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Age-dependent microglia inflammatory profile in the spinal cord of the ALS mSOD1G93A mice
Publication . Roberto, Afonso Sabino Ferreira; Botelho, Ana Rita Mendonça Vaz; Brites, Dora Maria Tuna de Oliveira
A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa fatal e heterogénea, caracterizada pela perda progressiva de neurónios motores e neuroinflamação. A microglia-associada-a-doença (MAD) desempenha um papel crucial, adotando fenótipos neurotóxicos ao longo da progressão de patologias. Além disso, existe evidência de que a disfunção microglial está relacionada com variações, dependentes da idade, em marcadores inflamatórios. Contudo, essa variação e os seus mecanismos moleculares subjacentes permanecem pouco compreendidos na patogénese da ELA.
Neste contexto, é essencial investigar os microRNAs (miRNAs) associados à inflamação, uma vez que são reguladores importantes da inflamação durante a progressão da doença e têm potencial terapêutico como alvos de modulação. O objetivo deste estudo é investigar o perfil inflamatório de miRNAs e os estádios reativos, dependentes da idade, na microglia espinhal de murganhos com mutação G93A no gene da superóxido dismutase-1, modelo mSOD1G93A de ELA. Assim, foram utilizadas culturas primárias de microglia espinhal de murganhos recém-nascidos mSOD1G93A (TG) e selvagens (WT) e analisados miRNAs inflamatórios (miR-21, miR-124, miR-125b, miR-146a e miR-155) e marcadores inflamatórios/fagocíticos (TNF-α, ARG1, iNOS, TREM2 e MFG-E8) aos 2, 10 e 16 dias in vitro (DIV), simulando as fases jovem/reativa, madura e envelhecida/senescentes, respetivamente. Os resultados obtidos revelam alterações dinâmicas na expressão de miRNAs com a idade, que se correlacionam com mudanças funcionais da microglia, de um estado reativo para um estado pouco responsivo. A microglia mSOD1 aos 2 DIV apresentou sobre-expressão do miR-124 e um fenótipo pró-inflamatório (sobre-expressão de iNOS e MFG-E8 e sub-expressão de ARG1). Aos 10 DIV, a microglia mSOD1 apresentou sub-expressão de miR-125b e miR-155a, associada à expressão aumentada de TNF-α e TREM2, possivelmente contribuindo para um perfil neurotóxico e inflamação crónica. Aos 16 DIV, encontrou-se um padrão misto na microglia mSOD1, caracterizado pela diminuição de miR-146a e sub-expressão de marcadores pró- e anti-inflamatórios, respetivamente TNF-α e ARG1.
Em suma, estes resultados demonstram um perfil complexo de miRNAs, dependente da idade, na microglia mSOD1, que transita de uma reatividade pró-inflamatória para um fenótipo mais disfuncional e de inflamação mista. A modulação específica destes miRNAs, regulando essas transições e restabelecendo a neuroprotecção, pode fornecer terapêuticas promissoras para a ELA.
Improving human mesenchymal stem cell-derived hepatic cell energy metabolism by manipulating glucose homeostasis and glucocorticoid signaling
Publication . Rodrigues, Joana S.; Faria-Pereira, Andreia; Camões, Sérgio P.; Serras, Ana S.; Morais, Vanessa A.; Ruas, Jorge Lira; Miranda, Joana P
Introduction: The development of reliable hepatic in vitro models may provide insights into disease mechanisms, linking hepatocyte dysmetabolism and related pathologies. However, several of the existing models depend on using high concentrations of hepatocyte differentiation-promoting compounds, namely glucose, insulin, and dexamethasone, which is among the reasons that have hampered their use for modeling metabolism-related diseases. This work focused on modulating glucose homeostasis and glucocorticoid concentration to improve the suitability of a mesenchymal stem-cell (MSC)-derived hepatocyte-like cell (HLC) human model for studying hepatic insulin action and disease modeling.
Methods: We have investigated the role of insulin, glucose and dexamethasone on mitochondrial function, insulin signaling and carbohydrate metabolism, namely AKT phosphorylation, glycogen storage ability, glycolysis and gluconeogenesis, as well as fatty acid oxidation and bile acid metabolism gene expression in HLCs. In addition, we evaluated cell morphological features, albumin and urea production, the presence of hepatic-specific markers, biotransformation ability and mitochondrial function.
Results: Using glucose, insulin and dexamethasone levels close to physiological concentrations improved insulin responsiveness in HLCs, as demonstrated by AKT phosphorylation, upregulation of glycolysis and downregulation of Irs2 and gluconeogenesis and fatty acid oxidation pathways. Ammonia detoxification, EROD and UGT activities and sensitivity to paracetamol cytotoxicity were also enhanced under more physiologically relevant conditions.
Conclusion: HLCs kept under reduced concentrations of glucose, insulin and dexamethasone presented an improved hepatic phenotype and insulin sensitivity demonstrating superior potential as an in vitro platform for modeling energy metabolism-related disorders, namely for the investigation of the insulin signaling pathway.
Spatiotemporal dysregulation of neuron-glia related genes and pro-/anti-inflammatory mirnas in the 5xFAD mouse model of Alzheimer’s diseaseouse model of Alzheimer’s disease
Publication . Ianni, Marta; Corraliza-Gomez, Miriam; Costa-Coelho, Tiago; Ferreira-Manso, Mafalda; Inteiro-Oliveira, Sara; Alemãn-Serrano, Nuno; Sebastião, Ana M; Garcia, Gonçalo; Diógenes, Maria José; Brites, Dora
Alzheimer's disease (AD), the leading cause of dementia, is a multifactorial disease influenced by aging, genetics, and environmental factors. miRNAs are crucial regulators of gene expression and play significant roles in AD onset and progression. This exploratory study analyzed the expression levels of 28 genes and 5 miRNAs (miR-124-3p, miR-125b-5p, miR-21-5p, miR-146a-5p, and miR-155-5p) related to AD pathology and neuroimmune responses using RT-qPCR. Analyses were conducted in the prefrontal cortex (PFC) and the hippocampus (HPC) of the 5xFAD mouse AD model at 6 and 9 months old. Data highlighted upregulated genes encoding for glial fibrillary acidic protein (Gfap), triggering receptor expressed on myeloid cells (Trem2) and cystatin F (Cst7), in the 5xFAD mice at both regions and ages highlighting their roles as critical disease players and potential biomarkers. Overexpression of genes encoding for CCAAT enhancer-binding protein alpha (Cebpa) and myelin proteolipid protein (Plp) in the PFC, as well as for BCL2 apoptosis regulator (Bcl2) and purinergic receptor P2Y12 (P2yr12) in the HPC, together with upregulated microRNA(miR)-146a-5p in the PFC, prevailed in 9-month-old animals. miR-155 positively correlated with miR-146a and miR-21 in the PFC, and miR-125b positively correlated with miR-155, miR-21, while miR-146a in the HPC. Correlations between genes and miRNAs were dynamic, varying by genotype, region, and age, suggesting an intricate, disease-modulated interaction between miRNAs and target pathways. These findings contribute to our understanding of miRNAs as therapeutic targets for AD, given their multifaceted effects on neurons and glial cells.
The blood-brain barrier in brain metastasization of breast cancer : a source of biomarkers, a target for modulation, and an obstacle to overcome
Publication . Pereira, Joana; Brito, Maria Alexandra de Oliveira Silva Braga Pedreira de; Videira, Mafalda de Castro Ascensão Marques; Carvalheiro, Manuela Colla
As of 2020, breast cancer (BC) has become the leading cause of neoplastic disease worldwide, with an estimated 3 million new cases, and projections indicate that it will result in 1 million fatalities by 2040. An alarming concern of this disease is its metastatic stage, responsible for most BC deaths. Among all BC subtypes, triple-negative BC (TNBC) stands out as the most aggressive with a poor prognosis. It is also particularly susceptible to developing brain metastases. BC brain metastases (BCBM) leave patients with limited treatment options and extremely low survival rates, due to lack of a timely diagnose owing to the absence of biomarkers, while blood-brain barrier (BBB) restricts most drugs’ brain access. Thus, it is crucial to understand the mechanism involved in the interaction between BBB endothelial cells and breast cancer cells (BCCs) prior to brain metastases formation, as well as the identification of early biomarkers of this process. Additionally, new therapeutics addressing BCCs extravasation process and eradicate BCBM should be investigated. This thesis stands out due to its multifaceted approach, which includes the 1) investigation of the key molecules involved in the interaction between BBB endothelial cells and BCCs during BCBM development; 2) identification of circulating biomarkers indicative of BCBM development, with potential diagnostic and prognostic value, utilizing liquid biopsy; 3) uncover molecules capable of preserving the BBB during extravasation, aiming to impede the transendothelial migration (TEM) of BCCs as a preventive measure; 4) and explore novel treatment strategies, such as the co-administration of a cytotoxic drug and a small interfering RNA (siRNA) to abrogate the already established BCBM.
Firstly, the players involved in TNBC cells (4T1 cell line) extravasation, as well as its temporal and spatial profile were disclosed in vivo and in vitro. Key mechanisms such as paracellular and transcellular BBB permeability, cell communication, cytoskeleton remodelling, proliferative and migratory BCC´s properties accompanied with invadopodium formation were revealed. Moreover, brain microvascular endotelial-derived extracellular vesicles and specific microRNAs (miR) were identified as biomarkers of advanced and precocious stages of BCBM, respectively.
Secondly, given the altered BBB permeability upon BCCs contact, a preventive strategy was explored aiming the improvement of BBB’ properties. A high-throughput microscopy screening was performed on a library of new and already approved drugs to assess their ability to preserve BBB properties and prevent BCCs from adhering and migrating across the endothelium. Among the tested drugs, minocycline hydrochloride (MH) was the most promising in preventing BBB disruption and 4T1 cells migration, thus rising as a potential modulator of BBB integrity preservation. To minimize side effects and ensure specific BBB-delivery, a trojan horse drug carrier approach was employed targeting the transferrin receptor (TfR), which is highly expressed in brain microvascular endothelial cells, using the RI7217 (Ri7), an anti-TfR antibody. A transferrin receptor-targeted liposome (Lip) carrying MH (Ri7-MH-Lip) was developed and characterized. Ri7-MH-Lip disclosed increased cellular uptake via clathrin-dependent pathway, with no endothelial safety concerns. The Ri7-MH-Lip treatment not only reduced in vitro BCC-induced junctional protein disruption but also decreased endothelial matrix metalloproteinase 9 (MMP-9) overexpression. Additionally, it hampered BCCs’ adhesion to the endothelial monolayer via downregulation of endothelial vascular cell adhesion molecule 1. Ri7-MH-Lip also showed a safe and efficient outcome in vivo. No alteration in the enzymes associated with cardiac, renal, and hepatic toxicity was observed. Moreover, a reduction in BCBM was demonstrated, with the decrease of paracellular permeability and immune-cell infiltration. Furthermore, modulation of circulant miR-802-5p and miR-194-5p expression revealed their potential to be used as biomarkers of therapeutic response.
Finally, given the persistent expression of platelet-derived growth factor subunit B (PDGF-B) in BCCs during the formation of brain metastases, coupled with the absence of specific therapies for this pathology, there is a pressing need for new therapeutic strategies. This thesis unveiled the potential of a BBB-permeant dual therapeutic strategy for the delivery of a cytotoxic molecule [salinomycin - SAL] and siRNA therapy [anti-PDGF-B - siPDGF-B] to induce BCBM eradication. Stable nucleic acid lipid particle (SNALP) for encapsulating siPDGF-B, as well as liposomes for incorporating SAL were developed and characterized. Both were conjugated with chlorotoxin (CTX) for to specifically target BCCs. The resulting formulations are denoted as CTX-siPDGF-B-SNALP and CTX-SAL-Lip, respectively. The co-administration of these systems in vitro inhibited BCCs’ proliferation (Ki-67) while the application of each formulation individually led to cell senescence in both cases. The formulations did not affect endothelial viability, although SAL liposomes impaired BBB integrity. In vivo, treatment with siPDGF-B lipid particles effectively reduced both the number of metastases and the levels of PDGF-B and Ki-67.
In conclusion, this study has unveiled the cellular and molecular processes involved in BCBM formation, it has also revealed how preventive and therapeutic strategies influence the pathological events associated with disease development. Notably, miR-802 and miR-194 have emerged as valuable diagnostic and prognostic biomarkers. While this study is merely the tip of the iceberg, it introduces exciting possibilities. It suggests repurposing MH as a preventive therapeutic approach and utilizing siPDGF-B as treatment. These findings pave the way for the development of personalized pharmacological strategies to combat this illness.
Thioredoxin reductase inhibitors in anticancer therapy : thimerosal efficacy in Glioblastoma
Publication . Bramatti, Isabella; Carvalho, Cristina; Branco, Vasco; Gonçalves, João Manuel Braz
Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor with a low life expectancy. The standard treatment includes resection surgery, radiotherapy and chemotherapy with temozolomide (TMZ). TMZ is considered the first choice and standard treatment, but therapeutic success is limited due to neoangiogenesis, intratumor heterogeneity and therapy resistance, with overall patient survival increasing by just 14 months. This poor outcome of current therapies increases the demand for new, more effective therapeutic options, including drug repurposing.
Tumor hypoxia involves the development of non-functional blood vessels within neoplastic tissue, leading to tumor survival, invasion and metastasis. Cancer growth and resistance to therapy are frequently related to overexpression of antioxidant systems such as the thioredoxin system. This system includes the disulfide reductase, thioredoxin (Trx), the selenoenzyme thioredoxin reductase (TrxR) and NADPH. This system is fundamental for cell survival and proliferation with a key role in maintaining redox homeostasis, being able to provide electrons to a large range of enzymes critical for DNA synthesis and defense against oxidative stress, regulating hypoxia inducible factor-1alpha (HIF-1α) activity, which in turn controls vascular endothelial growth factor (VEGF), an indispensable factor for tumor invasiveness, and microenvironment tumor maintenance. HIF-1α can also be regulated by the signal transducer and activator of transcription 3 (STAT3), an oncogene stimulated by pro-inflammatory cytokines and growth factors.
Indeed, targeting of antioxidant systems arises as a potential strategy to tackle tumors since they are more sensitive to loss of redox homeostasis than normal cells and those systems are involved in more than one of the considered hallmarks of cancer. The thioredoxin system has several known inhibitors including mercury compounds such as thimerosal (TmHg), an ethylmercury-containing compound, that readily crosses the blood-brain barrier (BBB) and accumulates in the brain. Despite having a history of human use, epidemiological evidence of TmHg neurotoxicity was never obtained.
Therefore, since elevation of oxidative stress triggers different pathways that will lead to apoptosis of glioma cells and the overall survival for this cancer is still low even with emerging therapies, the goal of this thesis is to study the use of thioredoxin’ inhibitors, in this case thimerosal, as a possible candidate for hard repurposing towards GBM as well as to overcome therapy resistance to TMZ.
Results showed that TmHg significantly decreased the cell viability (> 50%) of two different glioblastoma cell lines (GL261 and U87 cell line) at much lower concentrations than TMZ, and strongly inhibited the Trx system. In fact, in GL261 cells besides an inhibition of cytosolic TrxR1/Trx1 there was a strong inhibition of mitochondrial TrxR2/Trx2 expression, predominantly with TmHg. In U87 cells, exposure to TmHg decreased cell migration, HIF-1α expression (35%) and in co-exposure with TMZ reduced HIF-1α, VEGF, and phosphorylation of STAT3, showing that tumour invasiveness related factors are significantly hampered. Furthermore, TmHg elicit N9 microglial inflammation markers - increased levels of IL1β, iNOS and TNFα and the autophagic process. Moreover, in co-cultures of microglia and GBM cells, the cytotoxicity of TmHg towards GL261 was higher.
The effectiveness of EtHg and TmHg in reducing GBM viability and affecting the Trx system could also be related with its interaction with TrxR. We demonstrated that EtHg, unlike other Hg compounds such as methylmercury, is a partial inhibitor of this selenoenzyme, forming a selenium-compromised thioredoxin reductase-derived apoptotic proteins (SecTRAPs), which enhances ROS-production and cytotoxicity.
Also, a mouse (C57BL/6) model was used in a distribution study following TmHg administration (i.v.; 20, 40 and 100 µM) for 14 days. We observed a concentration-dependent Hg accumulation in the brain, kidney and liver. Nevertheless, no neurobehavioral alterations were noticed as well as no histopathological damage in the organs, which led to the optimization and testing in a GBM model encompassing stereotactic injection of GBM cells.
Overall, thimerosal arose as a good candidate for hard repurposing towards GBM and the results obtained indicate its potential to be used alone as well as to overcome therapy resistance to TMZ.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
Concurso de avaliação no âmbito do Programa Plurianual de Financiamento de Unidades de I&D (2017/2018) - Financiamento Programático
Número da atribuição
UIDP/04138/2020