Deciphering the mechanisms underlying the loss of BDNF neuroprotection in an Alzheimer’s disease model

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2016O fator neurotrófico derivado do cérebro (Brain-derived neurotrophic factor - BDNF) desempenha importantes funções no sistema nervoso central, nomeadamente diferenciação e sobrevivência neuronais e regulação da transmissão e plasticidade sinápticas. Em algumas doenças neurodegenerativas, como na doença de Alzheimer (Alzheimer’s disease - AD) que se caracteriza por declínio cognitivo e perda de memória, sabe-se que a sinalização mediada pelo BDNF se encontra diminuída. De facto, tanto em doentes como em modelos animais de AD, existem evidências de que os níveis proteicos de BDNF e da isoforma completa do seu recetor, TrkB-FL (full length – FL), se encontram diminuídos. O BDNF tem também a capacidade de se ligar a recetores TrkB truncados (truncated TrkB – TrkB-TC), porém estes recetores são moduladores negativos de TrkB-FL, uma vez que são incapazes de iniciar as vias de sinalização mediadas pelo BDNF. Estudos recentes revelaram que o recetor TrkB-FL é clivado por um grupo de proteases, designadas por calpaínas, resultando na formação de um novo recetor TrkB truncado (TrkB-T’) e de um fragmento intracelular (intracellular domain - ICD) que é translocado para o núcleo. Não se conhecem ainda em profundidade as ações destes novos fragmentos mas sabe-se que a função do BDNF fica severamente comprometida. As calpaínas são proteases dependentes de cálcio, sendo por isso ativadas por um aumento dos níveis intracelulares deste catião. Os recetores N-metil-D-aspartato (NMDARs), importantes mediadores da plasticidade sináptica, são permeáveis a cálcio e podem ser encontrados tanto na região sináptica como na extrassináptica. Enquanto a ativação dos recetores NMDAR sinápticos resulta em alterações que promovem a neuroprotecção, a ativação dos NMDARs extrassinápticos induz, preferencialmente, fenómenos de morte neuronal. Curiosamente, sabe-se que os eNMDARs se encontram sobreactivados em diversas condições patológicas, inclusivamente em modelos de AD, e que podem ter um importante papel na desregulação dos níveis de cálcio intracelular. Assim, o trabalho desenvolvido nesta tese teve como objetivo investigar se a ativação dos eNMDARs contribui para a ativação das calpaínas e consequente clivagem dos recetores TrkB-FL, assim como a perda da sinalização do BDNF. Em primeiro lugar, investigou-se se a prevenção da ativação dos eNMDARs, em neurónios expostos ao péptido β amilóide (amyloid β – Aβ), inibia a clivagem dos recetores TrkB-FL pelas calpaínas. Para testar esta hipótese, culturas primárias de neurónios de rato Sprague-Dawley com 14 dias in vitro (DIV14), foram incubadas durante 24 h com Aβ25-35 (25 μM), o mais pequeno fragmento tóxico do péptido Aβ, e memantina (1 μM), fármaco que bloqueia preferencialmente eNMDARs sobreactivados. Os resultados obtidos indicaram, como esperado, que Aβ25-35 induz um aumento dos níveis dos produtos específicos da clivagem da αII-espectrina mediada pelas calpaínas (specific spectrin breakdown products – SBDP150) sugerindo que existe uma forte ativação destas proteases. Esta alteração traduziu-se na diminuição significativa dos níveis proteicos de TrkB-FL e num aumento nos níveis de TrkB-ICD. Por outro lado, os resultados mostraram, pela primeira vez, que os efeitos de Aβ25-35 são prevenidos pela co-incubação com a memantina: i) a formação de SBDP150 diminuiu, ii) os níveis dos recetores TrkB-FL aumentaram e iii) os níveis de TrkB-ICD diminuíram. Assim, os resultados indicam que a ativação dos eNMDAR parece estar envolvida na ativação das calpaínas e, consequentemente, na clivagem dos recetores TrkB-FL. Uma vez que a formação de novas sinapses são processos que estão na base da formação de memória, pensa-se que as alterações que decorrem no número de espinhas dendríticas num neurónio de um doente de AD tem um papel preponderante nos défices cognitivos que se desenvolvem possivelmente adjacentes à perda neuronal. Sabe-se que o BDNF aumenta o número de espinhas dendríticas, protusões sinápticas através das quais a maioria das sinapses excitatórias ocorre e que correspondem à força de atividade sináptica de um neurónio. Assim, propusemo-nos avaliar, através de imunocitoquímica, se a ativação dos eNMDARs está relacionada com a perda de espinhas dendríticas num neurónio. Os resultados foram obtidos a partir de neurónios provenientes da cultura primária de rato a DIV14. O número de protusões (espinhas dendríticas e filopodia, protusões mais finas) foi quantificado em frações de 10 μm da dentrite-mãe a uma distância de 25 μm do corpo celular do neurónio. Os resultados indicam, como esperado, que Aβ25-35 diminui significativamente o número de protusões e que o BDNF aumenta o número de protusões per se. Na presença de Aβ25-35, os resultados sugerem que o BDNF perde a sua ação no aumento do número de protusões. Essa função é recuperada aquando bloqueio dos eNMDARs com memantina, assim como bloqueio da atividade das calpaínas com MDL28170 (20 μM). Estes resultados propõem que os mecanismos através dos quais Aβ interfere com as espinhas dendríticas envolvem não só a ativação das calpaínas, como visto anteriormente, como também a ativação dos eNMDARs. Por outro lado, foi também nosso propósito avaliar se o bloqueio dos eNMDARs restaurava o efeito do BDNF na potenciação de longa duração (long-term potentiation – LTP) na área CA1 do hipocampo, o mecanismo fisiológico da aprendizagem e memória, cuja magnitude se encontra diminuída na presença de Aβ. Para tal, foram preparadas fatias de hipocampo de rato Wistar com 8-12 semanas de vida e após incubação durante 3h com Aβ25-35 (25 μM) e/ou memantina (1 μM) procedemos ao registo extracelular dos potenciais pós-sinápticos excitatórios de campo (field excitatory postsynaptic potentials – fEPSP) e à indução de LTP na ausência ou presença de BDNF (20 ng/mL). Os dados sugerem, como esperado que, em fatias incubadas exclusivamente com líquido cefalorraquidiano artificial (artificial cerebrospinal fluid - aCSF), o BDNF induz um aumento significativamente da magnitude da LTP e que, na presença de Aβ, o BDNF perde a sua ação na LTP. Curiosamente, os nossos resultados indicam, pela primeira vez, que a co-incubação de memantina e Aβ25–35 restaura a capacidade do BDNF em facilitar a LTP. Estes resultados indicam que a sinalização mediada pelo BDNF na LTP se encontra diminuída na presença de Aβ e que esta diminuição pode ser mediada pela ativação dos eNMDARs. Em conclusão, os resultados sugerem que, na presença de Aβ, a sinalização mediada pelo BDNF se encontra severamente diminuída, afetando as suas ações sinápticas, através de um mecanismo que possivelmente é mediado pela ativação dos eNMDARs. Estas evidências realçam a consequência funcional da clivagem dos recetores TrkB-FL induzida pelo Aβ e propõem a modulação dos eNMDARs de modo a prevenir a desregulação dos níveis intracelulares de cálcio e, consequentemente, a perda dos mecanismos neuroprotetores mediados pelo BDNF.The brain-derived neurotrophic factor (BDNF) plays important functions in the central nervous system, such as cell survival, neuronal outgrowth, differentiation and plasticity. In contrast, BDNF signaling is known to be impaired in some neurodegenerative diseases, including Alzheimer’s disease (AD), which is characterized by cognitive decline and loss of memory. In fact, in AD patients and in several AD models a decrease in BDNF and its main receptor, TrkB-full length (TrkB-FL), has been reported. BDNF can also bind to truncated TrkB (TrkB-TC), however these receptors act as dominant negative inhibitor of TrkB-FL since they cannot initiate BDNF signaling. Recent evidences revealed that TrkB-FL is processed by calpains, which results in the formation of a new truncated TrkB (TrkB-T’) and in the formation of an intracellular domain (ICD) fragment. Thus, this cleavage culminates in the receptor loss of function. Calpains are Ca2+-dependent proteases that are activated by increased intracellular levels of this cation. N-methyl-d-aspartate receptors (NMDARs), which are known to be permeable to Ca2+, are essential mediators of brain synaptic plasticity and can be found at synaptic and extrasynaptic sites. Synaptic NMDARs are neuroprotective, whereas extrasynaptic NMDARs (eNMDARs) preferentially initiate cell death pathways. Importantly, eNMDARs are known to be over activated in AD. Furthermore, NMDARs have been proposed as one of the molecules that might be involved in intracellular Ca2+ deregulation. Thus, we purposed to investigate if, by preventing eNMDAR activation in primary rat neurons or hippocampal slices exposed to the active fragment of amyloid β (Aβ25-35) (25 μM), one of the main neurotoxic species that contribute to AD progression, we could inhibit the truncation of TrkB-FL by calpains, restoring the functions of BDNF. Our results have shown that the inhibition of eNMDAR by memantine (1 μM), which preferentially blocks extrasynaptic receptors over synaptic receptors, reduces significantly the activation of calpains. These findings are related with an increase in TrkB-FL levels and a decrease in ICD levels. Moreover, it is known that BDNF increases the number of spines in one neuron, which are synaptic protrusions where the majority of excitatory post-synaptic domains are localized and that can highly predict the strength of synaptic activity. Our results indicate that, in the presence of Aβ, BDNF loses its function upon spine density, which is prevented when calpains activity is inhibited with MDL28170 (20 μM) or while eNMDAR blockade. Finally, data suggest that the inhibition of eNMDAR restores the capacity of BDNF to enhance long-term potentiation in hippocampal slices, the physiological basis for learning and memory that is known to be impaired in the presence of Aβ. Finally, the focus of our work was to clarify the mechanism by which BDNF loss its function upon synapses. In conclusion, data suggest that, in primary neuronal cultures and hippocampal slices, Aβ severely impairs BDNF/TrkB-FL signaling affecting the synaptic actions of BDNF by a mechanism that is, at least in part, mediated by eNMDARs activation. These findings highlight the functional consequence of the Aβ-induced cleavage of TrkB receptors and propose eNMDAR modulation to prevent the disruption of Ca2+ homeostasis and, consequently, the loss of physiological mechanisms that depend on BDNF

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

Sustained NMDA receptor hypofunction impairs brain-derived neurotropic factor signalling in the PFC, but not in the hippocampus, and disturbs PFC-dependent cognition in mice

BACKGROUND: Cognitive deficits profoundly impact on the quality of life of patients with schizophrenia. Alterations in brain derived neurotrophic factor (BDNF) signalling, which regulates synaptic function through the activation of full-length tropomyosin-related kinase B receptors (TrkB-FL), are implicated in the aetiology of schizophrenia, as is N-methyl-D-aspartate receptor (NMDA-R) hypofunction. However, whether NMDA-R hypofunction contributes to the disrupted BDNF signalling seen in patients remains unknown. AIMS: The purpose of this study was to characterise BDNF signalling and function in a preclinical rodent model relevant to schizophrenia induced by prolonged NMDA-R hypofunction. METHODS: Using the subchronic phencyclidine (PCP) model, we performed electrophysiology approaches, molecular characterisation and behavioural analysis. RESULTS: The data showed that prolonged NMDA-R antagonism, induced by subchronic PCP treatment, impairs long-term potentiation (LTP) and the facilitatory effect of BDNF upon LTP in the medial prefrontal cortex (PFC) of adult mice. Additionally, TrkB-FL receptor expression is decreased in the PFC of these animals. By contrast, these changes were not present in the hippocampus of PCP-treated mice. Moreover, BDNF levels were not altered in the hippocampus or PFC of PCP-treated mice. Interestingly, these observations are paralleled by impaired performance in PFC-dependent cognitive tests in mice treated with PCP. CONCLUSIONS: Overall, these data suggest that NMDA-R hypofunction induces dysfunctional BDNF signalling in the PFC, but not in the hippocampus, which may contribute to the PFC-dependent cognitive deficits seen in the subchronic PCP model. Additionally, these data suggest that targeting BDNF signalling may be a mechanism to improve PFC-dependent cognitive dysfunction in schizophrenia

As políticas de acesso aberto: história, promessas e tensões

In Europe and Portugal, the Open Access movement has reached significant growth, with the development of institutional repositories and increasing initiatives that encourage open access publication. Most recently, these practices have been incorporated into science and technology policies by binding them to funding programs for research and innovation. This article seeks to recall the historical trajectory behind open access, currently in convergence with the discourses of citizen and open science. Thus, it is provided contextualization on the emergence and evolution of open access and its assumptions by science and technology policies. While addressing the challenges facing its implementation, contradictions have arisen, in view of the publishing market and the need to preserve ongoing business models. It is provided to the reader here a guiding reflection, discussing the implications that open access brings to academic publishing and data management

High-caloric diet Induces memory impairment and disrupts synaptic plasticity in aged rats

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).The increasing consumption of sugar and fat seen over the last decades and the consequent overweight and obesity, were recently linked with a deleterious effect on cognition and synaptic function. A major question, which remains to be clarified, is whether obesity in the elderly is an additional risk factor for cognitive impairment. We aimed at unravelling the impact of a chronic high caloric diet (HCD) on memory performance and synaptic plasticity in aged rats. Male rats were kept on an HCD or a standard diet (control) from 1 to 24 months of age. The results showed that under an HCD, aged rats were obese and displayed significant long-term recognition memory impairment when compared to age-matched controls. Ex vivo synaptic plasticity recorded from hippocampal slices from HCD-fed aged rats revealed a reduction in the magnitude of long-term potentiation, accompanied by a decrease in the levels of the brain-derived neurotrophic factor receptors TrkB full-length (TrkB-FL). No alterations in neurogenesis were observed, as quantified by the density of immature doublecortin-positive neurons in the hippocampal dentate gyrus. This study highlights that obesity induced by a chronic HCD exacerbates age-associated cognitive decline, likely due to impaired synaptic plasticity, which might be associated with deficits in TrkB-FL signaling.This research was funded by Santa Casa da Misericórdia de Lisboa—MB37-2017; Fundação para a Ciência e a Tecnologia (FCT)—IF/01227/2015; and it received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952455. Researchers were supported by the following: S.L.P.—FCT (PD/BD/150341/2019); C.M.-L.—FCT (SFRH/BD/118238/2016) and Universidade de Lisboa (BD2015); R.F.B.—FCT (PD/BD/114337/2016); R.S.R.—FCT (SFRH/BD/129710/2017); S.R.T.—FCT (SFRH/BD/128091/2016).info:eu-repo/semantics/publishedVersio

Inhibition of NMDA Receptors Prevents the Loss of BDNF Function Induced by Amyloid β

Brain-derived neurotrophic factor (BDNF) plays important functions in cell survival and differentiation, neuronal outgrowth and plasticity. In Alzheimer’s disease (AD), BDNF signaling is known to be impaired, partially because amyloid β (Aβ) induces truncation of BDNF main receptor, TrkB-full length (TrkB-FL). We have previously shown that such truncation is mediated by calpains, results in the formation of an intracellular domain (ICD) fragment and causes BDNF loss of function. Since calpains are Ca2+-dependent proteases, we hypothesized that excessive intracellular Ca2+ build-up could be due to dysfunctional N-methyl-d-aspartate receptors (NMDARs) activation. To experimentally address this hypothesis, we investigated whether TrkB-FL truncation by calpains and consequent BDNF loss of function could be prevented by NMDAR blockade. We herein demonstrate that a NMDAR antagonist, memantine, prevented excessive calpain activation and TrkB-FL truncation induced by Aβ25–35. When calpains were inhibited by calpastatin, BDNF was able to increase the dendritic spine density of neurons exposed to Aβ25135. Moreover, NMDAR inhibition by memantine also prevented Aβ-driven deleterious impact of BDNF loss of function on structural (spine density) and functional outcomes (synaptic potentiation). Collectively, these findings support NMDAR/Ca2+/calpains mechanistic involvement in Aβ-triggered BDNF signaling disruption

Purine nucleosides in neuroregeneration and neuroprotection

© 2015 Elsevier Ltd. All rights reserved.In the present review, we stress the importance of the purine nucleosides, adenosine and guanosine, in protecting the nervous system, both centrally and peripherally, via activation of their receptors and intracellular signalling mechanisms. A most novel part of the review focus on the mechanisms of neuronal regeneration that are targeted by nucleosides, including a recently identified action of adenosine on axonal growth and microtubule dynamics. Discussion on the role of the purine nucleosides transversally with the most established neurotrophic factors, e.g. brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), is also focused considering the intimate relationship between some adenosine receptors, as is the case of the A2A receptors, and receptors for neurotrophins. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.The authors work has been funded by Fundação para a Ciência e Tecnologia (FCT) Portugal (EXPL/BIM-MEC/0009/2013). Filipa F Ribeiro was in receipt of a fellowship (SFRH/BD/74662/2010) from FCTinfo:eu-repo/semantics/publishedVersio

Brain-derived neurotrophic factor mediates neuroprotection against Aβ-induced toxicity through a mechanism independent on adenosine 2A receptor activation

© 2015 Taylor & Francis.Brain-derived neurotrophic factor (BDNF) promotes neuronal survival through TrkB-FL activation. The activation of adenosine A2A receptors (A2AR) is essential for most of BDNF-mediated synaptic actions, such as synaptic plasticity, transmission and neurotransmitter release. We now aimed at evaluating the A2AR influence upon BDNF-mediated neuroprotection against Aβ25-35 toxicity in cultured neurons. Results showed that BDNF increases cell survival and reduces the caspase-3 and calpain activation induced by amyloid-β (Aβ) peptide, in a mechanism probably dependent on PLCγ pathway. This BDNF-mediated neuroprotection is not affected by A2AR activation or inhibition. Moreover neither activation nor inhibition of A2AR, per se, significantly influenced Aβ-induced neuronal death on calpain-mediated cleavage of TrkB induced by Aβ. In conclusion, these results suggest that, in opposition to the fast synaptic actions of BDNF, the neuroprotective actions of this neurotrophin against a strong Aβ insult do not require the activation of A2AR.The work was supported by Fundação para a Ciência e a Tecnologia (FCT) and Gabinete de Apoio à Investigação Científica, Tecnológica e Inovação (GAPIC), Faculty of Medicine of Lisbon, University of Lisbon, Portugal—16th Programme for Education and Science. AJS and RMR were supported by FCT (AJS: SFRH/BD/62828/2009; RMR: SFRH/BPD/94474/2013).info:eu-repo/semantics/publishedVersio

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<p>Brain-derived neurotrophic factor (BDNF) plays important functions in cell survival and differentiation, neuronal outgrowth and plasticity. In Alzheimer’s disease (AD), BDNF signaling is known to be impaired, partially because amyloid β (Aβ) induces truncation of BDNF main receptor, TrkB-full length (TrkB-FL). We have previously shown that such truncation is mediated by calpains, results in the formation of an intracellular domain (ICD) fragment and causes BDNF loss of function. Since calpains are Ca²⁺-dependent proteases, we hypothesized that excessive intracellular Ca²⁺ build-up could be due to dysfunctional N-methyl-d-aspartate receptors (NMDARs) activation. To experimentally address this hypothesis, we investigated whether TrkB-FL truncation by calpains and consequent BDNF loss of function could be prevented by NMDAR blockade. We herein demonstrate that a NMDAR antagonist, memantine, prevented excessive calpain activation and TrkB-FL truncation induced by Aβ_25–35. When calpains were inhibited by calpastatin, BDNF was able to increase the dendritic spine density of neurons exposed to Aβ₂₅₁₃₅. Moreover, NMDAR inhibition by memantine also prevented Aβ-driven deleterious impact of BDNF loss of function on structural (spine density) and functional outcomes (synaptic potentiation). Collectively, these findings support NMDAR/Ca²⁺/calpains mechanistic involvement in Aβ-triggered BDNF signaling disruption.</p