bardoxolone methyl and sulforaphane, protein-protein or DNA-protein interaction inhibitors, and even registered drugs such as metformin and statins, which activate NRF2 and may be repurposed for indications within the NRF2 cluster of disease phenotypes. Thus, NRF2 represents one of the first targets fully embraced by classic and systems medicine approaches to facilitate both drug development and drug repurposing by focusing on a set of disease phenotypes that appear to be mechanistically linked. The resulting NRF2 drugome may therefore rapidly advance several surprising clinical options for this subset of chronic diseases., This work was supported by Grants SAF2015-71304-REDT, SAF2016-76520-R, SAF2013-4874R, SAF2015-65267-R, and BIO2014-57518 of the Spanish Ministry of Economy and Competiveness; PII4/00372 from the Health Institute Carlos III; PROMETEOII/2014/071 of Generalitat Valenciana; P_37_732/2016 REDBRAIN of the European Regional Development Fund; Competitiveness Operational Program 2014-2020; and the ERC Advanced Grant RadMed 294683 and COST action 15120 OpenMultiMed (H.H.H.W.S.). M.P. is the recipient of a FPU fellowship of Autonomous University of Madrid. E.G. is supported by a European-cofunded Beatriu de Pinos fellowship. R.L. is supproted by the Miguel Servet II fellow (CPII16/00014).

Network medicine utilizes common genetic origins, markers and co-morbidities to uncover mechanistic links between diseases. These links can be summarized in the diseasome, a comprehensive network of disease–disease relationships and clusters. The diseasome has been influential during the past decade, although most of its links are not followed up experimentally. Here, we investigate a high prevalence unmet medical need cluster of disease phenotypes linked to cyclic GMP. Hitherto, the central cGMP-forming enzyme, soluble guanylate cyclase (sGC), has been targeted pharmacologically exclusively for smooth muscle modulation in cardiology and pulmonology. Here, we examine the disease associations of sGC in a non-hypothesis based manner in order to identify possibly previously unrecognized clinical indications. Surprisingly, we find that sGC, is closest linked to neurological disorders, an application that has so far not been explored clinically. Indeed, when investigating the neurological indication of this cluster with the highest unmet medical need, ischemic stroke, pre-clinically we find that sGC activity is virtually absent post-stroke. Conversely, a heme-free form of sGC, apo-sGC, was now the predominant isoform suggesting it may be a mechanism-based target in stroke. Indeed, this repurposing hypothesis could be validated experimentally in vivo as specific activators of apo-sGC were directly neuroprotective, reduced infarct size and increased survival. Thus, common mechanism clusters of the diseasome allow direct drug repurposing across previously unrelated disease phenotypes redefining them in a mechanism-based manner. Specifically, our example of repurposing apo-sGC activators for ischemic stroke should be urgently validated clinically as a possible first-in-class neuroprotective therapy., Systems based pharmacology: drug repurposing by diseasome Systems medicine utilizes common genetic origins and co-morbidities to uncover mechanistic links between diseases, which are summarized in the diseasome. Shared pathomechanisms may also allow for drug repurposing within these disease clusters. Here, Schmidt and co-workers show indeed that, based on this principle, a cardio-pulmonary drug can be surprisingly repurposed for a previously not recognised application as a direct neuroprotectant. They find that the cyclic GMP forming soluble guanylate cyclase becomes dysfunctional upon stroke but regains catalytic activity in the presence of specific activator compounds. This new mechanism-based therapy should be urgently validated clinically as a possible first-in-class treatment in stroke.

Growing evidence suggests a close relationship between Alzheimer's Disease (AD) and cerebral hypoxia. Astrocytes play a key role in brain homeostasis and disease states, while some of the earliest changes in AD occur in astrocytes. We have therefore investigated whether mutations associated with AD increase astrocyte vulnerability to ischemia. Two astroglioma cell lines derived from APPSWE /PS1A246E (APP, amyloid precursor protein; PS1, presenilin 1) transgenic mice and controls from normal mice were subjected to oxygen and glucose deprivation (OGD), an in vitro model of ischemia. Cell death was increased in the APPSWE /PS1A246E line compared to the control. Increasing extracellular calcium concentration ([Ca2+ ]) exacerbated cell death in the mutant but not in the control cells. In order to explore cellular Ca2+ homeostasis, the cells were challenged with ATP or thapsigargin and [Ca2+ ] was measured by fluorescence microscopy. Changes in cytosolic Ca2+ concentration ([Ca2+ ]c ) were potentiated in the APPSWE /PS1A246E transgenic line. Mitochondrial function was also altered in the APPSWE /PS1A246E astroglioma cells; mitochondrial membrane potential and production of reactive oxygen species were increased, while mitochondrial basal respiratory rate and ATP production were decreased compared to control astroglioma cells. These results suggest that AD mutations in astrocytes make them more sensitive to ischemia; Ca2+ dysregulation and mitochondrial dysfunction may contribute to this increased vulnerability. Our results also highlight the role of astrocyte dyshomeostasis in the pathophysiology of neurodegenerative brain disorders., This work was supported by the Spanish Government (partially funded by the European Union ERDF/FEDER) Ref. SAF2015-63935R to M.G.L.; Ref. SAF2015-65586 to R.M.M; Ref. PI15/ 00107 to A.M.R.; FPU/contract Ref. AP2009/0343 to A.J.M.O., the Spanish Ministry of Health (Instituto de Salud Carlos III) Ref. PI14/00372 to R.L and a grant from the Fundación Domingo Martínez (Spain) to A.M.R. and M.G.L. R.L. thanks the Instituto de Salud Carlos III and the European Regional Development’s funds (FEDER) for a research contract under the Miguel Servet II Program (CPII16/00014). We also thank the Fundación Teófilo Hernando for its continuous support.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed., Graphical abstract fx1, Highlights • RONS are chemical mediators and a communication tool. • RONS and disturbed redox balance play a role in a broad range of diseases and aging. • Bacteria and toxins are important stimulators of cellular RONS formation. • Drugs should preserve beneficial redox signaling and inhibit detrimental RONS sources. • Redox drugs may target the origin, identity, location and time of RONS formation.

Scope We have studied if curcumin can protect glial cells under an oxidative stress and inflammatory environment, which is known to be deleterious in neurodegeneration. Methods and results Primary rat glial cultures exposed to the combination of an oxidative (rotenone/oligomycin A) and a proinflammatory LPS stimuli reduced by 50% glial viability. Under these experimental conditions, curcumin afforded significant glial protection and reduction of reactive oxygen species; these effects were blocked by the HO-1 inhibitor tin protoporphyrin-IX (SnPP). These findings correlate with the observation that curcumin induced the antioxidative protein HO-1. Most interesting was the observation that the glial protective effects related to HO-1 induction were microglial specific as shown in glial cultures from LysMCreHmox∆/∆ mice where curcumin lost its protective effect. Under LPS conditions, curcumin reduced the microglial proinflammatory markers iNOS and tumor necrosis factor, but increased the anti-inflammatory cytokine IL4. Analysis of the microglial phenotype showed that curcumin favored a ramified morphology toward a microglial alternative activated state against LPS insult also by a HO-1-dependent mechanism. Conclusion The curry constituent curcumin protects glial cells and promotes a microglial anti-inflammatory phenotype by a mechanism that implicates HO-1 induction; these effects may have impact on brain protection under oxidative and inflammatory conditions.