Regulation of GABA transporter GAT-1 in neuronal cells : role of brain-derived neurotrophic factor and adenosine

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2011Gamma-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the central nervous system. Its activity at the synapse is terminated by re-uptake into nerve terminals and astrocytes, through membrane located specific GABA transporters (GATs), which therefore shape GABAergic transmission. There are three main high affinity subtypes of GATs, GAT-1, GAT-2 and GAT-3, and a low affinity one, the betaine transporter. GAT-1 is the predominant GABA transporter in the brain and is expressed in neurons and astrocytes. Several factors can regulate the continuous traffic of GATs to and from the neuronal plasma membrane. For instance surface expression of GAT-1 in cultured neurons and isolated nerve terminals is decreased by protein kinase C (PKC)-dependent phosphorylation. In contrast, surface expression of GAT-1 in neurons is enhanced by brain derived neurotrophic factor (BDNF)-mediated tyrosine kinase-dependent phosphorylation. Though reuptake of GABA might occur at different places of the neuronal membrane, its reuptake by the nerve terminal is the process that allows quick refilling of the released stores. On the other hand, uptake by astrocytes contributes to a fast removal of GABA from the synapse and delays its delivery to the neuronal release stores. To understand how GABAergic transmission can be shaped, it is therefore important to know how a single modulator can affect both processes of GABA removal from synapse. Thus, in the work presented on this thesis I evaluated the influence of BDNF upon GAT-1 transporters on presynaptic nerve terminals and cortical primary astrocyte culture. BDNF decreased GAT-1 mediated GABA uptake by isolated hippocampal rat nerve terminals (synaptosomes), an effect that occurred within 1 min of incubation with BDNF through activation of TrkB receptor. In contrast with what has been observed for other synaptic actions of BDNF, the inhibition of GABA transport by BDNF does not require tonic activation of adenosine A2A receptors, nevertheless is facilitated by activation of A2A receptors. On the other hand, BDNF enhances GAT-1 mediated GABA transport in cultured astrocytes, an effect mostly due to an increase in Vmax kinetic constant. This effect involves the truncated form of TrkB receptors (TrkB-t) coupled to a non-classic PLC-y/PKC-δ and Erk/MAP kinase pathway and requires active adenosine A2A receptors. To elucidate the trafficking of GAT-1 when astrocytes were treated with BDNF, a functional mutant of the rat GAT-1 was generated in which hemagglutinin epitope (HA) was incorporated into the second extracellular loop. By ELISA experiments, performed with astrocytes expressing HA-rGAT-1 transporter, it was possible to observe an exocytosis of HA-GAT-1 to plasma membrane when cells were treated with BDNF. In addition, cell surface biotinylation experiments, performed with astrocytes overexpressing the wild type rat GAT-1 (rGAT-1), also demonstrate an increase of GAT-1 2 transporter at plasma membrane when astrocytes were treated with BDNF. Results from experiments using selective inhibitors of endocytosis or selective inhibitors of recycling of molecules back to the plasma membrane allowed concluding that BDNF enhances GAT-1 expression at surface astrocytic membrane by slowing down exocytosis. A new role for BDNF is proposed whereby the effect of BDNF on GAT-1 transporter differs between pre-synaptic nerve terminals and astrocytes, suggesting that this neurotrophin operates in a much localized way, so that it may retard GABA uptake by the nerve terminal, enhancing synaptic actions of GABA, and accelerate its reuptake at extracellular neuronal areas allowing replenishment of neuronal pools of GABA. The results suggest that BDNF plays an active role in the regulation of GABAergic synaptic signalling, contributing to information processing.O ácido y-aminobutírico (GABA) é o principal neurotransmissor inibitório do sistema nervoso central. A rápida remoção do GABA presente na fenda sináptica, por transportadores de alta afinidade para o GABA, que se localizam quer a nível do terminal pré-sináptico dos neurónios, quer a nível das células da glia, nomeadamenrte dos astrócitos (Gether et al., 2006), é essencial para uma sinalização eficaz mediada por este neurotransmissor. Até ao momento quatro transportadores foram identificados para o GABA, três destes de alta afinidade, denominados de GAT-1, GAT-2 and GAT-3 e um quarto, de baixa afinidade, denominado betaine transporter. O transportador GAT-1 é o transportador de GABA predominante no sistema nervoso central e encontra-se expresso preferencialmente em neurónios, sendo, no entanto, também expresso em astrócitos. Relativamente ao transportador GAT-3, sabe-se que este é maioritariamente expresso em astrócitos, onde tem um predomínio de transporte de GABA relativamente ao transportador GAT-1. Assim a recaptação de GABA pode ocorrer em diferentes localizações celulares. Quando ocorre para o terminal nervoso pre-sináptico, tem como consequência uma rápida reposição do nível de GABA nas vesículas sinápticas. A ocorrência para os astrócitos contribui para uma remoção mais rápida do GABA da fenda sináptica, diminuindo assim a velocidade de reposição de GABA nas vesículas sinápticas. Para se entender como é que a transmissão GABAérgica é regulada, torna-se pois extremamente relevante compreeender como pode apenas uma molécula modular os dois locais onde ocorre transporte de GABA, nomeadamente o pré-sináptico e o astrocítico. Salienta-se também a importância dos transportadores para o controlo da excitabilidade e o seu eventual envolvimento em situação patológica, nomeadamente em doentes com epilepsia do lobo temporal que apresentam uma aumento da expressão dos transportadores de GABA nos astrócitos. Os transportadores de GABA são regulados de diversos modos, estando envolvidos diferentes factores e várias cascatas de transdução de sinal. Esta modulação pode ocorrer de dois modos distintos: por alteração do Km ou da Vmax do transportador. A regulação do tráfego dos transportadores de GABA de, e para a membrana plasmática neuronal, pode ocorrer por variações da velocidade de endocitose e exocitose e/ou por alteração da quantidade de transportadores disponíveis neste processo de tráfego contínuo. Uma molécula já identificada como reguladora do transportador GAT-1 é o Brain derived neurotrophic factor (BDNF). O BDNF é um factor neurotrófico com importantes funções na diferenciação, maturação e sobrevivência neuronal, levando a modificações estruturais e moleculares a longo-prazo que são cruciais para o desenvolvimento, mas também para a função 2 e plasticidade sináptica no indivíduo adulto (Vicario-Abejon et al., 2002). O BDNF exerce a sua acção através da activação de receptores tirosina cinase B (TrkB), que se apresentam em diferentes isoformas: uma isoforma “completa” (TrkB-fl) que apresenta domínios tirosina cinase e uma isoforma truncada (TrkB-t) que não apresenta estes domínios. O BDNF favorece a recaptação de GABA devido a um aumento da expressão de GAT-1 a nível da membrana plasmática em culturas primárias de neurónios, não se sabendo até ao início deste trabalho qual a função do BDNF no controlo da actividade do GAT-1 local a nível de terminais nervosos. Os astrócitos são a maior classe de células da glia encontrada no cérebro dos mamíferos e têm um papel extremamente relevante na transmissão sináptica, contribuindo para o processamento de informação a nível sináptico ao controlar quer a composição do meio extracelular, quer a quantidade de neurotransmissores presentes na fenda sináptica. Os astrócitos são assim células fundamentais a nível da comunicação existente entre astrócitos ou entre astrócitos-neurónios. No que diz respeito à regulação dos níveis extracelulares de GABA, estas células têm um papel muito importante uma vez que expressam transportadores específicos de GABA, que permitem, como foi anteriormente referido, o controlo dos níveis deste neurotransmissor na fenda sináptica. Todavia, pouco tem sido descrito em relação à regulação dos transportadores de GABA nos astrócitos. O trabalho que aqui se apresenta teve como objectivo estudar o efeito do BDNF sobre o transportador de GABA, em terminais nervosos pré-sinápticos e em astrócitos, bem como estudar os mecanismos subjacentes ao efeito do BDNF. Foi também abordado o possível envolvimento dos receptores A2A da adenosina, uma vez que a interacção entre o receptor do BDNF, TrkB e o receptor de adenosina A2A, tem sido descrita em vários sistemas biológicos. Verificou-se que em terminais nervosos pré-sinápticos o BDNF tem uma acção inibitória sobre o transportador exclusivo de GABA (GAT-1) nesta estrutura, levando a uma diminuição da recaptação de GABA através deste transportador. Este efeito depende da concentração de BDNF e ocorre num intervalo de tempo extremamente curto (1 minuto). O efeito do BDNF no transportador GAT-1 ocorre através da activação do receptor TrkB e, contrariamente a outros efeitos mediados pela activação deste receptor, não requer a activação tónica dos receptores A2A da adenosina. Em culturas primárias de astrócitos o BDNF aumentou a recaptação de GABA mediada pelo transportador GAT-1, não tendo qualquer efeito no transportador GAT-3, também presente nos astrócitos. Este efeito ocorre devido a um aumento da velocidade máxima do transportador. O efeito do BDNF envolve a forma truncada do receptor TrkB, estando esta acoplada a uma via não clássica da PLC-y/PKC-δ e da Erk/MAP cinases. O efeito descrito requer que os receptores 3 A2A da adenosina estejam activos, sendo que os níveis endógenos de adenosina extracelular são suficientes para desencadear o efeito do BDNF. Uma vez que um aumento do Vmax se correlaciona com um aumento do número de transportadores na membrana plasmática, procedeu-se seguidamente à avaliação de um possível aumento da expressão do transportador GAT-1 quando as células eram tratadas com BDNF. Para avaliar se o efeito do BDNF se correlacionava com o tráfego de GAT-1 de, e para a membrana celular, foi gerado um mutante funcional do transportador GAT-1 de rato (rGAT-1), no qual foi introduzido o epítopo hemaglutinina (HA) no segundo loop extracelular do transportador, procedendo-se à infecção dos astrócitos com o referido mutante. Após o tratamento das células com BDNF observou-se um aumento da expressão de HA-rGAT-1 na membrana plasmática. Também através de experiências de biotinilação, realizadas com astrócitos que sobreexpressavam rGAT-1, se pôde concluir que o BDNF aumenta a expressão de rGAT-1 na membrana plasmática. Estudos onde se usou um inibidor da endocitose (dynasore) ou um inibidor da reciclagem de moléculas internalizadas de volta para a membrana plasmática (monensin), permitiram concluir que o efeito do BDNF envolve inibição da internalização de GAT-1 nos astrócitos, tendo esta acção consequências na expressão do GAT-1 e na velocidade de transporte de GABA. Os resultados apresentados nesta tese mostram que o BDNF exerce a sua acção de um modo muito localizado, levando a uma diminuição da recaptação de GABA no terminal nervoso que favorece eventualmente as suas acções sinápticas, e a uma aceleração da recaptação de GABA em regiões extra-sinápticas, que contribui para uma redução da acção tónica deste neurotransmissor. Em última instância, este efeito do BDNF deverá determinar uma diminuição da velocidade de reposição de GABA nas vesículas sinápticas, conduzindo desta forma a um aumento da excitabilidade neuronal.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/27989/2006

Glutamate transporters in hippocampal LTD/LTP: not just prevention of excitotoxicity

Copyright © 2019 Gonçalves-Ribeiro, Pina, Sebastião and Vaz. 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.Glutamate uptake is a process mediated by sodium-dependent glutamate transporters, preventing glutamate spillover from the synapse. Typically, astrocytes express higher amounts of glutamate transporters, thus being responsible for most of the glutamate uptake; nevertheless, neurons can also express these transporters, albeit in smaller concentrations. When not regulated, glutamate uptake can lead to neuronal death. Indeed, the majority of the studies regarding glutamate transporters have focused on excitotoxicity and the subsequent neuronal loss. However, later studies have found that glutamate uptake is not a static process, evincing a possible correlation between this phenomenon and the efficiency of synaptic transmission and plasticity. In this review, we will focus on the role of the increase in glutamate uptake that occurs during long-term potentiation (LTP) in the hippocampus, as well as on the impairment of long-term depression (LTD) under the same conditions. The mechanism underpinning the modulatory effect of glutamate transporters over synaptic plasticity still remains unascertained; yet, it appears to have a more prominent effect over the N-methyl-D-aspartate receptor (NMDAR), despite changes in other glutamate receptors may also occur.This work was supported by UID/BIM/50005/2019 [project finnanced by Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado] and PTDC/BTM-SAL/32147/2017 (FCT). JG-R was in receipt of an FCT fellowship (iMM/BI/96-2018).info:eu-repo/semantics/publishedVersio

Editorial: Glial plasticity in depression

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hiPSC-based model of prenatal exposure to cannabinoids: effect on neuronal differentiation

Copyright © 2020 Miranda, Barata, Vaz, Ferreira, Quintas and Bekman. 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.Phytocannabinoids are psychotropic substances ofcannabis with the ability to bind endocannabinoid (eCB) receptors that regulate synaptic activity in the central nervous system (CNS). Synthetic cannabinoids (SCs) are synthetic analogs of Δ9-tetrahydrocannabinol (Δ9-THC), the psychotropic compound of cannabis, acting as agonists of eCB receptor CB1. SC is an easily available and popular alternative to cannabis, and their molecular structure is always changing, increasing the hazard for the general population. The popularity of cannabis and its derivatives may lead, and often does, to a child's exposure to cannabis both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with an altered rate of mental development and significant changes in nervous system functioning. However, the understanding of mechanisms of its action on developing the human CNS is still lacking. We investigated the effect of continuous exposure to cannabinoids on developing human neurons, mimicking the prenatal exposure by drug-consuming mother. Two human induced pluripotent stem cells (hiPSC) lines were induced to differentiate into neuronal cells and exposed for 37 days to cannabidiol (CBD), Δ9-THC, and two SCs, THJ-018 and EG-018. Both Δ9-THC and SC, at 10 μM, promote precocious neuronal and glial differentiation, while CBD at the same concentration is neurotoxic. Neurons exposed to Δ9-THC and SC show abnormal functioning of voltage-gated calcium channels when stimulated by extracellular potassium. In sum, all studied substances have a profound impact on the developing neurons, highlighting the importance of thorough research on the impact of prenatal exposure to natural and SC.This work was supported by the Fundação para a Ciência e a Tecnologia (FCT), Portugal (SFRH/BPD/81627/2011 to SV), by iBB — Institute for Bioengineering and Biosciences — project UIDB/04565/2020, and by Egas Moniz Higher Institute of Health Science (Egas Moniz, CRL). Funding was also received from the European Union’s Horizon 2020 Research and Innovation programme, under the Grant Agreement number 739572—The Discoveries Centre for Regenerative and Precision Medicine H2020-WIDESPREAD-01-2016-2017 to EB.info:eu-repo/semantics/publishedVersio

Interaction between cannabinoid type 1 and type 2 receptors in the modulation of subventricular zone and dentate Gyrus neurogenesis

Copyright © 2017 Rodrigues, Ribeiro, Ferreira, Vaz, Sebastião and Xapelli. 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) or licensor 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.Neurogenesis in the adult mammalian brain occurs mainly in two neurogenic niches, the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus (DG). Cannabinoid type 1 and 2 receptors (CB1R and CB2R) have been shown to differently modulate neurogenesis. However, low attention has been given to the interaction between CB1R and CB2R in modulating postnatal neurogenesis (proliferation, neuronal differentiation and maturation). We focused on a putative crosstalk between CB1R and CB2R to modulate neurogenesis and cultured SVZ and DG stem/progenitor cells from early postnatal (P1-3) Sprague-Dawley rats. Data showed that the non-selective cannabinoid receptor agonist WIN55,212-2 promotes DG cell proliferation (measured by BrdU staining), an effect blocked by either CB1R or CB2R selective antagonists. Experiments with selective agonists showed that facilitation of DG cell proliferation requires co-activation of both CB1R and CB2R. Cell proliferation in the SVZ was not affected by the non-selective receptor agonist, but it was enhanced by CB1R selective activation. However, either CB1R or CB2R selective antagonists abolished the effect of the CB1R agonist in SVZ cell proliferation. Neuronal differentiation (measured by immunocytochemistry against neuronal markers of different stages and calcium imaging) was facilitated by WIN55,212-2 at both SVZ and DG. This effect was mimicked by either CB1R or CB2R selective agonists and blocked by either CB1R or CB2R selective antagonists, cross-antagonism being evident. In summary, our findings indicate a tight interaction between CB1R and CB2R to modulate neurogenesis in the two major neurogenic niches, thus contributing to further unraveling the mechanisms behind the action of endocannabinoids in the brain.This work was supported by LISBOA-01-0145-FEDER-007391, project co-funded by FEDER through POR Lisboa 2020 (Programa Operacional Regional de Lisboa) from PORTUGAL 2020, and by Fundação para a Ciência e a Tecnologia (FCT). AS thanks the following supports: PTDC/DTP-FTO/3346/2014 from FCT and H2020 Twinning Action from EU (SynaNet 692340). SX is grateful for the support by the COST action BM1402. RR (IMM/BI/42-2016), FR (SFRH/BD/74662/2010), SV (SFRH/BPD/81627/2011), and SX (SFRH/BPD/76642/2011 and IF/01227/2015) were in receipt of a fellowship from FCT.info:eu-repo/semantics/publishedVersio

Recovery of depleted miR-146a in ALS cortical astrocytes reverts cell aberrancies and prevents paracrine pathogenicity on microglia and motor neurons

Copyright © 2021 Barbosa, Gomes, Sequeira, Gonçalves-Ribeiro, Pina, Carvalho, Moreira, Vaz, Vaz and Brites. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).Reactive astrocytes in Amyotrophic Lateral Sclerosis (ALS) change their molecular expression pattern and release toxic factors that contribute to neurodegeneration and microglial activation. We and others identified a dysregulated inflammatory miRNA profile in ALS patients and in mice models suggesting that they represent potential targets for therapeutic intervention. Such cellular miRNAs are known to be released into the secretome and to be carried by small extracellular vesicles (sEVs), which may be harmful to recipient cells. Thus, ALS astrocyte secretome may disrupt cell homeostasis and impact on ALS pathogenesis. Previously, we identified a specific aberrant signature in the cortical brain of symptomatic SOD1-G93A (mSOD1) mice, as well as in astrocytes isolated from the same region of 7-day-old mSOD1 mice, with upregulated S100B/HMGB1/Cx43/vimentin and downregulated GFAP. The presence of downregulated miR-146a on both cases suggests that it can be a promising target for modulation in ALS. Here, we upregulated miR-146a with pre-miR-146a, and tested glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS) for their immunoregulatory properties. VS was more effective in restoring astrocytic miR-146a, GFAP, S100B, HMGB1, Cx43, and vimentin levels than GUDCA, which only recovered Cx43 and vimentin mRNA. The miR-146a inhibitor generated typical ALS aberrancies in wild type astrocytes that were abolished by VS. Similarly, pre-miR-146a transfection into the mSOD1 astrocytes abrogated aberrant markers and intracellular Ca2+ overload. Such treatment counteracted miR-146a depletion in sEVs and led to secretome-mediated miR-146a enhancement in NSC-34-motor neurons (MNs) and N9-microglia. Secretome from mSOD1 astrocytes increased early/late apoptosis and FGFR3 mRNA in MNs and microglia, but not when derived from pre-miR-146a or VS-treated cells. These last strategies prevented the impairment of axonal transport and synaptic dynamics by the pathological secretome, while also averted microglia activation through either secretome, or their isolated sEVs. Proteomic analysis of the target cells indicated that pre-miR-146a regulates mitochondria and inflammation via paracrine signaling. We demonstrate that replenishment of miR-146a in mSOD1 cortical astrocytes with pre-miR-146a or by VS abrogates their phenotypic aberrancies and paracrine deleterious consequences to MNs and microglia. These results propose miR-146a as a new causal and emerging therapeutic target for astrocyte pathogenic processes in ALS.This work was supported by the Research Grant of the Santa Casa Scientific Research Program on ALS, by Santa Casa da Misericórdia de Lisboa (SCML), Portugal, Project Ref. ELA-2015-002 (to DB). Fundação para a Ciência e a Tecnologia (FCT) also supported the project PTDC/MED-NEU/31395/2017 (to DB), PTDC/MED-QUI/30021/2017 (to RM) and iMed. ULisboa (UIDB/04138/2020 and UIDP/04138/2020), together with Programa Operacional Regional de Lisboa and the Programa Operacional Competitividade e Internacionalização (LISBOA-01-0145-FEDER-031395 to DB). Individual fellowships MB (SFRH/BD/129586/2017), CG (SFRH/BD/102718/2014), AV (SFRH/BPD/76590/2011), and JG-R PD/BD/150342/2019 were from FCT. CS was a research fellowship recipient from SCML.info:eu-repo/semantics/publishedVersio

Changes in adenosine receptors and neurotrophic factors in the SOD1G93A mouse model of amyotrophic lateral sclerosis: modulation by chronic caffeine

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of corticospinal tract motor neurons. Previous studies showed that adenosine-mediated neuromodulation is disturbed in ALS and that vascular endothelial growth factor (VEGF) has a neuroprotective function in ALS mouse models. We evaluated how adenosine (A1R and A2AR) and VEGF (VEGFA, VEGFB, VEGFR-1 and VEGFR-2) system markers are altered in the cortex and spinal cord of pre-symptomatic and symptomatic SOD1G93A mice. We then assessed if/how chronic treatment of SOD1G93A mice with a widely consumed adenosine receptor antagonist, caffeine, modulates VEGF system and/or the levels of Brain-derived Neurotrophic Factor (BDNF), known to be under control of A2AR. We found out decreases in A1R and increases in A2AR levels even before disease onset. Concerning the VEGF system, we detected increases of VEGFB and VEGFR-2 levels in the spinal cord at pre-symptomatic stage, which reverses at the symptomatic stage, and decreases of VEGFA levels in the cortex, in very late disease states. Chronic treatment with caffeine rescued cortical A1R levels in SOD1G93A mice, bringing them to control levels, while rendering VEGF signaling nearly unaffected. In contrast, BDNF levels were significantly affected in SOD1G93A mice treated with caffeine, being decreased in the cortex and increased in spinal the cord. Altogether, these findings suggest an early dysfunction of the adenosinergic system in ALS and highlights the possibility that the negative influence of caffeine previously reported in ALS animal models results from interference with BDNF rather than with the VEGF signaling molecules.info:eu-repo/semantics/publishedVersio

Astrocytes in paper chips and their interaction with hybrid vesicles

© 2022 The Authors. Advanced Biology published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.The role of astrocytes in brain function has received increased attention lately due to their critical role in brain development and function under physiological and pathophysiological conditions. However, the biological evaluation of soft material nanoparticles in astrocytes remains unexplored. Here, the interaction of crosslinked hybrid vesicles (HVs) and either C8-D1A astrocytes or primary astrocytes cultured in polystyrene tissue culture or floatable paper-based chips is investigated. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) (P1) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine lipids are used for the assembly of HVs with crosslinked membranes. The assemblies show no short-term toxicity towards the C8-D1A astrocytes and the primary astrocytes, and both cell types internalize the HVs when cultured in 2D cell culture. Further, it is demonstrated that both the C8-D1A astrocytes and the primary astrocytes could mature in paper-based chips with preserved calcium signaling and glial fibrillary acidic protein expression. Last, it is confirmed that both types of astrocytes could internalize the HVs when cultured in paper-based chips. These findings lay out a fundamental understanding of the interaction between soft material nanoparticles and astrocytes, even when primary astrocytes are cultured in paper-based chips offering a 3D environment.This project was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 818890), the Lundbeck Foundation (R346-2020-1617), and the Carlsberg Foundation (CF 20–0418). The HRM Queen Margrethe II's travel grant (C.A.M.) is acknowledged for support. Tinfo:eu-repo/semantics/publishedVersio

Of adenosine and the blues: the adenosinergic system in the pathophysiology and treatment of major depressive disorder

© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Major depressive disorder (MDD) is the foremost cause of global disability, being responsible for enormous personal, societal, and economical costs. Importantly, existing pharmacological treatments for MDD are partially or totally ineffective in a large segment of patients. As such, the search for novel antidepressant drug targets, anchored on a clear understanding of the etiological and pathophysiological mechanisms underpinning MDD, becomes of the utmost importance. The adenosinergic system, a highly conserved neuromodulatory system, appears as a promising novel target, given both its regulatory actions over many MDD-affected systems and processes. With this goal in mind, we herein review the evidence concerning the role of adenosine as a potential player in pathophysiology and treatment of MDD, combining data from both human and animal studies. Altogether, evidence supports the assertions that the adenosinergic system is altered in both MDD patients and animal models, and that drugs targeting this system have considerable potential as putative antidepressants. Furthermore, evidence also suggests that modifications in adenosine signaling may have a key role in the effects of several pharmacological and non-pharmacological antidepressant treatments with demonstrated efficacy, such as electroconvulsive shock, sleep deprivation, and deep brain stimulation. Lastly, it becomes clear from the available literature that there is yet much to study regarding the role of the adenosinergic system in the pathophysiology and treatment of MDD, and we suggest several avenues of research that are likely to prove fruitful.This work was supported by project funding from Fundação para a Ciência e para a Tecnologia (FCT) to SHV (PTDC/BTM-SAL/32147/2017) and AMS (PTDC/MED-FAR/30933/2017). This project has received funding from H2020-WIDESPREAD-05-2017-Twinning (EpiEpinet) under grant agreement No. 952455. MF-F (SFRH/BD/147505/2019), JG-R (PD/BD/150342/2019), and NR (PD/BD/113463/2015) are supported by PhD fellowships from FCT.info:eu-repo/semantics/publishedVersio