Characterization of adenosinergic system in Rett syndrome

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2015A Síndrome de Rett (RTT) é uma doença rara do neurodesenvolvimento e de causa genética que afeta cerca de 1:10000 raparigas em todo o mundo. Esta doença caracteriza-se por um aparente normal desenvolvimento até aos 6 a 18 meses de idade, seguido de uma fase de regressão, na qual ocorre a perda das capacidades já adquiridas. Entre outros sintomas, destacam-se: severa disfunção cognitiva e motora, epilepsia e aparecimento de movimentos estereotipados e repetitivos das mãos com progressiva perda da sua funcionalidade. Estudos genéticos mostraram que esta síndrome se deve, maioritariamente, a mutações no gene methyl CpG binding protein 2 (MECP2) localizado no cromossoma X. Este gene codifica a proteína MeCP2, um modulador epigenético e regulador da estrutura da cromatina, com funções primordiais no desenvolvimento e maturação do Sistema Nervoso Central (central nervous system - CNS). Uma das proteínas cuja expressão é controlada pela MeCP2 é o fator neurotrófico derivado do cérebro (brain-derived neurotrophic factor - BDNF), conhecido pelas suas importantes funções na maturação e diferenciação celular, plasticidade sináptica e sobrevivência neuronal. Consequentemente, alterações na MeCP2 podem comprometer os níveis de expressão e função do BDNF. Estudos em modelos animais, que reproduzem a maioria dos sintomas característicos da RTT, demonstraram que o aumento da expressão do BDNF consegue reverter parcialmente algumas das disfunções e sintomas desta síndrome. Contudo, o uso terapêutico de BDNF não é ainda exequível uma vez que a barreira hematoencefálica (blood-brain barrier - BBB) é impermeável a este fator neurotrófico, impedindo-o de chegar ao cérebro e desempenhar adequadamente as suas funções. Na tentativa de facilitar os efeitos do BDNF, têm-se desenvolvido novas estratégias envolvendo, por exemplo, a utilização de fármacos que atravessando a BBB potenciem a ação neuroprotetora do BDNF. Um dos fármacos que tem merecido particular atenção é a adenosina. A adenosina é um neuromodulador do CNS que exerce as suas funções através da ativação de quatro recetores, A1, A2A, A3 e A2B. Em particular, a ativação dos recetores A2A é fulcral para a manutenção dos níveis de BDNF e do seu recetor, TrkB-FL, assim como para os seus efeitos sinápticos. É de realçar que, o sistema adenosinérgico, para além de ser crucial na sinalização mediada pelo BDNF, também tem um papel de destaque no controlo da excitabilidade sináptica através da ativação dos recetores inibitórios do tipo A1, reconhecidos como potenciais alvos terapêuticos no controlo da epilepsia. Estas ações da adenosina sugerem a possibilidade de este neuromodulador também estar afetado na RTT. De facto, estudos preliminares, realizados no nosso laboratório, apontam para a existência de uma disfunção do sistema adenosinérgico em associação à desregulação já conhecida da sinalização mediada pelo BDNF na RTT. Assim, este projeto teve como principal objetivo fazer a caracterização detalhada do sistema adenosinérgico e da sinalização mediada pelo BDNF, através da utilização de: 1) modelo animal, ratinho mutante Mecp2 knockout (KO); 2) modelo humano, neurónios derivados de células pluripotentes induzidas humanas (human induced pluripotent stem cells - hIPSCs) de pacientes com RTT e 3) tecido cerebral humano recolhido durante autópsia realizada a paciente com RTT. Ensaios de ligação anteriormente realizados já tinham mostrado um aumento dos níveis proteicos dos recetores A1 em amostras de córtex cerebral de ratinhos Mecp2 KO quando comparado com amostras de ratinhos WT, não havendo alterações na expressão do seu mRNA. No presente trabalho, ensaios de Western-Blot revelaram uma diminuição significativa dos recetores do tipo A2A no córtex cerebral dos ratinhos Mecp2 KO, não tendo sido contudo, detetadas alterações nos níveis de expressão do mRNA avaliados por polimerase chain reaction (PCR) quantitativo. Curiosamente, registos eletrofisiológicos realizados no hipocampo destes animais sugerem uma diminuição dos níveis de adenosina endógena, que não é atribuível a variações nos níveis proteicos de um dos enzimas responsáveis pela degradação da adenosina, adenosina cinase (Adenosine Kinase - ADK). No modelo animal, os resultados, obtidos por ELISA, confirmaram que os níveis proteicos de BDNF estão bastante diminuídos. Semelhante resultado foi obtido para os níveis dos seus recetores TrkB-FL quando avaliados pela técnica de Western Blot, não havendo, contudo, alterações significativas nos recetores truncados deste fator neurotrófico (TrkB-Tc). No entanto, os níveis de expressão de mRNA para as duas isoformas do recetor TrkB (TrkB-FL e TrkB-T1 – isoforma truncada) não mostraram alterações significativas. Em neurónios derivados de hIPSCs, a avaliação dos recetores adenosinérgicos (A1 e A2A), do BDNF e dos recetores TrkB, efetuada através de PCR quantitativo, demonstrou uma considerável variabilidade, não sendo por isso possível fazer uma comparação com os resultados obtidos no modelo animal. Relativamente ao estudo dos níveis proteicos de BDNF, neste modelo, observou-se uma tendência para aumento e, pelo contrário, registou-se uma tendência para diminuição dos níveis de expressão dos seus recetores. Os resultados obtidos a partir da amostra de córtex temporal de uma paciente com RTT mostraram um aumento da expressão de mRNA dos recetores A1 e uma diminuição da expressão de mRNA dos recetores A2A. No que respeita aos recetores TrkB, observou-se um aumento da expressão do mRNA que codifica para o recetor TrkB-FL. Não se observou contudo alteração no mRNA para o recetor TrkB-T1. Em associação, não foi observada alteração na expressão de mRNA que codifica para o BDNF. Neste trabalho, o maior problema encontrado foi a variabilidade observada, não só entre as linhas celulares provenientes de indivíduos do mesmo género, como também entre as rondas independentes de diferenciação da mesma linha parental. Parte dessa variabilidade pode estar relacionado com as múltiplas alterações genéticas e epigenéticas que ocorrem durante os procedimentos de reprogramação e de diferenciação, tais como a fixação de mutações aleatórias esporádicas e a inativação aleatória do cromossoma X em linhas femininas. Uma outra importante fonte de variabilidade é a eficiência da produção de neurónios corticais, que é propensa a flutuar comprometendo os resultados. Ainda que alguns resultados mostrem tendências concordantes entre os modelos estudados, é difícil retirar conclusões a partir destes, o que é ainda mais agravado pelo reduzido tamanho da amostra. Globalmente, os resultados apontam para uma disfunção na sinalização mediada quer pelo BDNF quer pelo sistema adenosinérgico, sugerindo um possível envolvimento de ambos na fisiopatologia da doença. Estas evidências abrem novas perspetivas para a intervenção farmacológica nesta patologia.Rett Syndrome (RTT) is a genetic neurodevelopmental disorder, with an incidence of 1:10,000 female live births. This disorder is the main genetic cause of intellectual disability in females and it is mainly caused by mutations in the methyl-CpG binding protein 2 (MECP2) gene. The MeCP2 protein, codified by the MECP2 gene, is an epigenetic modulator that controls chromatin structure. This protein is known to modulate the expression of brain-derived neurotrophic factor (BDNF), a neurotrophin with essential functions in cell differentiation, synaptic plasticity and survival. Furthermore, BDNF overexpression can partially ameliorate some RTT associated symptoms. Thus, therapeutic strategies designed at delivering BDNF to the brain could be a breakthrough for RTT. However, this strategy has been hampered by the inability of BDNF to cross the blood-brain barrier (BBB). The development of new therapeutic strategies is, therefore, of the outmost importance. The adenosine is a neuromodulator that acts through the activation of four different receptors: A1R, A2AR, A2BR and A3R. The two most well characterized receptors in the brain are the A1R and A2AR and their manipulation has been suggested for the treatment of several neurological pathologies. Given that, the activation of A2AR is known to potentiate BDNF synaptic actions in healthy animals, one could anticipate that the activation of these adenosine receptors could be a potential therapeutic strategy. On the other hand, most of RTT patients have epilepsy, a pathology where adenosine system might be affected. However, until recently, there was no available information about the contribution of the adenosinergic system to the pathophysiology of RTT. To overcome this gap in knowledge, our lab has developed a new line of research on this topic. The main goal of this project was to further characterize both the adenosinergic system and the BDNF signalling in RTT. This goal was achieved by using: i) a well-established animal model, Mecp2 KO mice; ii) human RTT model, neurons-derived from RTT patients induced pluripotent stem cells (IPSCs) and iii) post-mortem human brain samples from a RTT patient. Even though we found some concordant tendencies between the human and the mouse models, given the high variability observed in the human material, no clear conclusions can be drawn. This is certainly aggravated by the small sample size that, in the future, could be solved by increasing not only the number of patient-derived hIPSCs lines but also by increasing the amount of independent rounds of differentiation. The availability of more post-mortem samples would also be an important asset. Overall, the here presented results clearly show a dysfunction in BDNF signalling and in the adenosinergic system, suggesting that both systems are involved in the pathophysiology logy of the disorder. These evidences open new pharmacological avenues for the treatment of RTT

Portuguese Public Debt Management During The European Sovereign Debt Crisis - a case study

The presentcase study analyses the Portuguese publicdebt management during theEuropeanSovereign debtcrisis, namely the decisions undertook by the Government and by the Portuguese Debt Management Agency.On April 6th,2011, Portugal requestedofficial financial assistance, beginning a three-year period in which market access was severely constrained.This case studyfocus onthe behaviourof relevant parameters,such as the evolution of yieldsand spreads for Government bonds, the shareof public debt held by domestic and non-domestic investors through time, the progressionof Portuguese debt ratings and thecomposition of the stock of public debt

From cannabinoids and neurosteroids to statins and the ketogenic diet: new therapeutic avenues in Rett syndrome?

Copyright © 2019 Mouro, Miranda-Lourenço, Sebastião and Diógenes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused mainly by mutations in the MECP2 gene, being one of the leading causes of mental disability in females. Mutations in the MECP2 gene are responsible for 95% of the diagnosed RTT cases and the mechanisms through which these mutations relate with symptomatology are still elusive. Children with RTT present a period of apparent normal development followed by a rapid regression in speech and behavior and a progressive deterioration of motor abilities. Epilepsy is one of the most common symptoms in RTT, occurring in 60 to 80% of RTT cases, being associated with worsening of other symptoms. At this point, no cure for RTT is available and there is a pressing need for the discovery of new drug candidates to treat its severe symptoms. However, despite being a rare disease, in the last decade research in RTT has grown exponentially. New and exciting evidence has been gathered and the etiopathogenesis of this complex, severe and untreatable disease is slowly being unfolded. Advances in gene editing techniques have prompted cure-oriented research in RTT. Nonetheless, at this point, finding a cure is a distant reality, highlighting the importance of further investigating the basic pathological mechanisms of this disease. In this review, we focus our attention in some of the newest evidence on RTT clinical and preclinical research, evaluating their impact in RTT symptomatology control, and pinpointing possible directions for future research.FM was in receipt of a fellowship (IMM/CT/8-2018). The authors would like to thank the following organizations for their funding: AdoRett – LISBOA-01-0145-FEDER-031929; the Association Française du Syndrome de Rett Program “Educação pela Ciência” | Bolsas CHLN/FMUL; GAPIC. Project No. 20190017; Twinning action (SynaNet) from the EU H2020 Programme; and the UID/BIM/50005/2019, project financed by the FCT/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through the Fundos do Orçamento de Estado.info:eu-repo/semantics/publishedVersio

Results from a population-based cohort study

Funding Information: We have read the journal's policy and the authors of this manuscript have the following competing interests: ARF reports travel grants from Roche and advisory board fees from Daiichi Sankyo, Gilead, Merck Sharp & Dohme, Novartis and Roche, outside the submitted work. DMB reports travel grants from LEO Farmacêuticos, Merck Sharp & Dohme, Ipsen, Janssen, Roche, and Novartis, advisory board fees from Janssen, Pfizer, Merck Sharp & Dohme, Angelini, AstraZeneca, and Novartis, and institutional grants from F. Hoffmann-La Roche, outside the submitted work. The other authors have declared that no competing interests exist. Funding Information: The authors acknowledge the RON network that cooperated in providing up-to-date information on cases diagnosed and treated with the drug of interest (participating institutions: Centro Hospitalar Universit?rio de S?o Jo?o, Centro Hospitalar Universit?rio Lisboa Norte, Centro Hospitalar Universit?rio do Algarve, Hospital de Braga, Centro Hospitalar e Universit?rio de Coimbra, Centro Hospitalar de Tr?s-os-Montes e Alto Douro, Hospital Central do Funchal, Centro Hospitalar de Vila Nova de Gaia/Espinho, Centro Hospitalar Lisboa Ocidental, Hospital Garcia de Orta, Centro Hospitalar Universit?rio Lisboa Central, Hospital Distrital de Santar?m, Centro Hospitalar de Entre o Douro e Vouga, Hospital da Senhora da Oliveira Guimar?es, Centro Hospitalar de Set?bal, Centro Hospitalar e Universit?rio do Porto, Centro Hospitalar Tondela Viseu, Hospital do Esp?rito Santo de ?vora, Centro Hospitalar Barreiro Montijo, Hospital Beatriz ?ngelo, Hospital do Santo Esp?rito da Ilha Terceira, Hospital do Divino Esp?rito Santo de Ponta Delgada, Hospital Pedro Hispano ? ULS Matosinhos, Hospital do Litoral Alentejano ? Santiago do Cac?m ? ULS Litoral Alentejano, Centro Hospitalar do Oeste, Centro Hospitalar M?dio Tejo, Hospital Jos? Joaquim Fernandes ? Beja ? ULS Baixo Alentejo, Centro Hospitalar Universit?rio da Cova da Beira, Centro Cl?nico Champalimaud, Hospitais CUF, Hospitais da Luz, Hospitais dos Lus?adas, Hospital Particular do Algarve). Publisher Copyright: © 2022 The AuthorsBackground: Real-world (RW) data may provide valuable information on the effectiveness and safety of medicines, which is particularly relevant for clinicians, patients and third-party payers. Evidence on the effectiveness of palbociclib plus fulvestrant is scarce, which highlights the need of additional studies. The aim of this study was to evaluate the effectiveness of palbociclib plus fulvestrant in advanced breast cancer (ABC). Materials and methods: We conducted a population-based retrospective cohort study and cases of interest were identified through the Portuguese National Cancer Registry database and additional data sources. Patients aged≥18 years, diagnosed with ABC and exposed to palbociclib plus fulvestrant between May 31, 2017 and March 31, 2019 were included. Patients were followed-up until death or cut-off date (February 28, 2021). Primary outcome was rw-progression-free survival (rwPFS). Secondary outcomes were rw-overall survival (rwOS), rw-time to palbociclib failure (rwTPF) and rw-time to next treatment (rwTTNT). Results: A total of 210 patients were included. Median age was 58 years (range 29–83) and 99.05% were female. Median follow-up time was 23.22 months and, at cut-off date, treatment had been discontinued in 189 patients, mainly due to disease progression (n = 152). Median rwPFS was 7.43 months (95% confidence interval [CI] 6.28–9.05) and 2-year rwPFS was 16.65% (95%CI 11.97–22.00). Median rwOS was 24.70 months (95%CI 21.58–29.27), median rwTPF was 7.5 months (95%CI 6.51–9.08) and median rwTTNT was 11.74 months (95%CI 10.33–14.08). Conclusion: Palbociclib plus fulvestrant seems an effective treatment for ABC in real-world context. Compared to registrations studies, rwPFS and rwOS were shorter in real-life setting.publishersversionpublishe

Microglial Sirtuin 2 shapes long-term potentiation in hippocampal slices

Copyright © 2020 Sa de Almeida, Vargas, Fonseca-Gomes, Tanqueiro, Belo, Miranda-Lourenço, Sebastião, Diógenes and Pais. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Microglial cells have emerged as crucial players in synaptic plasticity during development and adulthood, and also in neurodegenerative and neuroinflammatory conditions. Here we found that decreased levels of Sirtuin 2 (Sirt2) deacetylase in microglia affects hippocampal synaptic plasticity under inflammatory conditions. The results show that long-term potentiation (LTP) magnitude recorded from hippocampal slices of wild type mice does not differ between those exposed to lipopolysaccharide (LPS), a pro-inflammatory stimulus, or BSA. However, LTP recorded from hippocampal slices of microglial-specific Sirt2 deficient (Sirt2-) mice was significantly impaired by LPS. Importantly, LTP values were restored by memantine, an antagonist of N-methyl-D-aspartate (NMDA) receptors. These results indicate that microglial Sirt2 prevents NMDA-mediated excitotoxicity in hippocampal slices in response to an inflammatory signal such as LPS. Overall, our data suggest a key-protective role for microglial Sirt2 in mnesic deficits associated with neuroinflammation.This study was supported by Santa Casa da Misericórdia de Lisboa (MB37-2017), GAPIC Research Program of the University of Lisbon Medical School (n° 2014002 and n° 2015028) and the following doctoral grants: PD/BD/128091/2016, SFRH/BD/118238/2016, PD/BD/114337/2016, and PD/BD/1144- 41/2016.info:eu-repo/semantics/publishedVersio

Unexpected short- and long-term effects of chronic adolescent HU-210 exposure on emotional behavior

© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/).Chronic adolescent cannabinoid receptor agonist exposure has been shown to lead to persistent increases in depressive-like behaviors. This has been a key obstacle to the development of cannabinoid-based therapeutics. However, most of the published work has been performed with only three compounds, namely Δ9-tetrahydrocannabinol, CP55,940 and WIN55,212-2. Hypothesizing that different compounds may lead to distinct outcomes, we herein used the highly potent CB1R/CB2R full agonist HU-210, and first aimed at replicating cannabinoid-induced long-lasting effects, by exposing adolescent female Sprague-Dawley rats to increasing doses of HU-210, for 11 days and testing them at adulthood, after a 30-day drug washout. Surprisingly, HU-210 did not significantly impact adult anxious- or depressive-like behaviors. We then tested whether chronic adolescent HU-210 treatment resulted in short-term (24h) alterations in depressive-like behavior. Remarkably, HU-210 treatment simultaneously induced marked antidepressant- and prodepressant-like responses, in the modified forced swim (mFST) and sucrose preference tests (SPT), respectively. Hypothesizing that mFST results were a misleading artifact of HU-210-induced behavioral hyperreactivity to stress, we assessed plasmatic noradrenaline and corticosterone levels, under basal conditions and following an acute swim-stress episode. Notably, we found that while HU-210 did not alter basal noradrenaline or corticosterone levels, it greatly augmented the stress-induced increase in both. Our results show that, contrary to previously studied cannabinoid receptor agonists, HU-210 does not induce persisting depressive-like alterations, despite inducing marked short-term increases in stress-induced reactivity. By showing that not all cannabinoid receptor agonists may induce long-term negative effects, these results hold significant relevance for the development of cannabinoid-based therapeutics.Work was supported by project funding from Fundação para a Ciência e para a Tecnologia (FCT) (PTDC/MED-FAR/30933/2017 and PTDC/MED-FAR/4834/2021) and by H2020-WIDESPREAD-05-2017-Twinning (EpiEpinet) under grant agreement No. 952455. MF-F (SFRH/BD/147505/2019), NR (PD/BD/113463/2015), JF-G (PD/BD/114441/2016) and CM-L (SFRH/BD/118238/2016) are supported by PhD fellowships from FCT. The funding sources had no involvement in study design, preparation of the manuscript, or decision regarding its submission.info:eu-repo/semantics/publishedVersio

Public Art Journal

info:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost