Publications by authors named "Paulo J Oliveira"

237 Publications

Mildbr. and (Gaertn.) Dunal Extracts Decrease Doxorubicin Cytotoxicity on H9c2 Cardiomyoblasts.

Evid Based Complement Alternat Med 2021 22;2021:8858165. Epub 2021 Feb 22.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech Building, Lot 8A, Biocant Park, Cantanhede 3060-197, Portugal.

Materials And Methods: Bark extracts of these plants (1 and 25 g/mL) were added 3 hours before coincubating H9c2 cardiomyoblasts with Dox (0.5 and 1 M) for 24 hours more. We measured cell mass and metabolic viability, mitochondrial transmembrane potential, superoxide anion content, and activity-like of caspase-3 and caspase-9 following treatment with the extracts and/or Dox. Also, selenium and vitamin C contents were measured in the plant extracts.

Results: The results confirmed that Dox treatment decreased cell mass, mitochondrial membrane potential and metabolic viability, increased mitochondrial superoxide anion, and stimulated caspase-3 and caspase-9-like activities. Pretreatment of the cells with the plant extracts significantly inhibited Dox cytotoxicity, with more significant results at the higher concentration. Measurements of selenium and vitamin C in the extracts revealed higher concentration of both when compared with other Cameroonian spices.

Conclusion: Both extracts of and were effective against Dox-induced cytotoxicity, most likely due to their content in antioxidants.
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http://dx.doi.org/10.1155/2021/8858165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920721PMC
February 2021

Fine-Tuning the Biological Profile of Multitarget Mitochondriotropic Antioxidants for Neurodegenerative Diseases.

Antioxidants (Basel) 2021 Feb 23;10(2). Epub 2021 Feb 23.

CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal.

Neurotransmitter depletion and mitochondrial dysfunction are among the multiple pathological events that lead to neurodegeneration. Following our previous studies related with the development of multitarget mitochondriotropic antioxidants, this study aims to evaluate whether the π-system extension on the chemical scaffolds of AntiOXCIN2 and AntiOXCIN3 affects their bioactivity and safety profiles. After the synthesis of four triphenylphosphonium (TPP) conjugates (compounds -), we evaluated their antioxidant properties and their effect on neurotransmitter-metabolizing enzymes. All compounds were potent equine butyrylcholinesterase (BChE) and moderate electric eel acetylcholinesterase (AChE) inhibitors, with catechols 4 and 5 presenting lower IC values than AntiOXCIN2 and AntiOXCIN3, respectively. However, differences in the inhibition potency and selectivity of compounds - towards non-human and human cholinesterases (ChEs) were observed. Co-crystallization studies with compounds - in complex with human ChEs (ChEs) showed that these compounds exhibit different binging modes to AChE and BChE. Unlike AntiOXCINs, compounds - displayed moderate human monoamine oxidase (MAO) inhibitory activity. Moreover, compounds and presented higher ORAC-FL indexes and lower oxidation potential values than the corresponding AntiOXCINs. Catechols 4 and 5 exhibited broader safety windows in differentiated neuroblastoma cells than benzodioxole derivatives 2 and 3. Compound 4 is highlighted as a safe mitochondria-targeted antioxidant with dual ChE/MAO inhibitory activity. Overall, this work is a contribution for the development of dual therapeutic agents addressing both mitochondrial oxidative stress and neurotransmitter depletion.
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http://dx.doi.org/10.3390/antiox10020329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926627PMC
February 2021

H9c2(2-1)-based sulforhodamine B assay as a possible alternative in vitro platform to investigate effluent and metals toxicity on fish.

Chemosphere 2021 Feb 24;275:130009. Epub 2021 Feb 24.

CNC - Center for Neuroscience and Cell Biology, UC Biotech, University of Coimbra, Biocant Park, 3060-197, Cantanhede, Portugal. Electronic address:

To overcome restrictions on the use of fish in toxicity testing, the present study proposes to compare the 50% growth inhibition potential (EC) of four types of effluents on the rat cardiomyoblast H9c2(2-1) cell line by using the sulforhodamine B (SRB) cell mass colorimetric assay, with the corresponding fish lethal test results. Our objective was to evaluate if H9c2(2-1) cells shows comparable sensitivities, in both relative and absolute terms, to those provided by fish. In parallel, this study also compared the results of the chemical characterization with the legislation in force for environmental protection against effluent release into the receiving environment. Moreover, we tested the H9c2(2-1)-based SRB assays with the metals of concern found in the effluent samples. Both fish and cell assays showed the same toxicity rank for effluents: Metal > Oil > Municipal > Paper, and it should be stressed that the complementarity of using chemical and biological data represents a step forward to guarantee both environmental and human safety, since the chemical characterization showed a different toxicity rank: Metal > Municipal > Oil > Paper. Regarding metal elements, the short-term fish results showed a toxicity rank non-comparable with the rank obtained for cells. Nevertheless, the gathered results reveal the potentiality of the in vitro H9c2(2-1) platform as an alternative for fish lethal testing to assess, in absolute terms, the toxicity of effluents, particularly municipal effluents, and metals.
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http://dx.doi.org/10.1016/j.chemosphere.2021.130009DOI Listing
February 2021

MOTS-c reduces myostatin and muscle atrophy signaling.

Am J Physiol Endocrinol Metab 2021 Apr 8;320(4):E680-E690. Epub 2021 Feb 8.

The Leonard Davis School of Gerontology, University of Southern California, Los Angeles, California.

Obesity and type 2 diabetes are metabolic diseases, often associated with sarcopenia and muscle dysfunction. MOTS-c, a mitochondrial-derived peptide, acts as a systemic hormone and has been implicated in metabolic homeostasis. Although MOTS-c improves insulin sensitivity in skeletal muscle, whether MOTS-c impacts muscle atrophy is not known. Myostatin is a negative regulator of skeletal muscle mass and also one of the possible mediators of insulin resistance-induced skeletal muscle wasting. Interestingly, we found that plasma MOTS-c levels are inversely correlated with myostatin levels in human subjects. We further demonstrated that MOTS-c prevents palmitic acid-induced atrophy in differentiated C2C12 myotubes, whereas MOTS-c administration decreased myostatin levels in plasma in diet-induced obese mice. By elevating AKT phosphorylation, MOTS-c inhibits the activity of an upstream transcription factor for myostatin and other muscle wasting genes, FOXO1. MOTS-c increases mTORC2 and inhibits PTEN activity, which modulates AKT phosphorylation. Further upstream, MOTS-c increases CK2 activity, which leads to PTEN inhibition. These results suggest that through inhibition of myostatin, MOTS-c could be a potential therapy for insulin resistance-induced skeletal muscle atrophy as well as other muscle wasting phenotypes including sarcopenia. MOTS-c, a mitochondrial-derived peptide reduces high-fat-diet-induced muscle atrophy signaling by reducing myostatin expression. The CK2-PTEN-mTORC2-AKT-FOXO1 pathways play key roles in MOTS-c action on myostatin expression.
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http://dx.doi.org/10.1152/ajpendo.00275.2020DOI Listing
April 2021

Glutaminolysis is a metabolic route essential for survival and growth of prostate cancer cells and a target of 5α-dihydrotestosterone regulation.

Cell Oncol (Dordr) 2021 Apr 19;44(2):385-403. Epub 2021 Jan 19.

CICS-UBI, Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506, Covilhã, Portugal.

Purpose: Resistance to androgen-deprivation therapies and progression to so-called castrate-resistant prostate cancer (CRPC) remain challenges in prostate cancer (PCa) management and treatment. Among other alterations, CRPC has been associated with metabolic reprogramming driven by androgens. Here, we investigated the role of androgens in regulating glutaminolysis in PCa cells and determined the relevance of this metabolic route in controlling the survival and growth of androgen-sensitive (LNCaP) and CRPC (DU145 and PC3) cells.

Methods: PCa cells (LNCaP, DU145 and PC3) and 3-month old rats were treated with 5α-dihydrotestosterone (DHT). Alternatively, LNCaP cells were exposed to the glutaminase inhibitor BPTES, alone or in combination with the anti-androgen bicalutamide. Biochemical, Western blot and extracellular flux assays were used to evaluate the viability, proliferation, migration and metabolism of PCa cells in response to DHT treatment or glutaminase inhibition.

Results: We found that DHT up-regulated the expression of the glutamine transporter ASCT2 and glutaminase, both in vitro in LNCaP cells and in vivo in rat prostate cells. BPTES diminished the viability and migration of PCa cells, while increasing caspase-3 activity. CRPC cells were found to be more dependent on glutamine and more sensitive to glutaminase inhibition. BPTES and bicalutamide co-treatment had an additive effect on suppressing LNCaP cell viability. Finally, we found that inhibition of glutaminolysis differentially affected glycolysis and lipid metabolism in both androgen-sensitive and CRPC cells.

Conclusion: Our data reveal glutaminolysis as a central metabolic route controlling PCa cell fate and highlight the relevance of targeting glutaminase for CRPC treatment.
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http://dx.doi.org/10.1007/s13402-020-00575-9DOI Listing
April 2021

Maternal high-fat high-sucrose diet and gestational exercise modulate hepatic fat accumulation and liver mitochondrial respiratory capacity in mothers and male offspring.

Metabolism 2021 03 7;116:154704. Epub 2021 Jan 7.

Laboratory of Metabolism and Exercise (LaMetEx), Research Centre in Physical Activity, Health and Leisure (CIAFEL), Faculty of Sport, University of Porto, 4200-450, Porto, Portugal.

Background: Maternal high-caloric nutrition and related gestational diabetes mellitus (GDM) are associated with a high-risk for developing metabolic complications later in life and in their offspring. In contrast, exercise is recognized as a non-pharmacological strategy against metabolic dysfunctions associated to lifestyle disorders. Therefore, we investigated whether gestational exercise delays the development of metabolic alterations in GDM mothers later in life, but also protects 6-week-old male offspring from adverse effects of maternal diet.

Methods: Female Sprague-Dawley rats were fed with either control (C) or high-fat high-sucrose (HFHS) diet to induce GDM and submitted to gestational exercise during the 3 weeks of pregnancy. Male offspring were sedentary and fed with C-diet.

Results: Sedentary HFHS-fed dams exhibited increased gestational body weight gain (p < 0.01) and glucose intolerance (p < 0.01), characteristic of GDM. Their offspring had normal glucose metabolism, but increased early-age body weight, which was reverted by gestational exercise. Gestational exercise also reduced offspring hepatic triglycerides accumulation (p < 0.05) and improved liver mitochondrial respiration capacity (p < 0.05), contributing to the recovery of liver bioenergetics compromised by maternal HFHS diet. Interestingly, liver mitochondrial respiration remained increased by gestational exercise in HFHS-fed dams despite prolonged HFHS consumption and exercise cessation.

Conclusions: Gestational exercise can result in liver mitochondrial adaptations in GDM animals, which can be preserved even after the exercise program cessation. Exposure to maternal GDM programs liver metabolic setting of male offspring, whereas gestational exercise appears as an important preventive tool against maternal diet-induced metabolic alterations.
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http://dx.doi.org/10.1016/j.metabol.2021.154704DOI Listing
March 2021

Refinement of a differentiation protocol using neuroblastoma SH-SY5Y cells for use in neurotoxicology research.

Food Chem Toxicol 2021 Mar 6;149:111967. Epub 2021 Jan 6.

CNC, Center for Neuroscience and Cell Biology, University of Coimbra, UC-Biotech Building, Biocant Park, Cantanhede, 3060-197, Portugal. Electronic address:

Since most models used to study neuronal dysfunction display disadvantages and ethical concerns, a fast and reproducible in vitro model to study mitochondria-related neurodegeneration is required. Here, we optimized and characterized a 3-day retinoic acid-based protocol to differentiate the SH-SY5Y cell line into a neuronal-like phenotype and investigated alterations in mitochondrial physiology and distribution. Differentiation was associated with p21-linked cell cycle arrest and an increase in cell mass and area, possibly associated with the development of neurite-like extensions. Notably, increased expression of mature neuronal markers (neuronal-specific nuclear protein, microtubule-associated protein 2, βIII tubulin and enolase 2) was observed in differentiated cells. Moreover, increased mitochondrial content and maximal area per cell suggests mitochondrial remodeling. To demonstrate that this model is appropriate to study mitochondrial dysfunction, cells were treated for 6 h with mitochondrial toxicants (rotenone, antimycin A, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and 6-hydroxydopamine (6-OHDA)). Differentiated cells were more susceptible to increasing concentrations of FCCP, antimycin A, and rotenone, while 6-OHDA showed a distinct dose-dependent neurotoxicity pattern. Even though differentiated cells did not exhibit a fully mature/differentiated neuronal phenotype, the protocol developed can be used to study neurotoxicity processes, mitochondrial dynamics, and bioenergetic impairment, representing an alternative to study mitochondrial impairment-related pathologies in vitro.
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http://dx.doi.org/10.1016/j.fct.2021.111967DOI Listing
March 2021

Mitochondria-targeted phenolic antioxidants induce ROS-protective pathways in primary human skin fibroblasts.

Free Radic Biol Med 2021 Feb 29;163:314-324. Epub 2020 Dec 29.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6500HB Nijmegen, the Netherlands. Electronic address:

Phytochemical antioxidants like gallic and caffeic acid are constituents of the normal human diet that display beneficial health effects, potentially via activating stress response pathways. Using primary human skin fibroblasts (PHSFs) as a model, we here investigated whether such pathways were induced by novel mitochondria-targeted variants of gallic acid (AntiOxBEN) and caffeic acid (AntiOxCIN). Both molecules reduced cell viability with similar kinetics and potency (72 h incubation, IC50 ~23 μM). At a relatively high but non-toxic concentration (12.5 μM), AntiOxBEN and AntiOxCIN increased ROS levels (at 24 h), followed by a decline (at 72 h). Further analysis at the 72 h timepoint demonstrated that AntiOxBEN and AntiOxCIN did not alter mitochondrial membrane potential (Δψ), but increased cellular glutathione (GSH) levels, mitochondrial NAD(P)H autofluorescence, and mitochondrial superoxide dismutase 2 (SOD2) protein levels. In contrast, cytosolic SOD1 protein levels were not affected. AntiOxBEN and AntiOxCIN both stimulated the gene expression of Nuclear factor erythroid 2-related factor 2 (NRF2; a master regulator of the cellular antioxidant response toward oxidative stress). AntiOxBEN2 and ANtiOxCIN4 differentially affected the gene expression of the antioxidants Heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1). Both antioxidants did not protect from cell death induced by GSH depletion and AntiOxBEN (but not AntiOxCIN) antagonized hydrogen peroxide-induced cell death. We conclude that AntiOxBEN and AntiOxCIN increase ROS levels, which stimulates NRF2 expression and, as a consequence, SOD2 and GSH levels. This highlights that AntiOxBEN and AntiOxCIN can act as prooxidants thereby activating endogenous ROS-protective pathways.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.12.023DOI Listing
February 2021

"Oncometabolism: The switchboard of cancer - An editorial".

Biochim Biophys Acta Mol Basis Dis 2021 02 2;1867(2):166031. Epub 2020 Dec 2.

Molecular Physical-Chemistry R&D Unit, Centre for Investigation in Environment, Genetics and Oncobiology (CIMAGO), Department of Life Sciences, University of Coimbra, Coimbra, Portugal. Electronic address:

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http://dx.doi.org/10.1016/j.bbadis.2020.166031DOI Listing
February 2021

Oxidative Stress in Amyotrophic Lateral Sclerosis: Pathophysiology and Opportunities for Pharmacological Intervention.

Oxid Med Cell Longev 2020 15;2020:5021694. Epub 2020 Nov 15.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech Building, Biocant Park, Cantanhede, Portugal.

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease or Charcot disease, is a fatal neurodegenerative disease that affects motor neurons (MNs) and leads to death within 2-5 years of diagnosis, without any effective therapy available. Although the pathological mechanisms leading to ALS are still unknown, a wealth of evidence indicates that an excessive reactive oxygen species (ROS) production associated with an inefficient antioxidant defense represents an important pathological feature in ALS. Substantial evidence indicates that oxidative stress (OS) is implicated in the loss of MNs and in mitochondrial dysfunction, contributing decisively to neurodegeneration in ALS. Although the modulation of OS represents a promising approach to protect MNs from degeneration, the fact that several antioxidants with beneficial effects in animal models failed to show any therapeutic benefit in patients raises several questions that should be analyzed. Using specific queries for literature search on PubMed, we review here the role of OS-related mechanisms in ALS, including the involvement of altered mitochondrial function with repercussions in neurodegeneration. We also describe antioxidant compounds that have been mostly tested in preclinical and clinical trials of ALS, also describing their respective mechanisms of action. While the description of OS mechanism in the different mutations identified in ALS has as principal objective to clarify the contribution of OS in ALS, the description of positive and negative outcomes for each antioxidant is aimed at paving the way for novel opportunities for intervention. In conclusion, although antioxidant strategies represent a very promising approach to slow the progression of the disease, it is of utmost need to invest on the characterization of OS profiles representative of each subtype of patient, in order to develop personalized therapies, allowing to understand the characteristics of antioxidants that have beneficial effects on different subtypes of patients.
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http://dx.doi.org/10.1155/2020/5021694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683149PMC
November 2020

Cancer cell metabolism: Rewiring the mitochondrial hub.

Biochim Biophys Acta Mol Basis Dis 2021 02 25;1867(2):166016. Epub 2020 Nov 25.

CNC-Center for Neuroscience and Cell Biology, UC-Biotech, University of Coimbra, Biocant Park, Cantanhede, Portugal. Electronic address:

To adapt to tumoral environment conditions or even to escape chemotherapy, cells rapidly reprogram their metabolism to handle adversities and survive. Given the rapid rise of studies uncovering novel insights and therapeutic opportunities based on the role of mitochondria in tumor metabolic programing and therapeutics, this review summarizes most significant developments in the field. Taking in mind the key role of mitochondria on carcinogenesis and tumor progression due to their involvement on tumor plasticity, metabolic remodeling, and signaling re-wiring, those organelles are also potential therapeutic targets. Among other topics, we address the recent data intersecting mitochondria as of prognostic value and staging in cancer, by mitochondrial DNA (mtDNA) determination, and current inhibitors developments targeting mtDNA, OXPHOS machinery and metabolic pathways. We contribute for a holistic view of the role of mitochondria metabolism and directed therapeutics to understand tumor metabolism, to circumvent therapy resistance, and to control tumor development.
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http://dx.doi.org/10.1016/j.bbadis.2020.166016DOI Listing
February 2021

Exposure to marine benthic dinoflagellate toxins may lead to mitochondrial dysfunction.

Comp Biochem Physiol C Toxicol Pharmacol 2021 Feb 7;240:108937. Epub 2020 Nov 7.

Centre for Neuroscience and Cell Biology, University of Coimbra, UC Biotech, Biocant Park, 3060-197 Cantanhede, Portugal; Department of Life Sciences, Calçada Martim de Freitas, University of Coimbra, 3000-456 Coimbra, Portugal. Electronic address:

Even though marine dinoflagellates are important primary producers, many toxic species may alter the natural equilibrium of aquatic ecosystems and even generate human intoxication incidents, as they are the major causative agents of harmful algal blooms. In order to deepen the knowledge regarding benthic dinoflagellate adverse effects, the present study aims to clarify the influence of Gambierdiscus excentricus strain UNR-08, Ostreopsis cf. ovata strain UNR-03 and Prorocentrum lima strain UNR-01 crude extracts on rat mitochondrial energetic function and permeability transition pore (mPTP) induction. Our results, expressed in number of dinoflagellate cell toxic compounds tested in a milligram of mitochondrial protein, revealed that 934 cells mg prot of G. excentricus, and 7143 cells mg prot of both O. cf. ovata and P. lima negatively affect mitochondrial function, including by decreasing ATP synthesis-related membrane potential variations. Moreover, considerably much lower concentrations of dinoflagellate extracts (117 cells mg prot of G. excentricus, 1429 cells mg prot of O. cf. ovata and 714 cells mg prot of P. lima) produced mPTP-induced swelling in Ca-loaded isolated mitochondria. The present study clearly demonstrates the toxicity of G. excentricus, O. cf. ovata and P. lima extracts at the mitochondrial level, which may lead to mitochondrial failure and consequent cell toxicity, and that G. excentricus always provide much more severe effects than O. cf. ovata and P. lima.
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http://dx.doi.org/10.1016/j.cbpc.2020.108937DOI Listing
February 2021

Western Diet Causes Obesity-Induced Nonalcoholic Fatty Liver Disease Development by Differentially Compromising the Autophagic Response.

Antioxidants (Basel) 2020 Oct 15;9(10). Epub 2020 Oct 15.

Nencki Institute of Experimental Biology of Polish Academy of Sciences, 02-093 Warsaw, Poland.

Nonalcoholic fatty liver disease (NAFLD) is characterized by the development of steatosis, which can ultimately compromise liver function. Mitochondria are key players in obesity-induced metabolic disorders; however, the distinct role of hypercaloric diet constituents in hepatic cellular oxidative stress and metabolism is unknown. Male mice were fed either a high-fat (HF) diet, a high-sucrose (HS) diet or a combined HF plus HS (HFHS) diet for 16 weeks. This study shows that hypercaloric diets caused steatosis; however, the HFHS diet induced severe fibrotic phenotype. At the mitochondrial level, lipidomic analysis showed an increased cardiolipin content for all tested diets. Despite this, no alterations were found in the coupling efficiency of oxidative phosphorylation and neither in mitochondrial fatty acid oxidation (FAO). Consistent with unchanged mitochondrial function, no alterations in mitochondrial-induced reactive oxygen species (ROS) and antioxidant capacity were found. In contrast, the HF and HS diets caused lipid peroxidation and provoked altered antioxidant enzyme levels/activities in liver tissue. Our work provides evidence that hepatic oxidative damage may be caused by augmented levels of peroxisomes and consequently higher peroxisomal FAO-induced ROS in the early NAFLD stage. Hepatic damage is also associated with autophagic flux impairment, which was demonstrated to be diet-type dependent. The HS diet induced a reduction in autophagosomal formation, while the HF diet reduced levels of cathepsins. The accumulation of damaged organelles could instigate hepatocyte injuries and NAFLD progression.
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http://dx.doi.org/10.3390/antiox9100995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602470PMC
October 2020

Letter from the president.

Authors:
Paulo J Oliveira

Eur J Clin Invest 2020 Sep;50 Suppl 1:e13393

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http://dx.doi.org/10.1111/eci.13393DOI Listing
September 2020

P-cadherin induces anoikis-resistance of matrix-detached breast cancer cells by promoting pentose phosphate pathway and decreasing oxidative stress.

Biochim Biophys Acta Mol Basis Dis 2020 12 10;1866(12):165964. Epub 2020 Sep 10.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; IPATIMUP - Institute of Molecular Pathology and Immunology, University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal; Medical Faculty of the University of Porto, Porto, Portugal. Electronic address:

Successful metastatic spreading relies on cancer cells with stem-like properties, glycolytic metabolism and increased antioxidant protection, allowing them to escape anoikis and to survive in circulation. The expression of P-cadherin, a poor prognostic factor in breast cancer, is associated with hypoxic, glycolytic and acidosis biomarkers. In agreement, P-cadherin-enriched breast cancer cell populations presents a glycolytic and an acid-resistance phenotype. Our aim was to evaluate whether P-cadherin expression controls the glycolytic and oxidative phosphorylation fluxes of matrix-detached breast cancer cells, acting as an antioxidant and enhancing their survival in anchorage-independent conditions. By using matrix-detached breast cancer cells, we concluded that P-cadherin increases glucose-6-phosphate dehydrogenase expression, up-regulating the carbon flux through the pentose phosphate pathway, while inhibiting pyruvate oxidation to acetyl-coA via pyruvate dehydrogenase kinase-4 (PDK-4) activation. Accordingly, P-cadherin expression conferred increased sensitivity to dichloroacetate (DCA), a PDK inhibitor. P-cadherin expression also regulates oxidative stress in matrix-detached breast cancer cells, through the control of antioxidant systems, such as catalase and superoxide dismutases (SOD)1 and 2, providing these cells with an increased resistance to doxorubicin-induced anoikis. Importantly, this association was validated in primary invasive breast carcinomas, where an enrichment of SOD2 was found in P-cadherin-overexpressing breast carcinomas. In conclusion, we propose that P-cadherin up-regulates carbon flux through the pentose phosphate pathway and decreases oxidative stress in matrix-detached breast cancer cells. These metabolic remodeling and antioxidant roles of P-cadherin can promote the survival of breast cancer cells in circulation and in metastatic sites, being a possible player in breast cancer therapeutic resistance to pro-oxidant-based interventions.
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http://dx.doi.org/10.1016/j.bbadis.2020.165964DOI Listing
December 2020

Development of a 96-well based assay for kinetic determination of catalase enzymatic-activity in biological samples.

Toxicol In Vitro 2020 Dec 6;69:104996. Epub 2020 Sep 6.

CNC- Center for Neuroscience and Cell Biology, UC-Biotech Building, Biocant Park, Cantanhede, Portugal; Research Centre in Physical Activity, Health and Leisure (CIAFEL), Faculty of Sport, University of Porto, Porto, Portugal. Electronic address:

Oxidative stress biomarkers are powerful endpoints in toxicological research. Cellular reductive/oxidative balance affects numerous signaling pathways involving HO. Detoxification and control of HO levels results mainly from catalase activity. The aim of this work was to develop a precise, simple, cost-effective microassay to measure catalase activity in small tissue samples and cell extracts. We developed a protocol that quantifies HO decomposition by intrinsic catalase in biological samples. Catalase activity was calculated based on rate of decomposition of HO, following absorbance at 240 nm. We developed a multi-well spectroscopic approach, reducing sample quantity requirements and allowing simultaneous assessment of large number of samples. The protocol is sensitive across a wide range of catalase activity (11.5-7575 U). The assay presents a 95% confidence interval with an intra-assay coefficient of variation of 3.7%, an inter-assay coefficient of variation of 6.2% and good correlation with a commercial kit. The assay was established and validated for different biological samples, including sheep hepatic tissue and human tumor and non-tumor cell lines. This high-throughput method is robust, sensitive, time-saving and cost-effective, generating highly reproducible results with precision and good correlation with a commercial kit reinforcing the method's validity for research and toxicological applications.
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http://dx.doi.org/10.1016/j.tiv.2020.104996DOI Listing
December 2020

Rat cardiomyocyte H9c2(2-1)-based sulforhodamine B assay as a promising in vitro method to assess the biological component of effluent toxicity.

J Environ Sci (China) 2020 Oct 30;96:163-170. Epub 2020 May 30.

University of Coimbra, Centre for Functional Ecology, Department of Life Sciences, Calçada Martim de Freitas, Coimbra 3000-456, Portugal.

The treatment of wastewaters is crucial to maintain the ecological status of receiving waters, and thereby guarantee the protection of aquatic life and human health. Wastewater quality evaluation is conventionally based on physicochemical parameters, but increasing attention has been paid to integrate physicochemical and biological data. Nevertheless, the regulatory use of fish in biological testing methods has been subject to various ethical and cost concerns, and in vitro cell-based assays have thus become an important topic of interest. Hence, the present study intends: (a) to evaluate the efficiency of two different sample pre-concentration techniques (lyophilisation and solid phase extraction) to assess the toxicity of municipal effluents on rat cardiomyoblast H9c2(2-1) cells, and (b) maximizing the use of the effluent sample collected, to estimate the environmental condition of the receiving environment. The gathered results demonstrate that the H9c2(2-1) sulforhodamine B-based assay is an appropriate in vitro method to assess biological effluent toxicity, and the best results were attained by lyophilising the sample as pre-treatment. Due to its response, the H9c2(2-1) cell line might be a possible alternative in vitro model for fish lethal testing to assess the toxicity of municipal effluents. The physicochemical status of the sample suggests a high potential for eutrophication, and iron exceeded the permissible level for wastewater discharge, possibly due to the addition of ferric chloride for wastewater treatment. In general, the levels of carbamazepine and sulfamethoxazole are higher than those reported for other countries, and both surpassed the aquatic protective values for long-term exposure.
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http://dx.doi.org/10.1016/j.jes.2020.04.029DOI Listing
October 2020

Maternal obesity in sheep impairs foetal hepatic mitochondrial respiratory chain capacity.

Eur J Clin Invest 2021 Feb 12;51(2):e13375. Epub 2020 Sep 12.

CNC-Center for Neuroscience and Cell Biology, UC-Biotech, University of Coimbra, Cantanhede, Portugal.

Background: Changes in the nutritional environment in utero induced by maternal obesity (MO) lead to foetal metabolic dysfunction predisposing offspring to later-life metabolic diseases. Since mitochondria play a crucial role in hepatic metabolism and function, we hypothesized that MO prior to conception and throughout pregnancy programmes foetal sheep liver mitochondrial phenotype.

Material And Methods: Ewes ate an obesogenic diet (150% requirements; MO), or 100% requirements (CTR), from 60 days prior to conception. Foetal livers were removed at 0.9 gestation. We measured foetal liver mitochondrial DNA copy number, activity of superoxide dismutase, cathepsins B and D and selected protein content, total phospholipids and cardiolipin and activity of mitochondrial respiratory chain complexes.

Results: A significant decrease in activities of mitochondrial complexes I, II-III and IV, but not aconitase, was observed in MO. In the antioxidant machinery, there was a significant increase in activity of total superoxide dismutase (SOD) and SOD2 in MO. However, no differences were found regarding autophagy-related protein content (p62, beclin-I, LC3-I, LC3-II and Lamp2A) and cathepsin B and D activities. A 21.5% decrease in total mitochondrial phospholipid was observed in MO.

Conclusions: The data indicate that MO impairs foetal hepatic mitochondrial oxidative capacity and affects total mitochondrial phospholipid content. In addition, MO affects the regulation of foetal liver redox pathways, indicating metabolic adaptations to the higher foetal lipid environment. Consequences of in utero programming of foetal hepatic metabolism may persist and compromise mitochondrial bioenergetics in later life, and increase susceptibility to metabolic diseases.
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http://dx.doi.org/10.1111/eci.13375DOI Listing
February 2021

Dihydroorotate dehydrogenase inhibitors in SARS-CoV-2 infection.

Eur J Clin Invest 2020 Oct 16;50(10):e13366. Epub 2020 Aug 16.

CNC-Center for Neuroscience and Cell Biology, UC-Biotech, University of Coimbra, Cantanhede, Portugal.

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http://dx.doi.org/10.1111/eci.13366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435507PMC
October 2020

High sensitivity of rat cardiomyoblast H9c2(2-1) cells to Gambierdiscus toxic compounds.

Aquat Toxicol 2020 Jun 7;223:105475. Epub 2020 Apr 7.

CFE-Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal. Electronic address:

Ciguatera fish poisoning is a frequently reported non-bacterial food-borne illness related to the consumption of seafood contaminated with ciguatoxins, and possibly maitotoxins. These toxins are synthesized by marine dinoflagellate species of Gambierdiscus and Fukuyoa genera, and their abundance is a matter of great concern due to their adverse effects to aquatic life and human health. The present study aims to assess the sensitivity of rat cardiomyoblast H9c2(2-1) cells to Gambierdiscus toxic compounds using concentration- and time-dependent sulforhodamine B (SRB) colorimetric assays. Low concentrations of Gambierdiscus extracts (corresponding to 1.3-2.3 cells mL) induced a concentration-dependent response. Specificity in time-dependent response of H9c2(2-1) cells was demonstrated for G. excentricus after a 180 min exposure compared to both G. cf. belizeanus and G. silvae species, with EC obtained after 720 and 360 min, respectively. The sensitivity of H9c2(2-1) cells to dinoflagellate toxic compounds was also tested with other genera from benthic (Coolia malayensis, Ostreopsis cf. ovata, Prorocentrum hoffmannianum and P. lima) and planktonic (Amphidinium carterae and Lingulodinium polyedrum) habitats. Amphidinium, Coolia and Lingulodinium data did not present any concentration-response relationships, and EC values could only be obtained after 720 and 1440 min of exposure to both Prorocentrum species and O. cf. ovata, respectively. This study demonstrated that the H9c2(2-1) SRB assay represents a promising and sensitive tool for the detection of Gambierdiscus toxic compounds present in water samples, particularly of G. excentricus at very low cell abundances.
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http://dx.doi.org/10.1016/j.aquatox.2020.105475DOI Listing
June 2020

Publicly stressing the role of mitochondria in NAFLD with(in) a sports event.

Eur J Clin Invest 2020 May 11;50(5):e13234. Epub 2020 May 11.

CNC-Center for Neuroscience and Cell Biology, Rua Larga, University of Coimbra, Coimbra, Portugal.

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http://dx.doi.org/10.1111/eci.13234DOI Listing
May 2020

Mitochondrial impairment and cytotoxicity effects induced by the marine epibenthic dinoflagellate Coolia malayensis.

Environ Toxicol Pharmacol 2020 Jul 30;77:103379. Epub 2020 Mar 30.

CNC-Centre for Neuroscience and Cell Biology, UC Biotech, University of Coimbra, Lot 8A, Biocant Park, 3060-197, Cantanhede, Portugal; Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456, Coimbra, Portugal. Electronic address:

Mitochondria was used to clarify the effects of Coolia malayensis strain UNR-02 crude extract by studying mitochondrial membrane potential (ΔΨ) generation and the fluctuations of ΔΨ associated with the induction of mitochondrial permeability transition (MPT). The cytoxicity of C. malayensis was also determined using both HepG2 and H9c2(2-1) cells. C. malayensis extract significantly depressed the oxidative phosphorylation efficiency, as was inferred from the perturbations in ΔΨ and in the phosphorylative cycle induced by ADP. Increased susceptibility to Ca-induced MPT was also observed. At the cellular level, the extract significantly decreased cell mass of both cell lines.
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http://dx.doi.org/10.1016/j.etap.2020.103379DOI Listing
July 2020

Mitochondrial Determinants of Doxorubicin-Induced Cardiomyopathy.

Circ Res 2020 03 26;126(7):926-941. Epub 2020 Mar 26.

CNC - Center for Neuroscience and Cell Biology, University of Coimbra, UC-Biotech, Biocant Park, Cantanhede, Portugal (V.A.S., P.J.O.).

Anthracycline-based chemotherapy can result in the development of a cumulative and progressively developing cardiomyopathy. Doxorubicin is one of the most highly prescribed anthracyclines in the United States due to its broad spectrum of therapeutic efficacy. Interference with different mitochondrial processes is chief among the molecular and cellular determinants of doxorubicin cardiotoxicity, contributing to the development of cardiomyopathy. The present review provides the basis for the involvement of mitochondrial toxicity in the different functional hallmarks of anthracycline toxicity. Our objective is to understand the molecular determinants of a progressive deterioration of functional integrity of mitochondria that establishes a historic record of past drug treatments (mitochondrial memory) and renders the cancer patient susceptible to subsequent regimens of drug therapy. We focus on the involvement of doxorubicin-induced mitochondrial oxidative stress, disruption of mitochondrial oxidative phosphorylation, and permeability transition, contributing to altered metabolic and redox circuits in cardiac cells, ultimately culminating in disturbances of autophagy/mitophagy fluxes and increased apoptosis. We also suggest some possible pharmacological and nonpharmacological interventions that can reduce mitochondrial damage. Understanding the key role of mitochondria in doxorubicin-induced cardiomyopathy is essential to reduce the barriers that so dramatically limit the clinical success of this essential anticancer chemotherapy.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.314681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7121924PMC
March 2020

Screening-level evaluation of marine benthic dinoflagellates toxicity using mammalian cell lines.

Ecotoxicol Environ Saf 2020 Jun 1;195:110465. Epub 2020 Apr 1.

CFE-Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456, Coimbra, Portugal. Electronic address:

Complementary studies at different levels of the biological organization are fundamental to fully link environmental exposure to marine benthic dinoflagellate toxins and their effects. In order to contribute to this transdisciplinary evaluation, and for the first time, the present study aims to study the effects of Gambierdiscus excentricus, Ostreopsis cf. ovata, Prorocentrum hoffmannianum and Prorocentrum lima extracts on seven functionally different mammalian cell lines: HEK 293, HepG2, HNDF, H9c2(2-1), MC3T3-E1, Raw 264.7 and SH-SY5Y. All the cell lines presented cell mass decrease in a concentration-dependence of dinoflagellate extracts, exhibiting marked differences in cell toxicity. Gambierdiscus excentricus presented the highest effect, at very low concentrations with EC (i.e., the concentration that gives half-maximal response after a 24-h exposure) between 1.3 and 13 cells mL, followed by O. cf. ovata (EC between 3.3 and 40 cells mL), and Prorocentrum species (P. lima: EC between 191 and 1027 cells mL and P. hoffmannianum: EC between 152 and 783 cells mL). Cellular specificities were also detected and rat cardiomyoblast H9c2(2-1) cells were in general the most sensitive to dinoflagellate toxic compounds, suggesting that this cell line is an animal-free potential model for dinoflagellate toxin testing. Finally, the sensitivity of cells expressing distinct phenotypes to each dinoflagellate extract exhibited low relation to human poisoning symptoms.
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http://dx.doi.org/10.1016/j.ecoenv.2020.110465DOI Listing
June 2020

Drp1-mediated mitochondrial fission regulates calcium and F-actin dynamics during wound healing.

Biol Open 2020 05 3;9(5). Epub 2020 May 3.

CEDOC, Chronic Diseases Research Center, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, 1169-056 Lisboa, Portugal

Mitochondria adapt to cellular needs by changes in morphology through fusion and fission events, referred to as mitochondrial dynamics. Mitochondrial function and morphology are intimately connected and the dysregulation of mitochondrial dynamics is linked to several human diseases. In this work, we investigated the role of mitochondrial dynamics in wound healing in the embryonic epidermis. Mutants for mitochondrial fusion and fission proteins fail to close their wounds, indicating that the regulation of mitochondrial dynamics is required for wound healing. By live-imaging, we found that loss of function of the mitochondrial fission protein Dynamin-related protein 1 (Drp1) compromises the increase of cytosolic and mitochondrial calcium upon wounding and leads to reduced reactive oxygen species (ROS) production and F-actin defects at the wound edge, culminating in wound healing impairment. Our results highlight a new role for mitochondrial dynamics in the regulation of calcium, ROS and F-actin during epithelial repair.
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http://dx.doi.org/10.1242/bio.048629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225088PMC
May 2020

Urine-Derived Stem Cells: Applications in Regenerative and Predictive Medicine.

Cells 2020 02 28;9(3). Epub 2020 Feb 28.

CNC-Center for Neuroscience and Cell Biology, UC-Biotech, University of Coimbra, 3030-789 Cantanhede, Portugal.

Despite being a biological waste, human urine contains a small population of cells with self-renewal capacity and differentiation potential into several cell types. Being derived from the convoluted tubules of nephron, renal pelvis, ureters, bladder and urethra, urine-derived stem cells (UDSC) have a similar phenotype to mesenchymal stroma cells (MSC) and can be reprogrammed into iPSC (induced pluripotent stem cells). Having simple, safer, low-cost and noninvasive collection procedures, the interest in UDSC has been growing in the last decade. With great potential in regenerative medicine applications, UDSC can also be used as biological models for pharmacology and toxicology tests. This review describes UDSC biological characteristics and differentiation potential and their possible use, including the potential of UDSC-derived iPSC to be used in drug discovery and toxicology, as well as in regenerative medicine. Being a new cellular platform amenable to noninvasive collection for disease stratification and personalized therapy could be a future application for UDSC.
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http://dx.doi.org/10.3390/cells9030573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140531PMC
February 2020

Insights into the Discovery of Novel Neuroprotective Agents: A Comparative Study between Sulfanylcinnamic Acid Derivatives and Related Phenolic Analogues.

Molecules 2019 Dec 2;24(23). Epub 2019 Dec 2.

CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal.

Exogenous antioxidants may be beneficial therapeutic tools to tackle the oxidative damage in neurodegenerative diseases by regulation of the redox state that is critical for cell viability and organ function. Inspired by natural plant polyphenols, a series of cinnamic acid-based thiophenolic and phenolic compounds were synthesized and their antioxidant and neuroprotective properties were studied. In general, our results showed that the replacement of the hydroxyl group (OH) by a sulfhydryl group (SH) increased the radical scavenging activity and enhanced the reaction rate with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and galvinoxyl radical (GO). These results correlated well with the lower oxidation potential () values of thiophenols. However, a lower peroxyl radical (ROO) scavenging activity was observed for thiophenols in oxygen radical absorbance capacity (ORAC-FL) assay. Furthermore, the introduction of 5-methoxy and 5-phenyl groups in the aromatic ring of 4-thioferulic acid (TFA) and ferulic acid (FA) did not significantly improve their antioxidant activity, despite the slight decrease of observed for compounds , and . Concerning cinnamic acid amides, the antioxidant profile was similar to the parent compounds. None of the compounds under study presented significant cytotoxic effects in human differentiated neuroblastoma cells. Thiophenolic amide stands out as the most promising thiophenol-based antioxidant, showing cellular neuroprotective effects against oxidative stress inducers (hydrogen peroxide and iron).
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http://dx.doi.org/10.3390/molecules24234405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930627PMC
December 2019

Mitochondria-Lysosome Crosstalk: From Physiology to Neurodegeneration.

Trends Mol Med 2020 01 29;26(1):71-88. Epub 2019 Nov 29.

Institute of Cellular Biochemistry, University Medical Center Goettingen, 37073 Goettingen, Germany. Electronic address:

Cellular function requires coordination between different organelles and metabolic cues. Mitochondria and lysosomes are essential for cellular metabolism as major contributors of chemical energy and building blocks. It is therefore pivotal for cellular function to coordinate the metabolic roles of mitochondria and lysosomes. However, these organelles do more than metabolism, given their function as fundamental signaling platforms in the cell that regulate many key processes such as autophagy, proliferation, and cell death. Mechanisms of crosstalk between mitochondria and lysosomes are discussed, both under physiological conditions and in diseases that affect these organelles.
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http://dx.doi.org/10.1016/j.molmed.2019.10.009DOI Listing
January 2020

Doxorubicin persistently rewires cardiac circadian homeostasis in mice.

Arch Toxicol 2020 01 25;94(1):257-271. Epub 2019 Nov 25.

Mitochondrial Toxicology and Experimental Therapeutics Laboratory (MitoXT), CNC, Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech Building (Lote 8A), Biocant Park, 3060-197, Cantanhede, Portugal.

Circadian rhythms disruption can be the cause of chronic diseases. External cues, including therapeutic drugs, have been shown to modulate peripheral-circadian clocks. Since anthracycline cardiotoxicity is associated with loss of mitochondrial function and metabolic remodeling, we investigated whether the energetic failure induced by sub-chronic doxorubicin (DOX) treatment in juvenile mice was associated with persistent disruption of circadian regulators. Juvenile C57BL/6J male mice were subjected to a sub-chronic DOX treatment (4 weekly injections of 5 mg/kg DOX) and several cardiac parameters, as well as circadian-gene expression and acetylation patterns, were analyzed after 6 weeks of recovery time. Complementary experiments were performed with Mouse Embryonic Fibroblasts (MEFs) and Human Embryonic Kidney 293 cells. DOX-treated juvenile mice showed cardiotoxicity markers and persistent alterations of transcriptional- and signaling cardiac circadian homeostasis. The results showed a delayed influence of DOX on gene expression, accompanied by changes in SIRT1-mediated cyclic deacetylation. The mechanism behind DOX interference with the circadian clock was further studied in vitro, in which were observed alterations of circadian-gene expression and increased BMAL1 SIRT1-mediated deacetylation. In conclusion, DOX treatment in juvenile mice resulted in disruption of oscillatory molecular mechanisms including gene expression and acetylation profiles.
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http://dx.doi.org/10.1007/s00204-019-02626-zDOI Listing
January 2020

Mitochondrial remodeling in human skin fibroblasts from sporadic male Parkinson's disease patients uncovers metabolic and mitochondrial bioenergetic defects.

Biochim Biophys Acta Mol Basis Dis 2020 03 20;1866(3):165615. Epub 2019 Nov 20.

CNC - Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech, Biocant Park, 3060-197 Cantanhede, Portugal; IIIUC - Institute for Interdisciplinary Research, University of Coimbra, 3030-789 Coimbra, Portugal. Electronic address:

Parkinson's Disease (PD) is characterized by dopaminergic neurodegeneration in the substantia nigra. The exact mechanism by which dopaminergic neurodegeneration occurs is still unknown; however, mitochondrial dysfunction has long been implicated in PD pathogenesis. To investigate the sub-cellular events that lead to disease progression and to develop personalized interventions, non-neuronal cells which are collected in a minimally invasive manner can be key to test interventions aimed at improving mitochondrial function. We used human skin fibroblasts from sporadic PD (sPD) patients as a cell proxy to detect metabolic and mitochondrial alterations which would also exist in a non-neuronal cell type. In this model, we used a glucose-free/galactose- glutamine- and pyruvate-containing cell culture medium, which forces cells to be more dependent on oxidative phosphorylation (OXPHOS) for energy production, in order to reveal hidden metabolic and mitochondrial alterations present in fibroblasts from sPD patients. We demonstrated that fibroblasts from sPD patients show hyperpolarized and elongated mitochondrial networks and higher mitochondrial ROS concentration, as well as decreased ATP levels and glycolysis-related ECAR. Our results also showed that abnormalities of fibroblasts from sPD patients became more evident when stimulating OXPHOS. Under these culture conditions, fibroblasts from sPD cells presented decreased basal respiration, ATP-linked OCR and maximal respiration, and increased mitochondria-targeting phosphorylation of DRP1 when compared to control cells. Our work validates the relevance of using fibroblasts from sPD patients to study cellular and molecular changes that are characteristic of dopaminergic neurodegeneration of PD, and shows that forcing mitochondrial OXPHOS uncovers metabolic defects that were otherwise hidden.
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http://dx.doi.org/10.1016/j.bbadis.2019.165615DOI Listing
March 2020