Biomarkers of inflammation in chronic kidney disease staging, progression and outcome

Irina Lousa, Maria João Valente, Susana Rocha, Susana Coimbra, Cristina Catarino, Petronila Rocha-Pereira, Elsa Bronze-da-Rocha, Maria do Sameiro-Faria, Luís Belo, Alice Santos-Silva

Chronic kidney disease (CKD) is characterized by a persistent, low-grade inflammatory state, regardless of its etiology, that is known to underlie disease progression and outcome. Inflammation starts early in the onset of renal diseases and worsens with disease progression, being particularly elevated in end-stage renal disease (ESRD) patients.

We analysed the circulating levels of several acute phase proteins and inflammatory molecules, in pre-dialysis and in hemodialysis patients. We studied their association with parameters of kidney function and kidney injury, to evaluate their potential as early markers of the disease and of its worsening, as well as markers of CKD outcome and mortality.

Among the studied inflammatory biomarkers, tumor necrosis factor receptor 2 (TNFR2) revealed to be promising in detecting CKD onset and progression. Furthermore, acute phase proteins and cytokines (such as PTX3 and IL-6) as well as cell-free DNA (cfDNA), seem to be important predictors of CKD mortality in ESRD patients under regular hemodialysis therapy.

Our group has focused on traditional and emergent inflammatory biomarkers, proposed as CKD biomarkers. However, it is not completely elucidated whether they might be useful for CKD detection and progression. Furthermore, there is an emerging interest in the study of biomarkers of inflammation as potential therapeutic targets.

References

[1] Valente MJ, Rocha S, Coimbra S, Catarino C, Rocha-Pereira P, Bronze-da-Rocha E, Oliveira JG, Madureira J, Fernandes JC, do Sameiro-Faria M, Miranda V, Belo L, Santos-Silva A. Long Pentraxin 3 as a Broader Biomarker for Multiple Risk Factors in End-Stage Renal Disease: Association with All-Cause Mortality. Mediators Inflamm. 2019 Jun 16;2019:3295725. doi: 10.1155/2019/3295725.

[2] Coimbra S, Rocha S, Nascimento H, Valente MJ, Catarino C, Rocha-Pereira P, Sameiro-Faria M, Oliveira JG, Madureira J, Fernandes JC, Miranda V, Belo L, Bronze-da-Rocha E, Santos-Silva A. Cell-free DNA as a marker for the outcome of end-stage renal disease patients on haemodialysis. Clin Kidney J. 2020 Aug 24;14(5):1371-1378. doi: 10.1093/ckj/sfaa115.

[3] Rocha S, Valente MJ, Coimbra S, Catarino C, Rocha-Pereira P, Oliveira JG, Madureira J, Fernandes JC, do Sameiro-Faria M, Miranda V, Belo L, Santos-Silva A, Bronze-da-Rocha E. Interleukin 6 (rs1800795) and pentraxin 3 (rs2305619) polymorphisms-association with inflammation and all-cause mortality in end-stage-renal disease patients on dialysis. Sci Rep. 2021 Jul 20;11(1):14768. doi: 10.1038/s41598-021-94075-x.

[4] Lousa I, Belo L, Valente MJ, Rocha S, Preguiça I, Rocha-Pereira P, Beirão I, Mira F, Alves R, Reis F, Santos-Silva A. Inflammatory biomarkers in staging of chronic kidney disease: Elevated TNFR2 levels accompanies renal function decline. Inflam Res. Aceite para publicação em abril de 2022

Modulation of protein aggregation with mitochondria-targeted drugs in models of Huntington’s disease

Brígida R. Pinho1, Michael P. Murphy2, Michael R. Duchen3,4, Jorge M. A. Oliveira1,4 

1) UCIBIO-REQUIMTE – Applied Molecular Biosciences Unit; i4HB – Institute for Health and Bioeconomy, Dep. Drug Sciences, Pharmacology Lab, FFUP, U.Porto, PT 

2) MRC Mitochondrial Biology Unit, University of Cambridge, UK 

3) Department of Cell and Developmental Biology, University College London, UK 

4) Consortium for Mitochondrial Research (CfMR), University College London, UK 

Mitochondrial dysfunction and the accumulation of misfolded and aggregated proteins are common pathophysiological mechanisms in neurodegenerative disorders. Key open questions are how mitochondrial dysfunction influences the protein homeostasis (proteostasis) network [1] - including chaperones and protein degradation pathways - and whether proteotoxicity can be attenuated by mitochondria-targeted drugs. To address these questions, we tested mitochondria-targeted drugs in models of Huntington’s disease (HD) - a neurodegenerative disorder caused by polyglutamine expansion mutations in the huntingtin (Htt) protein. We investigated if the modulation of mitochondrial reactive oxygen species (ROS) with the mitochondrial redox cycler Mitoparaquat and with the mitochondrial antioxidant MitoQ would modify HD phenotypes. We also investigated if YM-1, a mitochondria-targeted activator of the chaperone Hsp70, modifies Htt proteostasis and the role of mitochondria in the mechanism of action of YM-1. 

Mitoparaquat, which selectively produces superoxide in mitochondria, promoted mutant (mut-) Htt aggregation in a HD cell model [2]. The antioxidant MitoQ ameliorated fine motor control of HD mice (R6/2). MitoQ maintained unchanged the levels of mut-Htt but reduced the oxidative damage and attenuated the overactive autophagy in muscle – tissue where the levels of MitoQ were higher [3]. We studied the role of mitochondria in modulation of Htt levels by the Hsp70 chaperone activator YM-1 in HD cell models. YM-1, which is cationic and accumulates in negatively charged mitochondria, reduced mut-Htt levels and aggregation of its N-terminal fragments. During the study of YM-1 mechanism of action, we found that YM-1 increased the levels of mitochondrial Hsp70 (a chaperone involved in mitochondrial protein import) and of NRF1 (a master regulator of proteasome genes) and reduced the levels of ubiquitinated proteins [4]. We propose that YM-1 can act on the mitochondrial Hsp70, delaying the release of substrates, interfering with the mitochondrial protein import, increasing NRF1 levels and boosting proteasomal degradation. Altogether these results show that mitochondria via ROS or NRF1 can regulate protein aggregation and clearance pathways in cytosol. These studies also support that MitoQ and YM-1 hold therapeutic potential in HD.  

References: 

1 - Soares TR, Reis SD, Pinho BR, Duchen MR, Oliveira JMA (2019) Targeting the proteostasis network in Huntington's disease. Ageing Research Reviews 49:92-103 https://doi.org/10.1016/j.arr.2018.11.006 

2 - Pinho BR, Reis SD, Hartley RC, Murphy MP, Oliveira JMA (2019) Mitochondrial superoxide generation induces a parkinsonian phenotype in zebrafish and huntingtin aggregation in human cells. Free Radical Biology and Medicine 130:318-327 https://doi.org/10.1016/j.freeradbiomed.2018.10.446 

3 - Pinho BR, Duarte AI, Canas PM, Moreira P, Murphy MP, Oliveira JMA (2020) The interplay between redox signalling and proteostasis in neurodegeneration: In vivo effects of a mitochondria-targeted antioxidant in Huntington’s disease mice. Free Radical Biology and Medicine 146:372-382 https://doi.org/10.1016/j.freeradbiomed.2019.11.021 

4 - Pinho BR, Almeida LM, Duchen MR, Oliveira JMA (2021) Allosteric activation of Hsp70 reduces mutant huntingtin levels, the clustering of N-terminal fragments, and their nuclear accumulation. Life Sciences 285:120009 https://doi.org/10.1016/j.lfs.2021.120009 

Acknowledgements:

Fundação para a Ciência e a Tecnologia: PTDC/NEU-NMC/0412/2014, DL57/2016/CP1346/CT0016, LA/P/0140/2020, UIDP/04378/2020 and UIDB/04378/2020.  

Carbon monoxide-mediated pro-regenerative and non-inflammatory microglial phagocytosis: towards clearance of dead neurons

Catarina G. Simões1,2, Nuno L. Soares2, Raquel Domingues1,2,3, Helena L.A. Vieira1,2,4

1 UCIBIO, Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, Portugal 2 CEDOC – Faculdade de Ciências Médicas / NOVA Medical School, Universidade Nova de Lisboa, Portugal 3 Univ Coimbra, Faculty of Medicine 4 Associate Laboratory i4HB - Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade Nova de Lisboa, Portugal

Chronic neurodegenerative diseases and acute stroke lead to neuronal cell death and neuroinflammation. Dead neurons must be rapidly removed in these pathologies to avoid an amplification loop of neurotoxicity and neuroinflammation.

Microglia are the resident immune cells of the central nervous system (CNS). They patrol the brain parenchyma searching for any homeostasis disturbance that may endanger CNS integrity. When microglia detect a pathological stimulus, they become activated and initiate an immune response, secreting cytokines and phagocyting pathogens, protein aggregates, and dead cells. Microglial activation may be reparative, but it needs to be under tight regulation since exacerbated or chronic neuroinflammation become neurotoxic. Thus, our strategy is to stimulate microglial non-inflammatory phagocytosis for the clearance of dead neurons, which is a pro-regenerative process by eliminating toxic factors.

Carbon monoxide (CO) is an endogenous cytoprotective molecule generated by heme oxygenase (HO), which has two isoforms: the constitutive HO-2 and inducible HO-1, whose activation or expression occurs in response to stress [1–3]. Furthermore, CO presents anti- neuroinflammatory role in microglia by modulating cell metabolism [4,5] and is neuroprotective by modulating microglia-neurons communication [6].

In order to assess the CO effect on microglial phagocytosis a CO-releasing molecule ALF826 [4,6] is used. ALF826 modulates microglial phagocytosis at both engulfment and cargo digestion processes. ALF826 decreases excessive engulfment and cargo digestion in response to inflammation (lipopolysaccharide treatment) whenever latex beads and fluorescent BSA were used as cargo to follow phagocytosis. While, CO increases microglial engulfment of dead neurons under inflammatory and non-inflammatory conditions; and improves digestion of dead neurons under inflammatory conditions. Further work is in progress to identify which are the key players involved in “eat-me” signaling, namely the microglial CD200R and neuronal CD200 pair, whose both expressions enhance in the presence of CO.

References

1. Queiroga CSF, Vercelli A, Vieira HLA. Carbon monoxide and the CNS: challenges and achievements. Br J Pharmacol. GB; 2015;172:1533–45.

2. Figueiredo-Pereira C, Dias-Pedroso D, Soares NL, Vieira HLA. CO-mediated cytoprotection is dependent on cell metabolism modulation. Redox Biol. 2020.

3. Soares NL, Vieira HLA. Microglia at the Centre of Brain Research: Accomplishments and Challenges for the Future. Neurochem Res [Internet]. 2022;47:218–33. Available from: https://link.springer.com/10.1007/s11064-021-03456-1 4. Dias-Pedroso D, Ramalho JS, Sardão VA, Jones JG, Romão CC, Oliveira PJ, et al. Carbon Monoxide-Neuroglobin Axis Targeting Metabolism Against Inflammation in BV-2 Microglial Cells. Mol Neurobiol [Internet]. 2022;59:916–31. Available from: https://link.springer.com/10.1007/s12035-021-02630-4

5. Wilson JLJL, Bouillaud F, Almeida ASAS, Vieira HL, Ouidja MOMO, Dubois-Randé JLJ-L, et al. Carbon monoxide reverses the metabolic adaptation of microglia cells to an inflammatory stimulus. Free Radic Biol Med. 2017;104:311–23.

6. Soares NL, Paiva I, Bravo J, Queiroga CSF, Melo BF, Conde S V., et al. Carbon Monoxide Modulation of Microglia- Neuron Communication: Anti-Neuroinflammatory and Neurotrophic Role. Mol Neurobiol [Internet]. 2022;59:872– 89. Available from: https://link.springer.com/10.1007/s12035-021-02643-z

Inflammatory Pathways in Granulosa Cells: Exploring Pharmacological Significance of Chalcones

Thaise Martins1,2, Bruno M. Fonseca1,2, Irene Rebelo1,2

1UCIBIO, REQUIMTE, Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto

2Associate Laboratory i4HB - Institute for Health and Bioeconomy, Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

Ovulation features of inflammation are associated with neutrophil and macrophage recruitment and production of mediators, such as reactive species (RS) and bioactive lipids. The maintenance of these activated pathways can trigger reproductive pathologies, being the most prominent cause of female infertility [1]. Granulosa cells (GC) are the ovarian somatic cells that surround the oocyte and play a key role in its growth and maturation, being relevant in predicting follicular health [2]. Considering that neutrophils are the first cells recruited during an inflammatory response to produce RS, our initial aim was to investigate the ability of 34 chalcones to scavenge HOCl and/or inhibit the human neutrophils oxidative burst. In a second stage, considering the role of macrophages in the ovary, the following aim was to elucidate the inflammatory pathways that activated in human GCs when exposed to different macrophage phenotypes. As result, it was possible to observe that the ability of chalcones to scavenge HOCl depended on the position and number of hydroxy groups present in their aromatic rings. Inhibition of neutrophils' oxidative burst depended on the presence of a hydroxy group at 2 position on A-ring and methoxy, hydroxy, and nitro groups and/or chlorine atom at 2, 3, and 4 positions on B-ring [3]. In GCs, it was observed that M1 and M2 macrophages were able to distinctly influence the expression of COX-2, 5-, 12-, and 15-LOX, suggesting that the inflammatory environment within human ovary may, in part, influence the oocyte quality and fertilization. Overall, the studied chalcones showed high activity, indicating their potential as antioxidant agents to be used as a molecular structural scaffold for the design of novel compounds as well as polarized macrophages differently modulate the expression of the key enzymes of lipid signaling by GC.

 References

1.     Jabbour, H.N., et al., Inflammatory pathways in female reproductive health and disease. Reproduction, 2009. 138(6): p. 903-19.

2.     Alam, M.H. and T. Miyano, Interaction between growing oocytes and granulosa cells in vitro. Reprod Med Biol, 2020. 19(1): p. 13-23.

3.     Martins, T., et al., Chalcones as Scavengers of HOCl and Inhibitors of Oxidative Burst: Structure-Activity Relationship Studies. Med Chem, 2022. 18(1): p. 88-96

Motor activity and sleep phenotypes in a zebrafish model of sporadic Amyotrophic Lateral Sclerosis 

Nuno A. S. Oliveira, Rui F. O. Silva, Brígida R. Pinho, Jorge M. A. Oliveira 

UCIBIO-REQUIMTE – Applied Molecular Biosciences Unit; i4HB – Institute for Health and Bioeconomy, Dep. Drug Sciences, Pharmacology Lab, FFUP, U.Porto, PT 

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that poses a major burden to global health. It leads to progressive disability and motor impairment, through the degeneration of motor neurons. Most ALS cases are idiopathic, and environmental exposures likely contribute to sporadic disease. The small vertebrate zebrafish is a leading organism to model neurodegenerative diseases, and previous studies have proposed Bisphenol A (BPA) exposure to model sporadic ALS in zebrafish, inducing motor neuron damage and reduced motor responses [1]. Traditionally, the behavioural characterization of such models has been primarily focused on spontaneous locomotion and reflexes; however, sleep and circadian rhythm disruptions are also common in ALS patients, contributing to disease progression and burden. Here we aim to extend the phenotypic characterisation of the BPA-induced zebrafish model of ALS, including its sleep and circadian phenotypes. We combined an infrared-based Locomotion Activity Monitor (LAM) with our recently developed Rtivity behavioural analysis software [2] to characterize zebrafish motor activity, sleep and circadian rhythms in BPA-exposed animals versus non-exposed controls. A survival study was first conducted to select the highest concentrations that did not induce mortality in each time point (20 µM for 7 days, or 60 µM for 72 hours). BPA exposure induced concentration-dependent reductions in motor activity (tail coiling at 24 hours post-fertilization, swimming at 4-6 days post-fertilization) and sleep disruptions (e.g. increased sleep ratio and sleep latency). Activity and sleep disruptions induced by BPA were found in the absence of effects on muscle tissue, touch-evoked escape responses (sensorimotor reflexes), or heart rate at 6 days post-fertilization. These results evidence that the BPA-induced zebrafish model shows selective sleep and activity phenotypes akin to those of ALS patients. The extended characterization of sleep and circadian rhythm phenotypes in ALS models can assist future studies addressing pathogenic pathways and experimental therapeutics. 

References:

1. Morrice JR, Gregory-Evans CY, Shaw CA (2018) Modeling Environmentally-Induced Motor Neuron Degeneration in Zebrafish. Scientific reports 8(1):4890-4890. https://doi.org/10.1038/s41598-018-23018-w 

2. Silva RFO, Pinho BR, Monteiro NM, Santos MM, Oliveira JMA (2022) Automated analysis of activity, sleep, and rhythmic behaviour in various animal species with the Rtivity software. Scientific Reports 12:4179 https://doi.org/10.1038/s41598-022-08195-z 

Mechanisms of action of aromatase inhibitors used in the clinic: challenges for the treatment of breast cancer

Cristina Amaral (1,2), Tiago V. Augusto (1,2), Cristina Ferreira Almeida (1,2) Georgina Correia-da-Siva (1,2), Natércia Teixeira (1,2)

(1) UCIBIO.REQUIMTE, Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal;

(2) Associate Laboratory i4HB - Institute for Health and Bioeconomy - Faculty of Pharmacy, University of Porto, Porto, Portugal.

Introduction: Around 70-85% of all breast cancer cases are estrogen receptor-positive (ER+). The third-generation of aromatase inhibitors (AIs), anastrozole (Ana), letrozole (Let) and exemestane (Exe) are the first-line treatment option for these tumors, in postmenopausal women or in pre-menopausal women, after ovarian function suppression. Despite their therapeutic success, acquired resistance may occur after AI-treatment, causing tumor relapse [1]. Thus, to improve treatment it is crucial to understand the biological mechanisms induced by these AIs on cancer cells in order to find new targets/strategies. In the last years, our group has been studying the effects of Exe in sensitive and resistant breast cancer cells and we observed that autophagy [2, 3] or androgen receptor (AR) [4] play pro-survival roles, being potential targets to improve treatment. Aim: Considering that the mechanism of action of the non-steroidal AIs Ana and Let is not fully understood, it was investigated their biological effects in Ana-/Let-treated sensitive (MCF-7aro) ER+ breast cancer cells. Results: Results demonstrated that contrary to Exe, Ana and Let did not induce autophagy in sensitive [5] cancer cells but instead cause senescence. Moreover, Ana and Let act as pure AIs, whereas Exe besides acting as an AI also has the ability to modulate both ER and AR. Conclusion: Our work provides new insights on the mechanisms induced by the different AIs on cancer cells, highlighting new targets to improve treatment. Furthermore, our results corroborate the lack of cross-resistance between the AIs verified in clinic and it discourages the sequential use of Exe as second-line therapy in postmenopausal breast cancer, highlighting their beneficial use as first-line. It may also support the ongoing clinical trials with AIs and AR antagonists.

Acknowledgements: FCT for C. Amaral contract (DL 57/2016 Norma Transitória, Post-doc grant SFRH/BPD/98304/2013), T. Augusto PhD grant (BD/128333/2017) and C. Almeida PhD grant (UI/BD/151314/2021). This work was supported by UCIBIO (UIDP/04378/2020, UIDB/04378/2020) and i4HB (LA/P/0140/2020), by national funds from FCT.

References:

[1] Augusto, T.V., et al., Acquired resistance to aromatase inhibitors: where we stand! Endocr Relat Cancer, 2018 25 (5): R283-R301. doi: 10.1530/ERC-17-0425 

[2] Amaral, C., et al., Apoptosis and autophagy in breast cancer cells following exemestane treatment. PLoS One, 2012. 7(8): e42398. doi: 10.1371/journal.pone.0042398

[3] Amaral, C., et al., Hormone-dependent breast cancer: Targeting autophagy and PI3K overcomes Exemestane-acquired resistance. J Steroid Biochem Mol Biol, 2018. 183: 51-61. doi: 10.1016/j.jsbmb.2018.05.006

[4] Amaral, C., et al., The potential clinical benefit of targeting androgen receptor (AR) in estrogen-receptor positive breast cancer cells treated with Exemestane. Biochim Biophys Acta Mol Basis Dis, 2020. 1866(5): 165661. doi: 10.1016/j.bbadis.2019.165661

[5] Augusto, T.V., et al., Differential biological effects of aromatase inhibitors: Apoptosis, autophagy, senescence and modulation of the hormonal status in breast cancer cells. Mol Cell Endocrinol, 2021. 537: 111426. doi: 10.1016/j.mce.2021.111426

Erythrocyte Peroxidases in Non-Immune Hemolytic Anemias – oxidative stress and antioxidant response

Daniela Melo1, 2, Fátima Ferreira3, Maria José Teles4, 5, 6, 7, Graça Porto5, 7, 8, 9, Susana Coimbra1, 2, 10, Susana Rocha1, 2, Alice Santos-Silva1, 2

1UCIBIO, REQUIMTE, Laboratory of Biochemistry, Faculty of Pharmacy, University of Porto; 2Associate Laboratory i4HB - Institute for Health and Bioeconomy, FFUP; 3Hematology Service, CHU de São João, Porto; 4Clinical Pathology, CHU de São João, Porto;  5Clinical Hematology, Santo António Hospital – CHUP; 6Anemia Working Group Portugal; 7Institute for the Health Research and Innovation (i3S), University of Porto; 8Center for Predictive and Preventive Genetics (CGPP), University of Porto; 9Abel Salazar Institute of Biomedical Sciences, University of Porto; 10TOXRUN – Toxicology Research Unit, University Institute of Health Sciences, CESPU, CRL, Gandra

Non-immune hemolytic anemias (NIHAs) are associated to oxidative stress (OS). Pathologic red blood cells (RBCs) are metabolically stressed to maintain cell integrity, accumulating OS-induced damage within the cells. To counteract the oxidative modifications, the RBCs are equipped with an efficient antioxidant system, in which peroxiredoxin 2 (Prx2), glutathione peroxidase (GPx) and catalase (CAT) are known as the main H2O2 scavengers. The role of these cytosolic RBC peroxidases in the RBC membrane and their contribution for the clinical outcome of NIHAs has been one of the focus of our group’s research [1, 2]. Our aim is to study the crosstalk between Prx2, GPx and CAT in association with RBC’s OS biomarkers in NIHAs (in vivo models), as well as, in in vitro models using healthy RBCs and in a human erythroleukemia cell line (HEL). Incubating healthy RBCs with increasing H2O2 concentrations while inhibiting methemoglobin formation, Prx2 appears to shift its action from peroxidase to chaperone of hemoglobin [4]; while inhibiting CAT, we observed GPx binding to the RBC membrane probably to protect the membrane, contributing to lipid peroxidation prevention [3]. Moreover, inhibition of CAT in HEL cells induced the most significant effects on viability and proliferation. In a preliminary study with NIHA patients versus healthy individuals [5], we found a significant reduction in RBC total antioxidant status alongside with an increase in RBC’s ascorbic acid (AA), which may be an adaptive mechanism to OS, resulting from mobilization of plasma’s AA. Our work contributes to unfold the interplay between RBC peroxidases in the erythrocyte antioxidant system and their importance in the biology and clinical outcome of NIHAs, which can be useful in the development of potential therapeutic targets and in the establishment of biomarkers for prognosis and clinical management of these anemias.

Acknowledgments: Financial support from FCT/MCTES through national funds to Applied Molecular Biosciences Unit (UCIBIO), UIDP/04378/2020 and UIDB/04378/2020 and the project LA/P/0140/2020 of Associate Laboratory Institute for Health and Bioeconomy (i4HB) and Daniela Melo’s PhD Grant (SFRH/BD/139622/2018).

References:

[1] S. Rocha et al., Biochim Biophys Acta Biomembr., 2020, DOI: 10.1016/j.bbamem.2019.183172

[2] S. Rocha et al., Free Radic. Res., 2015, DOI: https://doi.org/10.3109/10715762.2015.1028402

[3] Melo D. et al., Free Radic. Biol, 2019, DOI: https://doi.org/10.1016/j.freeradbiomed.2019.05.021

[4] Melo D. et al., Free Radic. Biol, 2018, DOI:10.1016/j.freeradbiomed.2018.04.274

[5] Melo D. et al. Free Radic. Biol, 2020, DOI: 10.1016/j.freeradbiomed.2020.10.084

Development of an in vivo model of Wolcott-Rallison syndrome using the small vertebrate zebrafish

Liliana M. Almeida, Leonor P. Lima, Rui F. O. Silva, Brígida R. Pinho, Jorge M. A. Oliveira 

UCIBIO-REQUIMTE – Applied Molecular Biosciences Unit; i4HB - Institute for Health and Bioeconomy, Dep. Drug Sciences, Pharmacology Lab, FFUP, U.Porto, PT 

Wolcott-Rallison syndrome (WRS) is recognised as the most frequent genetic cause of neonatal diabetes in patients with consanguineous parents. Missense mutations in the gene EIF2AK3 that codes for the kinase PERK – an endoplasmic reticulum protein that decreases protein synthesis in response to proteotoxic stress - can cause partial or total loss of its kinase activity and the development of WRS [1]. WRS manifest during the embryonic development by neonatal diabetes, skeletal and cardiac abnormalities, and delayed motor or cognitive development [1]. WRS is a rare disease (ORPHA:1667) that has few models available for studying disease mechanisms and potential treatments. Our goal was to investigate if an inhibitor of PERK (GSK2606414, GSK) mimics key human WRS features in zebrafish, which is commonly used to study embryonic development due to high fecundity rates, fast ex utero development and good homology with humans [3]. We also characterized the toxicity of selective (CCT020312, CCT) and non-selective (thapsigargin, Tg) PERK activators since this strategy has been proposed to benefit individuals suffering with mutations that partially decrease PERK activity [4]. We treated zebrafish at 4 hours post fertilization (hpf) with solvent or drugs and, at 28, 52 and 76 hpf, we classified zebrafish as alive or dead, and scored their detectable abnormalities as skeletal, cardiac, or others. At 76hpf, for live zebrafish, we evaluated heart rate (bpm), sensorimotor reflexes (% positive responses after head and tail touches) and development of diabetic phenotype (labelling with a fluorescent glucose analogue). PERK inhibition with GSK2606414 (10 μM) induced concentration-dependent toxicity and death at 76 hpf, while also mimicking main WRS phenotypes: diabetes, skeletal defects, cardiac oedema, bradycardia and decreased sensorimotor reflexes. Regarding PERK activators, the No Observed Adverse Effect Level (NOAEL) for Tg within the evaluated parameters was 0.1 μM since higher concentrations reduced sensorimotor reflexes and induced skeletal abnormalities. NOAEL for CCT was 3 μM, while higher concentrations induced bradycardia and death. These results indicate that the pharmacological inhibition of PERK in zebrafish larvae mimics main developmental defects present in human WRS, supporting the genetic disruption of PERK in zebrafish as a potential model for WRS. The NOAEL concentrations of CCT or Tg could be used to test if PERK activation recovers WRS phenotypes.  

References: [1] Julier C et al. Orphanet J Rare Dis. 2010; 4; 5:29; 

[2] Hetz C et al. Nat Rev Mol Cell Biol. 2020; 21(8):421-438; 

[3] Basnet RM, et al. Biomedicines. 2019; 7(1):23; 

[4] Yuan SH et al. Hum Mol Genet. 2018 Nov 15;27(22):3951-3963.

Acknowledgments: FCT: UIDB/04378/2020; UIDP/04378/2020; LA/P/0140/2020; SFRH/BD/138451/2018; DL57/2016/CP1346/CT0016; SFRH/BD/145364/2019; PTDC/CTA-AMB/31544/2017. 

Searching for a vaccine against toxoplasmosis

Carina Brito1, Daniela Teixeira1, 2, Alexandra Correia3, 4, 5, Manuel Vilanova3, 4, 5, Salette Reis6, Natércia Teixeira7,8, Margarida Borges7, 8

1.         Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Porto, Portugal

2.         Departamento de Ciências Médicas, Universidade de Aveiro, Aveiro, Portugal

3.         Immunobiology Group, Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal

4.         Instituto de Biologia Molecular (IBMC), Universidade do Porto, Porto, Portugal

5.         Departamento de Imuno-Fisiologia e Farmacologia, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal.

6.         REQUIMTE, Department of Chemical Sciences - Applied Chemistry Lab, Faculty of Pharmacy, University of Porto, Porto, Portugal

7.         Associate Laboratory i4HB—Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto, Porto, Portugal

8.         UCIBIO/REQUIMTE, Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal

Toxoplasma gondii is a zoonotic parasite infecting about one-third of the world population. Humans can be infected through the ingestion of contaminated meat, vegetables and water, organ transplantation, blood transfusion, or vertical transmission. In immunocompromised hosts, the infection can originate neurological, ocular, or systemic disease. Primary infection during pregnancy may cause abortion or congenital disease. At present, there is no vaccine to prevent human disease caused by T. gondii [1, 2]. We have established a murine model of congenital toxoplasmosis, in which new potential vaccines could be tested. In this model, maternal T. gondii infection induced profound alterations at the maternal-foetal interface, such as inflammatory cell infiltration and tissue necrosis [3].

Our work aims to obtain an effective vaccine against toxoplasmosis. Since mucosal vaccination induces systemic and mucosal immunity, we are performing in vivo experiments, using an intranasal immunization protocol targeting T. gondii membrane proteins (TgMP) plus CpG adjuvant, a strategy that proved to be effective against the related protozoan Neospora caninum [4]. To overcome limitations, such as the high cost of CpG adjuvant, and the need for a high amount of TgMP to induce protection, work is being undertaken to achieve a nanoformulation loading TgMP, suitable for nasal administration and to increase the immunogenic properties of TgMP.

Our data showed that intranasal immunization with TgMP plus CpG conferred significant protection against murine T. gondii infection and a significant increase and stable levels of TgMP-specific IgG1, IgG2a and IgA in the short and long term. The synthesis of a chitosan/carrageenan delivery nanosystem containing TgMP was optimised and the encapsulation efficiency of TgMP was determined.

The results are promising and set the basis for optimization and validation of the nanoformulation in future in vivo experiments.

References

1. Borges, M., et al., How does toxoplasmosis affect the maternal-foetal immune interface and pregnancy? Parasite Immunol, 2019. 41(3): p. e12606.

2. Barros, M., et al., Vaccines in Congenital Toxoplasmosis: Advances and Perspectives. Frontiers in Immunology, 2021. 11(3900). 

3. Brito, C., et al., Toxoplasma gondii infection reduces serum progesterone levels and adverse effects at the maternal-foetal interface. Parasite Immunol, 2020. 42(2): p. e12690.

4. Ferreirinha, P., et al., Protective effect of intranasal immunization with Neospora caninum membrane antigens against murine neosporosis established through the gastrointestinal tract. Immunology, 2014. 141(2): p. 256-67.

The role of glutathione signaling in mitophagy induced by oxidative stress: cytoprotective role in stroke?

Diana S. F. Tavares 1,2,3, Cláudia Figueiredo-Pereira2 , Pedro Cipriano 1,2 , Beatriz Villarejo 4, Patricia Boya 4 and Helena L. A. Vieira 1,2,3

1 UCIBIO, Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, Caparica, Portugal, 2 CEDOC - Faculdade de Ciências Médicas / NOVA Medical School, Universidade NOVA de Lisboa, Portugal, 3 Associate Laboratory i4HB - Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, Caparica, Portugal,4 Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas Margarita Salas, CSIC,28040 Madrid, Spain

Mitochondria are key organelles for several cellular processes, such as production of bioenergy and biomolecules, and control of cell death. Because of oxidative phosphorylation, mitochondria are also the main cellular source of reactive oxygen species (ROS). Mitochondrial ROS are signaling molecules, but whenever mitochondria are dysfunctional, ROS are generated in excess leading to oxidative stress and eventually cell death, which amplifies tissue damage in stroke. Thus, the maintenance of mitochondrial homeostasis is critical and occurs by mitochondrial quality control processes, namely: fusion-fission, mitophagy and mitochondrial biogenesis. Mitophagy is a type of selective autophagy promoting degradation of excessive or damaged mitochondria. The accumulation of ROS induces mitophagy through PTEN-induced kinase 1 (PINK1)-PARKIN dependent pathway. Moreover, excessive ROS are also counteracted by glutathione (GSH), the main cellular non-enzymatic antioxidant molecule. GSH reduces oxidative species, generating oxidized glutathione (GSSG). Increased levels of GSSG leads to protein glutathionylation, which is a post-transcriptional protein modification that alters protein function. The aim is to disclose the molecular mechanisms underlying mitophagy activation in response to ROS in neurons and astrocytes.

Herein, we hypothesize that mild uncoupling of mitochondria (induced by carbonyl cyanide m- chlorophenylhydrazone - CCCP) generates low amounts of ROS, that in turn stimulate mitophagy via protein glutathionylation. Mitophagy reporter mitoQC transgene expressed in the human neuroblastoma SH-SY5Y cell line and in mice (for isolating primary culture of astrocytes) have been used for mitophagy assessment in a more accurate manner.

In primary culture of astrocytes and in the neuron-like cell line SH-SY5Y, CCCP-induced mitophagy increased ROS levels and the expression of γ-glutamyl-cysteine synthetase (GCLC), which is the rate limiting enzyme for GSH synthesis. Accordingly, CCCP induced mitophagy via PINK1/PARKIN pathway in neuron-like cells and astrocytes. Using immunoprecipitation techniques, PINK1 appears to be glutathionylated during CCCP-induced mitophagy in astrocytes. Revealing the role of protein glutathionylation in ROS-induced mitophagy is a step forward for the development of cytoprotective strategies against stroke.

References:

1. Mauro-Lizcano M, Esteban-Martínez L, Seco E, Serrano-Puebla A, Garcia-Ledo L, Vieira HLA, et al. New method to assess mitophagy flux by flow cytometry. Autophagy. Taylor and Francis Inc.; 2015;11:833–43.

2. Rosignol I, Villarejo-Zori B, Teresak P, Sierra-Filardi E, Pereiro X, Rodríguez-Muela N, et al. The mito-QC Reporter for Quantitative Mitophagy Assessment in Primary Retinal Ganglion Cells and Experimental Glaucoma Models. Int J Mol Sci [Internet]. 2020;21:1882. Available from: https://www.mdpi.com/1422-0067/21/5/1882

3. Queiroga CSF, Almeida AS, Martel C, Brenner C, Alves PM, Vieira HLA. Glutathionylation of Adenine Nucleotide Translocase Induced by Carbon Monoxide Prevents Mitochondrial Membrane Permeabilization and Apoptosis. J Biol Chem. 2010;285:17077–88.

4. Figueiredo-Pereira C, Menezes R, Ferreira S, Santos CN, Vieira HLA. Carbon monoxide released by CORM-A1 prevents yeast cell death via autophagy stimulation. FEMS Yeast Res. England; 2019;19.

5. Almeida AS, Soares NL, Sequeira CO, Pereira A, Sonnewald U, Vieira HLA. Redox Biology Improvement of neuronal differentiation by carbon monoxide : Role of pentose phosphate pathway. 2018;17:338–47.

The role of cannabinoids in human placenta development: impact on angiogenesis and endocrine function

João Maia1,2, Bruno Fonseca1,2, Natércia Teixeira1,2, Georgina Correia-da-Silva1,2

1UCIBIO – Applied Molecular Biosciences Unit, Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

2 Associate Laboratory i4HB - Institute for Health and Bioeconomy, Faculty of Pharmacy, University of Porto, Portugal.

Introduction: In recent years, cannabis consumption has increased for both medical and recreational purposes. However, its impact in pregnancy and placental development is still understudied. Despite this, an increase in cannabis consumption during gestation has been observed, which is associated to poor reproductive outcomes, such as low birth weight, prematurity, and intrauterine growth restriction. These effects may be due to the main psychoactive compound of cannabis, Δ9-Tetrahydrocannabinol (THC). It activates the cannabinoid receptors CB1 and CB2 and together with their main endogenous ligands the endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) and their metabolic enzymes constitute the endocannabinoid system (ECS). This system modulates several physiological functions including reproductive events as decidualization and placentation.

Aim: To elucidate the role of cannabinoids in placenta development, the effect of  THC and 2-AG in placental angiogenesis and trophoblast cell migration were examined.

Results: Both cannabinoid receptors and endocannabinoids are integrated components of the networks controlling endocrine function [1] and differentiation and apoptosis of trophoblast [2,3]. In placental explants, THC caused major alterations in estrogen signaling and 2-AG levels and expression of its main metabolic enzymes. Moreover, these fluctuations allowed us to report, for the first time, the presence of two non-canonical 2-AG metabolic enzymes ABHD6 and ABHD12 in the placenta. As 2-AG effects on angiogenesis and invasion, are yet unexplored, we examined its impact on cell migration and angiogenesis on extravillous trophoblasts, the HTR8/SVneo cells. The results showed that 2-AG affects the expression of angiogenic transcription factors, like PlGF, as well as cell migration and tube formation.

Conclusion: THC impacts 2-AG enzymatic machinery and placental endocrine function, while 2-AG itself modulates placental angiogenesis and cell migration.

Funding

This work is financed by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. João Maia also thanks FCT for his PhD grant SFRH/BD/136105/2018.

References

1.   Costa, M.A., et al., The endocannabinoid 2-arachidonoylglycerol dysregulates the synthesis of proteins by the human syncytiotrophoblast. Biochim Biophys Acta, 2016. 1861(3): p. 205-12.

2.   Costa, M.A., et al., The psychoactive compound of Cannabis sativa, Δ(9)-tetrahydrocannabinol (THC) inhibits the human trophoblast cell turnover. Toxicology, 2015. 334: p. 94-103.

3.   Costa, M.A., et al., 2-arachidonoylglycerol effects in cytotrophoblasts: metabolic enzymes expression and apoptosis in BeWo cells. Reproduction, 2014. 147(3): p. 301-11. 

4.   Maia, J., et al., Effects of cannabis tetrahydrocannabinol on endocannabinoid homeostasis in human placenta. Archives of Toxicology, 2019. 93(3): p. 649-658.

5.   Maia, J., et al., Impact of tetrahydrocannabinol on the endocannabinoid 2-arachidonoylglycerol metabolism: ABHD6 and ABHD12 as novel players in human placenta. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2020. 1865(12): p. 158807. 

6.   Maia, J., et al., The Cannabinoid Delta-9-tetrahydrocannabinol Disrupts Estrogen Signaling in Human Placenta. Toxicological Sciences, 2020. 177(2): p. 420-430.

Remote Ischemic Conditioning against Ischemic Stroke: Identification of Circulating Signaling Factors

Catarina C. Pires 1,2,4, Inês Mollet 1,2,4, Ricardo Viana-Soares 2, Marcelo Mendonça 2, João Pedro Marto 2,3, Cláudia S.F. Queiroga 2, Miguel Viana Baptista 2,3 e Helena L. A. Vieira 1,2,4

1 UCIBIO, Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, Caparica, Portugal 2 CEDOC - Faculdade de Ciências Médicas / NOVA Medical School, Universidade NOVA de Lisboa, Portugal 3 Department of Neurology, Hospital de Egas Moniz, Centro Hospitalar Lisboa Ocidental, Lisboa Portugal 4 Associate Laboratory i4HB - Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, Caparica, Portugal

This project focuses on remote ischemic conditioning (RIC) and its potential use as a novel therapy against ischemic stroke. Stroke is the first cause of death and disability in Portugal and the second in Europe. The available therapeutic approaches to stroke treatment, thrombolysis and mechanical thrombectomy, promote blood reperfusion, but do not directly target the brain parenchyma.

RIC is a specific sub-type of conditioning that triggers our endogenous mechanisms of defense through the application of a stimulus, below the damage threshold, such as ischemia. In this particular case, non-vital organ (arm) is submitted to 4 cycles of 5 minutes ischemic stimuli, the RIC procedure, which may stimulate protective signaling to remote target organs (such as brain)[1]. To further clarify this potential mechanisms underlying the neuroprotective effects of RIC, healthy volunteers were submitted to RIC followed by: (i) assessment of autonomic nervous system [2], analysis of circulating immune cells [3] and biochemical characterization of plasma (ongoing work). The timepoints for the characterization of circulating factors are: before RIC, immediately, 4h and 22h after RIC.

Proteomic assessment of RIC-derived plasma proteins by mass spectrometry has identified 30 significantly differently expressed proteins. Moreover, carboxyhemoglobin, nitric oxide and glutathione metabolites were also analyzed, being COHb the single factor increasing in response to RIC. Finally functional tests of conditioned plasma were also performed for assessing: (i) neuroinflammation using human microglia cell line HMC3, (ii) neuroprotection using differentiated and non-differentiated human neuroblastoma SH-SY5Y cell line and (iii) BBB integrity using human cerebral microvasculature endothelial cells hCMEC/D3. Preliminary data of conditioned plasma do not show any significant change in the analyzed parameters. One may speculate that being a mild response and activation of endogenous protection, RIC should be applied in a more chronic manner.

Ultimately, the aim of this project is to explore what is activated in the arm that acts on the brain and how this inter-organ communication is established.

References:

1. Mollet I, Marto JP, Mendonça M, Baptista MV, Vieira HLA. Remote but not Distant: a Review on Experimental Models and Clinical Trials in Remote Ischemic Conditioning as Potential Therapy in Ischemic Stroke. Mol Neurobiol [Internet]. 2022;59:294–325. Available from: https://link.springer.com/10.1007/s12035-021-02585-6

2. Noronha Osório D, Viana-Soares R, Marto JP, Mendonça MD, Silva HP, Quaresma C, et al. Autonomic nervous system response to remote ischemic conditioning: Heart rate variability assessment. BMC Cardiovasc Disord. BioMed Central Ltd.; 2019;19.

3. Mollet I, Martins C, Ângelo-Dias M, Carvalho AS, Aloria K, Matthiesen R, et al. Pilot study in human healthy volunteers on the mechanisms underlying remote ischemic conditioning (RIC) – Targeting circulating immune cells and immune-related proteins. J Neuroimmunol [Internet]. 2022;577847. Available from: https://linkinghub.elsevier.com/retrieve/pii/S016557282200042X