SFRR-E ECR 1 [YIA]:
Changes in NADPH oxidase activity in E-cigarette vapor condensate exposed cultured cells and the role of acrolein
Ivana Djordjevic | Germany
Even though electronic cigarettes (Ecig) are marketed as a healthier substitute for tobacco cigarettes, evidence is arising, that Ecig vapor could cause adverse health effects. It is assumed that Ecig liquid components that are degraded thermally are the ones responsible for the observed effects on human health, with toxic aldehydes being the most prominent group. The aim of this study is to evaluate the mechanistic effects of Ecig vapor toxicity on NADPH oxidase activation in immune and vascular cells, as well as to test if acrolein, a byproduct of Ecig liquid heating, is the main malefactor. My group previously showed that Ecig vapor exposure causes oxidative stress, inflammation, apoptosis, endothelial dysfunction and high blood pressure in a mouse model, through activation of NADPH oxidase [Kuntic et al. Eur. Heart J. 2020]. To understand the mechanism of NAPDH oxidase activation better, we have now exposed cultured endothelial cells (EA.hy 925) and macrophages (RAW 264.7) to condensed Ecig vapor (EcigCon). We observed that incubation of EA.hy 925 and RAW 264.7 cells with EcigCon leads to concentration-dependent cell death. In both EA.hy 925 and RAW 264.7 cells, EcigCon incubation promoted the transfer of the cytosolic NADPH oxidase subunits (p47phox, p67phox and Rac1) to the plasma membrane, hence showing the activation of the enzyme complex. Recent studies have shown that among toxic aldehydes found in Ecig vapor, acrolein plays a prominent role. Thus, we incubated both EA.hy 925 and RAW 264.7 cells with increasing concentrations of acrolein. It was again observed that p47phox, p67phox and Rac1 translocate to the plasma membrane. These data show that acrolein could be a significant part of Ecig vapor induced oxidative stress and cell death. More understanding is still needed to disclose the full mechanism of the negative effects of consumption of Ecig and the possible long-term toxicity.
SFRR-E ECR 2 [YIA]:
Proteasome activation in C. elegans causes mild mitochondrial defects; Is this the link to lifespan extension?
Dr. Anna Gioran | National Hellenic Research Foundation | Greece
Protein homeostasis is extensively regulated by a variety of proteostatic mechanisms that are considered the guardians of the proteome and ensure precise synthesis, maintenance, function and elimination of proteins. These mechanisms tend to fail with ageing, thus contributing to loss of proteostasis, one of the ageing hallmarks. Our group and others have previously shown that activation of the proteasome, that is responsible for approximately 80% of protein degradation, can prolong the lifespan of C. elegans and D. melanogaster. However, how exactly proteasome activation facilitates lifespan extension, remains unknown. In this study, we have attempted to shed light onto this open question by focusing on the underlying molecular effects of proteasome activation. A proteomic analysis of C. elegans with an activated proteasome revealed differentially regulated glycolysis, a metabolic shift compatible with mitochondrial deficiency. Evaluation of various mitochondrial parameters showed that mitochondria were depolarized and fragmented in nematodes with an activated proteasome compared to control. Although further investigation is needed, our data may suggest that a mild mitochondrial defect accounts for the lifespan extension found in nematodes with an activated proteasome. Our future work will focus on determining how proteasome activation causes mitochondrial defects and if indeed the latter are responsible for the observed lifespan extension.
SFRR-E ECR 3 [YIA]:
Evidence of ferroptosis involvement in Rett syndrome pathogenesis
PhD student Anna Guiotto | University of Ferrara, Italy | Italy
Rett syndrome (RTT) is a rare neurodevelopmental disorder caused in 90% of the cases by mutation in the X-linked gene encoding for MeCP2, an important epigenetic regulator. RTT patients show compromised metabolic processes including redox imbalance, dysfunctional mitochondrial bioenergetics and altered lipid metabolism.
Since several molecular aspects involved in the pathophysiological mechanisms of RTT could suggest a possible role of ferroptosis, an iron-dependent cell death characterized by excessive lipid peroxidation, the aim of our study was to evaluate RTT susceptibility to this type of cell death using primary fibroblasts obtained from RTT patients.
As a first step, we observed an increase of cell death rate in RTT compared to controls after treatment with several concentrations of two ferroptosis inducers: erastin (GPX4 inhibitor) or RSL3 (inhibitor of the cystine/glutamate antiporter). At the same time, the co-treatment with ferrostatin-1, a well know inhibitor of ferroptosis, reduced the levels of cell death. In addition, we found changes in GPx and GR activity after 3h treatment with 10 μM erastin or 5 nM RSL3, while Western blot analysis also showed an alteration in GPX4 protein levels and in formation of 4HNE protein adducts, after the treatment with the same doses of erastin and RSL3 for 3 and 6h.
Finally, both the mitochondrial ROS production and lipid peroxidation levels were higher in RTT after the induction of ferroptosis with the two molecules, while ferrostatin-1 co-treatment significantly prevented these processes.
In conclusion, our results indicate an increased vulnerability of RTT cells to ferroptosis that could contribute to the clinical features of RTT phenotypes, also suggesting this process as a possible therapeutic target to improve the quality of life of RTT patients.
SFRR-E ECR 4 [YIA]:
SC-Nanophytosomes formulation promotes benefits on Parkinson’s disease models: a mitochondria-targeted therapy approach
Daniela Mendes | REQUIMTE/LAQV, Laboratory of Pharmacognosy, Department of Chemistry, Faculty of Pharmacy, University of Porto | Portugal
Mitochondrial dysfunction is a common pathological hallmark of many degenerative diseases, including Parkinson’s disease (PD). Thus, developing therapeutic strategies to modulate mitochondrial function is a great challenge. In this work, SC-Nanophytosomes, assembled with polar lipids from Codium tomentosum Stackhouse and elderberry anthocyanin-enriched extract (EAE-extract) from Sambucus nigra L, were used to address this challenge. The algae polar lipids were chosen considering their richness in the anionic phosphatidylglycerol (cardiolipin precursor) and omega-3 polyunsaturated fatty acids, while the elderberry anthocyanins were by their mitochondriotropic properties and ability to overcome the complex I-related mitochondrial dysfunction. The competence of SC-Nanophytosomes to modulate the mitochondria functionality, by improving the activity of the mitochondrial respiratory chain complexes and protecting the cells against mitochondria-specific toxic and/or oxidant stimuli (e.g., rotenone and glutamate) was unveiled in cellular assays. SC-Nanophytosomes, engineered with 600 µM algae phospholipids and 0.5 mg/L of EAE-extract, are nanosized vesicles with high negative surface charge and versatile shapes that preserve their properties under conditions that mimics the gastrointestinal tract pH changes. The oral administration of SC-Nanophytosomes (delivery by drinking water for 3 weeks at the concentration of 3 µM in phospholipid) to a rotenone-induced PD-like pathology rat model showed positive outcomes disabling motor symptoms associated with rotenone neurotoxicity. Ex vivo brain biochemical assays showed that SC-Nanophytosomes-treatment induced benefits on mitochondrial functionality, revealed by respiratory control index and by redox complexes activities. Cell redox state also shows benefits, indicated by the activity of superoxide dismutase, catalase, and glutathione reductase enzymes. While these data point out SC-Nanophytosomes as promising tool to support a mitochondria-targeted therapy for neurodegenerative diseases, additional assays with other doses and animal models are needed to evaluate their effectiveness.
ACKNOWLEDGMENTS: The work was supported by UIDB/50006/2020 with funding from FCT/MCTES. Daniela Mendes thanks FCT and ESF through POCH for her PhD grant (SFRH/BD/138206/2018).
SFRR-E ECR 5 [YIA]:
Generation of genetically-encoded redox biosensors for super-resolution imaging of hydrogen peroxide production
Dr. Brandán Pedre | KU Leuven | Belgium
In cells, hydrogen peroxide (H2O2) is deliberately produced and is key for a healthy cellular metabolism, and an adequate H2O2 production and delivery is required for correct cellular and tissue functions. The majority of this H2O2 production occurs in cellular microdomains, and the signalling actions in these microdomains are restricted to a very confined area. Most of these discoveries were made possible thanks to the use of genetically-encoded fluorescent biosensors that detect the changes in H2O2 concentration, including the HyPer family of biosensors. However, the nanoscale details of these microdomains are missing due to the spatial resolution limitation (~200nm) of conventional fluorescence microscopy methods.
To overcome this resolution limitation, we have generated HyPer variants that can be reversibly switched off and on upon illumination with cyan and violet light, respectively, while keeping their H2O2-sensing abilities. The photoswitching ability allows the use of super-resolution microscopy techniques, such as photochromic stochastic optical fluctuation imaging (pcSOFI) or reversible optically-linear fluorescence transitions (RESOLFT). Combining these techniques with the photoswitchable HyPers has enabled a high-resolution mapping of mitochondrial H2O2 production upon hypoxia-reoxygenation.
SFRR-E ECR 6 [YIA]:
Avenanthramides: antioxidant and anti-inflammatory activities from oats to human therapy
Dr. Andrea Perrelli | University of Rochester Medical Center | United States
The Avena sativa is a cereal known since antiquity as a useful grain with abundant nutritional and
health benefits. It contains distinct molecular components with high antioxidant activity, such as
tocopherols, tocotrienols, and flavonoids. Among them, avenanthramides (Avns), polyphenols found
exclusively in oats, are particularly emerging as promising therapeutic candidates for the treatment
of several human diseases. Avenanthramides are phenolic amides containing anthranilic acid and
hydroxycinnamic acid moieties, and endowed with major beneficial health properties because of their
antioxidant, anti-inflammatory, and antiproliferative effects. Several studies have clearly shown the
beneficial biological activities of avenanthramides, including analogs produced in recombinant yeast
(YAvns), with a major focus on the therapeutic potential of these secondary metabolites in the
treatment of oxidative stress-related human diseases. In particular, we have demonstrated that the
antioxidant and anti-inflammatory activities of avenanthramides were effective in rescuing major
molecular and cellular dysfunctions underlying the pathogenesis of cerebral cavernous malformation
(CCM) disease, a major cerebrovascular disorder affecting up to 0.5% of the human population.
Indeed, the treatment of cellular and animal models of CCM disease with avenanthramides has been
shown to enhance the expression level of fundamental scavengers of reactive oxygen species (ROS),
such as superoxide dismutase 2 (SOD2), through the upregulation of the master transcription factor
FoxO1, as well as to reduce the NF-κB signaling pathway. Using nanotechnology methods, we have
recently implemented these findings by developing cytocompatible nanosystems loaded with
avenanthramides, which were specifically designed for the treatment of CCM disease and
neurological comorbidities though targeted administration routes that bypass the systemic circulation,
such as the nose-to-brain delivery route. This nanomedicine approach may allow the selective
delivery of avenanthramides to the brain, thus increasing their local therapeutic effectiveness and
reducing their potential systemic side-effects, and is currently being tested in cellular and animal
models of CCM disease.
SFRR-E ECR 7 [YIA]:
Assessment of enzyme-like activity in metal-free nanomaterials
Andreia D. Veloso | Chemistry Center – Vila Real (CQ-VR), Chemistry Department, ECVA, University of Trás-os-Montes e Alto Douro, Vila Real, Portugal | Portugal
Antioxidant nanozymes are promising therapeutic tools for pathologies associated with oxidative stress. Fullerenes, carbon nanotubes, graphene, and carbon dots show the ability to mimic antioxidant enzymes, but most of these nanomaterials have low water solubility and only exhibit antioxidant enzyme-like activities after functionalization and/or doping. Electrogenerated hydrophilic carbon (EHC) nanomaterials produced from graphite in one single step may emerge as a potential alternative to overcome these limitations. This work aimed to evaluate if three sets of EHC nanomaterials, synthesized using three biological compatible buffers, namely, citrate, malate, and tartrate, could mimic the activity of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). TEM and AFM analysis demonstrated that all nanomaterials exhibit a string-assembly organization dominated by amorphous carbon nanoparticles. Cyclic voltammetry indicated that they also possess electron-donating properties in the same potential range. The SOD-like activity was assessed using the hypoxanthine-xanthine oxidase system as the source of O2•- and NBT as the detector. The results revealed that, under physiological-like aqueous buffer conditions, only EHC@malic exhibited SOD-like activity. The CAT-like ability was appraised in the presence of H2O2 following the rate of O2 formation using a Clark-type electrode system. Results showed that all nanomaterials have a semi-CAT-like activity since they can react with H2O2 without O2 generation. The POD-like activity appraised spectrophotometrically by the TMB method indicated that none of the EHC nanomaterials exhibit POD-like activity. Since EHC@malic is the only nanomaterial that exhibits enzyme mimic ability, its effects on cell viability were investigated. Results showed that EHC@malic displays no toxicity for human neuronal and keratinocyte cells as well as for mouse preadipocytes cells. Overall, despite the similar structure and redox behavior, only EHC@malic emerge as a new SOD mimic with the potential to be considered for therapeutic applications development.
Acknowledgments: FCT (SFRH/BD/138425/2018, UIDB/00616/2020, UIDP/00616/2020, UIDB/50006/2020) and NORTE2020 (project OBTAIN, NORTE-01-0145-FEDER-000084).
SFRR-E ECR 8 [YIA]:
Regulation of metastasis suppressor NME1 by a key metabolic cofactor coenzyme A
Bess Yi Kun Yu | University College London | United Kingdom
The metastasis suppressor protein NME1 is an evolutionarily conserved and multifunctional enzyme that plays an important role in suppressing the invasion and metastasis of tumour cells. The nucleoside diphosphate kinase (NDPK) activity of NME1 is well recognized in balancing the intracellular pools of nucleotide diphosphates and triphosphates to regulate cytoskeletal rearrangement and cell motility, endocytosis, intracellular trafficking, and metastasis. In addition, NME1 was found to function as a protein-histidine kinase, 3’-5’ exonuclease and geranyl/farnesyl pyrophosphate kinase. These diverse cellular functions are regulated at the level of expression, post-translational modification, and regulatory interactions. The NDPK activity of NME1 has been shown to be inhibited in vitro and in vivo under oxidative stress, and the inhibitory effect mediated via redox-sensitive cysteine residues. In this study, affinity purification followed by mass spectrometric analysis revealed NME1 to be a major coenzyme A binding protein in cultured cells and rat tissues. NME1 is also found covalently modified by CoA (CoAlation) at Cys109 in the CoAlome analysis of HEK293/Pank1β cells treated with the disulfide-stress inducer, diamide. Further analysis showed that recombinant NME1 is efficiently CoAlated in vitro and in cellular response to oxidising agents and metabolic stress. In vitro CoAlation of recombinant wild type NME1, but not the C109A mutant, results in the inhibition of its NDPK activity. Moreover, CoA also functions as a competitive inhibitor of the NME1 NDPK activity by binding non-covalently to the nucleotide binding site. Taken together, our data reveal metastasis suppressor protein NME1 as a novel binding partner of the key metabolic regulator CoA, which inhibits its nucleoside diphosphate kinase activity via non-covalent and covalent interactions. Further studies on the functional modulation of NME1 by CoA will be presented.