Neuroprotective effects of fluorophore-labelled manganese complexes: Determination of ROS production, mitochondrial membrane potential and confocal fluorescence microscopy studies in neuroblastoma cells

In this work, four manganese(II) complexes derived from the ligands H2L1-H2L4, that incorporate dansyl or tosyl fluorescent dyes, have been investigated in term of their antioxidant properties. Two of the manganese(II) complexes have been newly prepared using the asymmetric half-salen ligand H2L2 and the thiosemicarbazone ligand H2L3. The four organic strands and the manganese complexes have been characterized by different analytical and spectroscopic techniques. The study of the antioxidant behaviour of these two new complexes and other two fluorophore-labelled analogues was tested in SH-SY5Y neuroblastoma cells. These four model complexes 1–4 were found to protect cells from oxidative damage in this human neuronal model, by reducing the release of reactive oxygen species. Complexes 1–4 significantly improved cell survival, with levels between 79.1 ±0.8% and 130.9 ±4.1%. Moreover, complexes 3 and 4 were able to restore the mitochondrial membrane potential at 1 μM, with 4 reaching levels higher than 85%, similar to the percentages obtained by the positive control agent cyclosporin A. The incorporation of the fluorescent label in the complexes allowed the study of their ability to enter the human neuroblastoma cells by confocal microscopy.The research leading to these results has received funding from the following FEDER cofunded-grants. From Consellería de Cultura, Educaci´ on e Ordenaci´ on Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01), 2018 GRC GI-1584 (ED431C 2018/13), MetalBIO Network (ED431D 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad IISCIII/PI19/001248. From Ministerio de Ciencia, Innovaci´ on y Universidades, MULTIMETDRUGS (RED2018-102471-T). From European Union, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA- 998-2018, and H2020 778069-EMERTOX.S

Anhydroexfoliamycin, a Streptomyces Secondary Metabolite, Mitigates Microglia-Driven Inflammation

Anhydroexfoliamycin, a secondary metabolite from Streptomyces, has shown antioxidant properties in primary cortical neurons reducing neurodegenerative hallmarks diseases, both in vitro and in vivo models. Activated microglia, in the central nervous system, plays a crucial role in neuroinflammation and is associated with neurodegeneration. Therefore, the aim of the present study was to determine the anti-inflammatory and antioxidant potential of the anhydroexfoliamycin over microglia BV2 cells. Neuroinflammation was simulated by incubation of microglia cells in the presence of lipopolysaccharide to activate proinflammatory transduction pathways. Moreover, a coculture of neuron SH-SY5Y and microglia BV2 cells was used to evaluate the neuroprotective properties of the Streptomyces metabolite. When microglia cells were preincubated with anhydroexfoliamycin, proinflammatory pathways, such as the translocation of the nuclear factor κB, the phosphorylation of c-Jun N-terminal kinase, and the inducible nitric oxide synthase expression, were inhibited. In addition, intracellular reactive oxygen species generation and the liberation of nitric oxide, interleukin 6, and tumor necrosis factor α were also decreased. Besides, the Streptomyces-derived compound showed antioxidant properties promoting the translocation of the factor erythroid 2-related factor 2 and protecting the SH-SY5Y cells from the neurotoxic mediators released by activated microglia. The effects of this compound were at the same level as the immunosuppressive drug cyclosporine A. Therefore, these results indicate that anhydroexfoliamycin is a promising tool to control microglia-driven inflammation with therapeutic potential in neuroinflammationThe research leading to these results has received funding from the following FEDER cofunded grants: Conselleria de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (Grant ED431C 2017/01); Ministerio de Ciencia e Innovación Grants ISCIII/PI16/01830 and ISCIII/PI19/01248; European Union Interreg AlertoxNet Grant EAPA-317-2016, Interreg Agritox Grant EAPA-998-2018, and Grant H2020 778069-EMERTOX. S.G. was supported by a fellowship from FIDIS, SpainS

Neuroprotective effects of fluorophore-labelled manganese complexes: determination of ROS production, mitochondrial membrane potential and confocal fluorescence microscopy studies in neuroblastoma cells

In this work, four manganese(II) complexes derived from the ligands H2L1-H2L4, that incorporate dansyl or tosyl fluorescent dyes, have been investigated in term of their antioxidant properties. Two of the manganese(II) complexes have been newly prepared using the asymmetric half-salen ligand H2L2 and the thiosemicarbazone ligand H2L3. The four organic strands and the manganese complexes have been characterized by different analytical and spectroscopic techniques. The study of the antioxidant behaviour of these two new complexes and other two fluorophore-labelled analogues was tested in SH-SY5Y neuroblastoma cells. These four model complexes 1–4 were found to protect cells from oxidative damage in this human neuronal model, by reducing the release of reactive oxygen species. Complexes 1–4 significantly improved cell survival, with levels between 79.1 ± 0.8% and 130.9 ± 4.1%. Moreover, complexes 3 and 4 were able to restore the mitochondrial membrane potential at 1 μM, with 4 reaching levels higher than 85%, similar to the percentages obtained by the positive control agent cyclosporin A. The incorporation of the fluorescent label in the complexes allowed the study of their ability to enter the human neuroblastoma cells by confocal microscopyThe research leading to these results has received funding from the following FEDER cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01), 2018 GRC GI-1584 (ED431C 2018/13), MetalBIO Network (ED431D 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad IISCIII/PI19/001248. From Ministerio de Ciencia, Innovación y Universidades, MULTIMETDRUGS (RED2018-102471-T). From European Union, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOXS

Cyclophilins A, B, and C Role in Human T Lymphocytes Upon Inflammatory Conditions

Cyclophilins (Cyps) are a group of peptidyl-prolyl cis/trans isomerases that play crucial roles in regulatory mechanisms of cellular physiology and pathology in several inflammatory conditions. Their receptor, CD147, also participates in the development and progression of the inflammatory response. Nevertheless, the main function of Cyps and their receptor are yet to be deciphered. The release of CypA and the expression of the CD147 receptor in activated T lymphocytes were already described, however, no data are available about other Cyps in these cells. Therefore, in the present work intra and extracellular CypA, B and C levels were measured followed by induced inflammatory conditions. After activation of T lymphocytes by incubation with concanavalin A, both intra and extracellular Cyps levels and the CD147 membrane receptor expression were increased leading to cell migration towards circulating CypA and CypB as chemoattractants. When CypA was modulated by natural and synthetic compounds, the inflammatory cascade was avoided including T cell migration. Our results strengthen the relationship between CypA, B, and C, their receptor, and the inflammatory process in human T lymphocytes, associating CypC with these cells for the first timeThe research leading to these results has received funding from the following FEDER cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, Spain, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, Spain, ISCIII/PI19/01248, ISCIII/PI19/00879. In European Union, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX. SG was supported by a fellowship from FIDIS, SpainS

Biological Activities of Cyclic and Acyclic B-Type Laxaphycins in SH-SY5Y Human Neuroblastoma Cells

Laxaphycins are a family of non-ribosomal lipopeptides that have been isolated from several cyanobacteria. Some of these compounds have presented cytotoxic activities, but their mechanism of action is poorly understood. In this work, the already described laxaphycins B and B3, and acyclolaxaphycins B and B3 were isolated from the marine cyanobacteria Anabaena torulosa. Moreover, two new acyclic compounds, [des-(Ala4-Hle5)] acyclolaxaphycins B and B3, were purified from the herviborous gastropod Stylocheilus striatus, with this being the first description of biotransformed laxaphycins. The structure of these new compounds was elucidated, together with the absolute configuration of acyclolaxaphycins B and B3. The bioactivities of the six peptides were determined in SH-SY5Y human neuroblastoma cells. Laxaphycins B and B3 were cytotoxic (IC50: 1.8 and 0.8 µM, respectively) through the induction of apoptosis. In comparison, acyclic laxaphycins did not show cytotoxicity but affected mitochondrial functioning, so their effect on autophagy-related protein expression was analyzed, finding that acyclic peptides affected this process by increasing AMPK phosphorylation and inhibiting mTOR. This work confirms the pro-apoptotic properties of cyclic laxaphycins B and is the first report indicating the effects on autophagy of their acyclic analogs. Moreover, gastropod-derived compounds presented ring opening and amino-acids deletion, a biotransformation that had not been previously describedThe research leading to these results has received funding from the following FEDER cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2, ITC-20161072. From European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOXS

Detection of mycotoxins in cheese using an optimized analytical method based on a QuEChERS extraction and UHPLC-MS/MS quantification

Mycotoxins can produce toxic effects on humans; hence, it is of high importance to determine their presence in food products. This work presents a reliable method for the quantification of 32 mycotoxins in cheese. The analysis procedure was optimized based on a QuEChERS extraction process and the ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) detection. The analysis method was validated for four cheese varieties (emmental, blue, brie and camembert) in terms of linearity, sensitivity, matrix effect, accuracy and precision. Satisfactory precision and accuracy values were achieved, with recoveries above 70% for most mycotoxins. The developed method was applied to the analysis of 38 commercial cheese samples. A high occurrence of beauvericin and enniatins were found, ranging from 31% for enniatin A to 100% for enniatin B. The ochratoxin A was detected in three samples at concentrations that may pose a risk to human healthThe research leading to these results has received funding from the following FEDER cofunded-grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01). From Ministerio de Ciencia e Innovación IISCIII/PI19/001248, PID 2020-11262RB-C21. From European Union Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX. R. A. is supported by a postdoctoral fellowship from Xunta de Galicia (ED481B-2021-038), SpainS

Caniferolide A, a Macrolide from Streptomyces caniferus, Attenuates Neuroinflammation, Oxidative Stress, Amyloid-Beta, and Tau Pathology in Vitro

The macrolide caniferolide A was isolated from extracts of a culture of the marine-derived actinomyceteStreptomyces caniferus, and its ability to ameliorate Alzheimer’s disease (AD) hallmarks was determined. The compound reducedneuroinflammatory markers in BV2 microglial cells activated with lipopolysaccharide (LPS), being able to block NFκB-p65translocation to the nucleus and to activate the Nrf2 pathway. It also produced a decrease in pro-inflammatory cytokines (IL-1β,IL-6, and TNF-α), reactive oxygen species (ROS) and nitric oxide release and inhibited iNOS, JNK, and p38 activities.Moreover, the compound blocked BACE1 activity and attenuated Aβ-activation of microglia by drastically diminishing ROSlevels. The phosphorylated state of the tau protein was evaluated in SH-SY5Y tau441 cells. Caniferolide A reduced Thr212 andSer214 phosphorylation by targeting p38 and JNK MAPK kinases. On the other side, the antioxidant properties of themacrolide were determined in an oxidative stress model with SH-SY5Y cells treated with H2O2. The compound diminishedROS levels and increased cell viability and GSH content by activating the nuclear factor Nrf2. Finally, the neuroprotectiveability of the compound was confirmed in two trans-well coculture systems with activated BV2 cells (both with LPS and Aβ)and wild type and transfected SH-SY5Y cells. The addition of caniferolide A to microglial cells produced a significant increase inthe survival of neuroblastoma in both cases. These results indicate that the compound is able to target many pathologicalmarkers of AD, suggesting that caniferolide A could be an interesting drug lead for a polypharmacological approach to theillnessThe research leading to these results has received funding from the following FEDER cofunded-grants, Consellería de Cultura, Educación e Ordenación Universitaria Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01); CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830, RTC-2016-5507-2, and ITC-20161072; and European Union POCTEP 0161-Nanoeaters-1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, H2020 778069-EMERTOX, and FP7 PharmaSea (Grant Agreement 312184).This document is the Accepted Manuscript version of a Published Work that appeared in final form in Molecular pharmaceutics, copyright © 2019 American Chemical Society, after peer review and technical editing by the publisher.S

Streptocyclinones A and B ameliorate Alzheimer's disease pathological processes in vitro

Alzheimer's disease (AD) is a pathology characterized by the abnormal accumulation of amyloid-beta (Aβ) and hyperphosphorylated tau. Oxidative stress and neuroinflammation are also strongly related to this disease. The ability of two new glycosylated angucyclinones, streptocyclinones A and B (1 and 2), isolated from Streptomyces sp to improve AD hallmarks was evaluated. Compounds were able to protect SH-SY5Y neuroblastoma cells from H2O2-induced oxidative injury by activating the nuclear factor E2-related factor (Nrf2). Their capacity to modulate neuroinflammation was tested in lipopolysaccharide-activated BV2 microglial cells. Compounds reduced the release of pro-inflammatory factors, inhibited the activation of NFκB and mitogen activated kinases (MAPK), and induced the translocation of Nrf2 to the nucleus of microglial cells. A trans-well co-culture was established to determine the effect of microglia treated with streptocyclinones on the survival of SH-SY5Y cells. The cell viability of neuroblastoma cells increased when the compounds were added to BV2 cells. SH-SY5Y-TMHT441 cells were used to determine the effect of compounds on tau phosphorylation. Both compounds reduced tau hyperphophorylation by targeting MAPK kinases. Moreover, streptocyclinone B (2) was able to inhibit the activity of β-secretase 1 and decrease the release of reactive oxygen species in BV2 cells stimulated with Aβ. With the same co-culture trans-well system, the treatment of Aβ-stimulated microglia with compound 2 augmented the viability of SH-SY5Y-TMHT441 cells. The results presented in this work provide evidences of the multitarget activities displayed by these new Streptomyces compounds, making them good candidates for further studies in the treatment of ADThe research leading to these results has received funding from the following FEDER cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2, ITC-20161072. From European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, H2020 778069-EMERTOX and FP7 PharmaSea (Grant Agreement 312184).S

Single and combined effects of regulated and emerging mycotoxins on viability and mitochondrial function of SH-SY5Y cells

Co-occurrence of emerging and regulated mycotoxins in contaminated samples has been widely documented, but studies about their combined toxicity are scarce. In this report, the regulated mycotoxins deoxynivalenol, fumonisin B1 and zearalenone, and the emerging ones enniatin A, enniatin B and beauvericin were tested in SH-SY5Y human neuroblastoma cells. Their individual and binary combined effects on cell viability and mitochondrial function were evaluated. The results with individual mycotoxins revealed that deoxynivalenol and emerging mycotoxins were the most damaging to neuronal cells, presenting IC50 values between 0.35 and 2.4 μM. Interestingly, non-regulated mycotoxins triggered apoptosis by affecting to mitochondrial membrane potential. However, when regulated and non-regulated mycotoxins were binary mixed, antagonistic effects were found in all cases. Finally, cow feed and milk extracts were analysed by UHPLC-MS/MS, detecting the presence of several mycotoxins included in this study. These extracts were tested in neuroblastoma cells, and damaging effects on cell viability were found. Although binary combinations of mycotoxins produced antagonistic effects, their mixture in natural matrixes induces greater effects than expected. Therefore, it would be interesting to explore the matrix influence on mycotoxin toxicity, and to continue studying the neurotoxic mechanism of action of emerging mycotoxins, as they could be a health hazardThe research leading to these results has received funding from the following FEDER cofunded grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From Ministerio de Ciencia e Innovación IISCIII/PI19/001248. From European Union Interreg Alertox-Net EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOXS

Enniatins A1 and B1 alter calcium homeostasis of neuronal cells leading to apoptotic death

Enniatins (ENNs) A1 and B1 are non-regulated mycotoxins produced by Fusarium spp. that commonly occur in different types of food. These toxins are cytotoxic in several cell lines, but their mechanism of action is unclear. In this study, the cytotoxic effects of ENNs A1 and B1 in SH-SY5Y human neuroblastoma cells were analysed. Moreover, to better understand their mechanism of action, mitochondrial function, reactive oxygen species (ROS) levels and calcium fluxes were monitored. ENNs A1 and B1 reduced cell viability, presenting IC50 values of 2.0 and 2.7 μM, respectively. Both toxins induced caspase-dependent apoptosis, but only ENN A1 increased ROS production. Apoptotic cell death seems to be triggered by the increase in cytosolic calcium produced by both ENNs, since the toxins altered Ca2+ homeostasis by depleting intracellular reservoirs. Finally, binary combinations of ENN A1, ENN B1, ENN A and ENN B were tested. All mixtures resulted in an antagonistic effect, with the exception of ENN A and ENN B1 combination, which produced an additive effect. The results presented in this study provide the first evidence of ENNs A1 and B1 effects on calcium fluxes, providing new insights into the mechanism of action of these mycotoxinsThe research leading to these results has received funding from the following FEDER cofunded-grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01). From Ministerio de Ciencia e Innovación IISCIII/PI19/001248 and PID 2020-11262RB-C21. From European Union Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX. R. Alvariño is supported by a postdoctoral fellowship from Xunta de Galicia (ED481B-2021-038), Spain. N.P–F. is supported by a fellowship from FIDIS, SpainS