FM-IFN-Artigos em Revistas Internacionais
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- Impairment of spatial working memory but preservation of recognition memory in female rats with spontaneous absence seizuresPublication . Neuparth-Sottomayor, Mariana; Morais, Tatiana P.; Good, Mark; Sebastião, Ana M; Di Giovanni, Giuseppe; Crunelli, Vincenzo; Vaz, Sandra H.Epidemiological studies reveal gender-specific differences in epilepsy. Childhood absence epilepsy (CAE), which is more prevalent in females, is characterized by typical absence seizures (ASs) consisting of brief periods of unconsciousness, associated with 2.5-4 Hz spike-wave discharges (SWDs) in the electroencephalogram (EEG). Children with CAE often present neuropsychological comorbidities, including deficits in attention and executive function. In this study, we investigated anxiety-like behaviour and memory in female Genetic Absence Epilepsy Rat from Strasbourg (GAERS), a validated model of ASs, compared to Non-Epileptic Control (NEC) and Wistar rats. We found that female GAERS generally showed normal anxiety-like behaviour relative to both control strains, although some tests suggested a reduction in anxiety. Importantly, female GAERS showed impaired spatial working memory, while recognition memory was preserved. These findings when compared with previous data in males indicate that while anxiety levels in female GAERS are preserved as those of male GAERS, memory performance differs, with males showing impairments in both spatial working memory and recognition memory. These findings emphasize the importance of considering gender differences in both clinical and preclinical epilepsy research to better understand the neuropsychological comorbidities associates with ASs. This knowledge is crucial for the identification of gender-specific mechanism, as well as the development of gender-sensitive, personalized therapies targeting both seizures and associated cognitive impairments.
- Cognitive comorbidities of experimental absence seizures are independent of anxietyPublication . Neuparth-Sottomayor, Mariana; Pina, Carolina; Morais, Tatiana P.; Farinha Ferreira, Jorge Miguel; Abreu, Daniela Sofia; Solano, Filipa; Mouro, Francisco; Good, Mark; Sebastião, Ana M; Di Giovanni, Giuseppe; Crunelli, Vincenzo; Vaz, Sandra H.Typical absence seizures (ASs) are brief periods of lack of consciousness, associated with 2.5-4 Hz spike-wave discharges (SWDs) in the EEG, which are highly prevalent in children and teenagers. The majority of probands in these young epileptic cohorts show neuropsychological comorbidities, including cognitive, memory and mood impairments, even after the seizures are pharmacologically controlled. Similar cognition and memory deficits have been reported in different, but not all, genetic animal models of ASs. However, since these impairments are subtle and highly task-specific their presence may be confounded by an anxiety-like phenotype and no study has tested anxiety and memory in the same animals. Moreover, the majority of studies used non-epileptic inbred animals as the only control strain and this may have contributed to a misinterpretation of these behavioural results. To overcome these issues, here we used a battery of behavioural tests to compare anxiety and memory in the same animals from the well-established inbred model of Genetic Absence Epilepsy Rats from Strasbourg (GAERS), their inbred strain of Non-Epileptic Control (NEC) strain (that lack ASs) and normal outbred Wistar rats. We found that GAERS do not exhibit increased anxiety-like behavior and neophobia compared to both NEC and Wistar rats. In contrast, GAERS show decreased spontaneous alternation, spatial working memory and cross-modal object recognition compared to both NEC and Wistar rats. Furthermore, GAERS preferentially used egocentric strategies to perform spatial memory tasks. In summary, these results provide solid evidence of memory deficits in GAERS rats that do not depend on an anxiety or neophobic phenotype. Moreover, the presence of differences between NEC and Wistar rats stresses the need of using both outbred and inbred control rats in behavioural studies involving genetic models of ASs.
- Adenosinergic system and BDNF signaling changes as a cross-sectional feature of RTT: characterization of Mecp2 heterozygous mouse femalesPublication . Miranda-Lourenço, Catarina; Rosa, Jéssica; Rei, Nádia; Belo, Rita F.; Lopes, Ana Luísa; Silva, Diogo; Vieira, Cátia; Magalhães-Cardoso, Teresa; Viais, Ricardo; Correia-de-Sá, Paulo; Sebastião, Ana M; Diógenes, Maria JoséRett Syndrome is an X-linked neurodevelopmental disorder (RTT; OMIM#312750) associated to MECP2 mutations. MeCP2 dysfunction is seen as one cause for the deficiencies found in brain-derived neurotrophic factor (BDNF) signaling, since BDNF is one of the genes under MeCP2 jurisdiction. BDNF signaling is also dependent on the proper function of the adenosinergic system. Indeed, both BDNF signaling and the adenosinergic system are altered in Mecp2-null mice (Mecp2-/y), a representative model of severe manifestation of RTT. Considering that symptoms severity largely differs among RTT patients, we set out to investigate the BDNF and ADO signaling modifications in Mecp2 heterozygous female mice (Mecp2+/-) presenting a less severe phenotype. Symptomatic Mecp2+/- mice have lower BDNF levels in the cortex and hippocampus. This is accompanied by a loss of BDNF-induced facilitation of hippocampal long-term potentiation (LTP), which could be restored upon selective activation of adenosine A2A receptors (A2AR). While no differences were observed in the amount of adenosine in the cortex and hippocampus of Mecp2+/- mice compared with healthy littermates, the density of the A1R and A2AR subtype receptors was, respectively, upregulated and downregulated in the hippocampus. Data suggest that significant changes in BDNF and adenosine signaling pathways are present in an RTT model with a milder disease phenotype: Mecp2+/- female animals. These features strengthen the theory that boosting adenosinergic activity may be a valid therapeutic strategy for RTT patients, regardless of their genetic penetrance.
- Astrocytes control hippocampal synaptic plasticity through the vesicular-dependent release of D-serinePublication . Abreu, Daniela Sofia; Gomes, Joana I.; Ribeiro, Filipa; Diógenes, Maria José; Sebastião, Ana M; Vaz, Sandra H.Astrocytes, the most abundant glial cells in the central nervous system (CNS), sense synaptic activity and respond through the release of gliotransmitters, a process mediated by intracellular Ca2+ level changes and SNARE-dependent mechanisms. Ionotropic N-methyl-D-aspartate (NMDA) receptors, which are activated by glutamate along with D-serine or glycine, play a crucial role in learning, memory, and synaptic plasticity. However, the precise impact of astrocyte-released D-serine on neuronal modulation remains insufficiently characterized. To address this, we have used the dominant negative SNARE (dnSNARE) mouse model, which selectively inhibits SNARE-dependent exocytosis from astrocytes. We recorded field excitatory postsynaptic potentials (fEPSPs) in CA3-CA1 synapses within hippocampal slices obtained from dnSNARE mice and wild-type (Wt) littermates. Our results demonstrate that hippocampal θ-burst long-term potentiation (LTP), a critical form of synaptic plasticity, is impaired in hippocampal slices from dnSNARE mice. Notably, this LTP impairment was rescued upon incubation with D-serine. To further investigate the involvement of astrocytes in D-serine-mediated mechanisms of LTP maintenance, we perfused hippocampal slices with L-serine - a substrate used by both neurons and astrocytes for D-serine production. The enhancement in LTP observed in dnSNARE mice was exclusively associated with D-serine presence, with no effects evident in the presence of L-serine. Additionally, both D- and L-serine reduced basal synaptic strength in the hippocampal slices of both Wt and dnSNARE mice. These results provide compelling evidence that distinct processes underlie the modulation of basal synaptic transmission and LTP through D-serine. Our findings underscore the pivotal contribution of astrocytes in D-serine-mediated processes that govern LTP establishment and basal transmission. This study not only provides essential insights into the intricate interplay between neurons and astrocytes but also emphasizes their collective role in shaping hippocampal synaptic function.
- Age-related impact of social isolation in mice: young vs middle-agedPublication . Magalhães, Daniela M; Mampay, Myrthe; Sebastião, Ana M; Sheridan, Graham K.; Valente, Cláudia A.Social isolation is a chronic mild stressor and a significant risk factor for mental health disorders. Herein we explored the impact of social isolation on depression- and anxiety-like behaviours, as well as spatial memory impairments, in middle-aged male mice compared to post-weaning mice. We aimed to quantify and correlate social isolation-induced behaviour discrepancies with changes in hippocampal glial cell reactivity and pro-inflammatory cytokine levels. Post-weaning and middle-aged C57BL7/J6 male mice were socially isolated for a 3-week period and behavioural tests were performed on the last five days of isolation. We found that 3 weeks of social isolation led to depressive-like behaviour in the forced swim test, anxiety-like behaviour in the open field test, and spatial memory impairment in the Morris water maze paradigm in middle-aged male mice. These behavioural alterations were not observed in male mice after post-weaning social isolation, indicating resilience to isolation-mediated stress. Increased Iba-1 expression and NLRP3 priming were both observed in the hippocampus of socially isolated middle-aged mice, suggesting a role for microglia and NLRP3 pathway in the detrimental effects of social isolation on cognition and behaviour. Young socially isolated mice also demonstrated elevated NLRP3 priming compared to controls, but no differences in Iba-1 levels and no significant changes in behaviour. Ageing-induced microglia activation and enhancement of IL-1β, TNF-α and IL-6 proinflammatory cytokines, known signs of a chronic low-grade inflammatory state, were also detected. Altogether, data suggest that social isolation, in addition to inflammaging, contributes to stress-related cognitive impairment in middle-aged mice.
- 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 diseasePublication . 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, DoraAlzheimer'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.
- Brain-derived neurotrophic factor modulation in response to oxidative stress and corticosterone: role of scopolamine and mirtazapinePublication . Correia, Ana Salomé; Torrado, Marília; Costa-Coelho, Tiago; Carvalho, Eva Daniela; Inteiro-Oliveira, Sara; Diógenes, Maria José; Pêgo, Ana Paula; Santos, Sofia Duque; Sebastião, Ana M; Vale, NunoMajor Depressive Disorder (MDD) is a very complex disease, challenging to study and manage. The complexities of MDD require extensive research of its mechanisms to develop more effective therapeutic approaches. Crucial in the context of this disease is the role of brain-derived neurotrophic factor (BDNF) signaling pathway. Aim: This manuscript aims to explore the complex relationship between MDD and BDNF signaling pathway, focusing on how BDNF is modulated in response to oxidative stress and corticosterone, known to be altered in MDD and contributing to the pathology of the disorder, when treated with scopolamine and mirtazapine. Methods: To assess BDNF levels after the different treatment conditions, rat hippocampal slices and mice primary hippocampus and cortical cell culture were analyzed by immunofluorescence and Western blot. Key findings: Both mirtazapine and scopolamine under stress conditions induced by hydrogen peroxide (H2O2) and corticosterone, had a significant impact on BDNF levels, and this was distinct in different neuronal models. Mirtazapine, especially when combined with H2O2, altered BDNF expression. Scopolamine when combined with both stressors also altered BDNF levels. However, its effects varied depending on the specific neuronal model and stress condition. In accordance with BDNF results, phosphorylated tropomyosin receptor kinase B (pTrkB) presented increased activation when neuronal cells subjected to stress were treated with mirtazapine or scopolamine. Significance: Collectively, this study highlights the complex connection between these compounds, stress conditions, and BDNF/TrkB modulation, supporting the potential therapeutic effects of scopolamine and mirtazapine in modulating BDNF levels, even in stressful conditions.
- Chemogenetics with PSAM4-GlyR decreases excitability and epileptiform activity in epileptic hippocampusPublication . Gonzalez-Ramos, Ana; Berglind, Fredrik; Kudláček, Jan; Ribeiro Rocha, Elza; Melin, Esbjörn; Sebastião, Ana M; Valente, Cláudia A.; Ledri, Marco; Andersson, My; Kokaia, MerabDespite the availability of new drugs on the clinics in recent years, drug-resistant epilepsy remains an unresolved challenge for healthcare, and one-third of epilepsy patients remain refractory to anti-seizure medications. Gene therapy in experimental models has emerged as effective treatment targeting specific neuronal populations in the epileptogenic focus. When combined with an external chemical activator using chemogenetics, it also becomes an "on-demand" treatment. Here, we evaluate a targeted and specific chemogenetic therapy, the PSAM/PSEM system, which holds promise as a potential candidate for clinical application in treating drug-resistant epilepsy. We show that the inert ligand uPSEM817, which selectively activates the chloride-permeable channel PSAM4-GlyR, effectively reduces the number of depolarization-induced action potentials in vitro. This effect is likely due to the shunting of depolarizing currents, as evidenced by decreased membrane resistance in these cells. In organotypic slices, uPSEM817 decreased the number of bursts and peak amplitude of events of spontaneous epileptiform activity. Although administration of uPSEM817 in vivo did not significantly alter electrographic seizures in a male mouse model of temporal lobe epilepsy, it did demonstrate a strong trend toward reducing the frequency of interictal epileptiform discharges. These findings indicate that PSAM4-GlyR-based chemogenetics holds potential as an anti-seizure strategy, although further refinement is necessary to enhance its efficacy.
- Unravelling a novel role for cannabidivarin in the modulation of subventricular zone postnatal neurogenesisPublication . Lourenço, Diogo M.; Soares, Rita; Sá Santos, Sónia; Mateus, Joana; Rodrigues, Rui S.; Moreira, João B.; Vaz, Sandra H.; Sebastião, Ana M; Solá, Susana; Xapelli, SaraPostnatal neurogenesis has been shown to rely on the endocannabinoid system. Here we aimed at unravelling the role of Cannabidivarin (CBDV), a non-psychoactive cannabinoid, with high affinity for the non-classical cannabinoid receptor TRPV1, on subventricular zone (SVZ) postnatal neurogenesis. Using the neurosphere assay, SVZ-derived neural stem/progenitor cells (NSPCs) were incubated with CBDV and/or 5'-Iodoresinferotoxin (TRPV1 antagonist), and their role on cell viability, proliferation, and differentiation were dissected. CBDV was able to promote, through a TRPV1-dependent mechanism, cell survival, cell proliferation and neuronal differentiation. Furthermore, pulse-chase experiments revealed that CBDV-induced neuronal differentiation was a result of cell cycle exit of NSPCs. Regarding oligodendrocyte differentiation, CBDV inhibited oligodendrocyte differentiation and maturation. Since our data suggested that the CBDV-induced modulation of NSPCs acted via TRPV1, a sodium-calcium channel, and that intracellular calcium levels are known regulators of NSPCs fate and neuronal maturation, single cell calcium imaging was performed to evaluate the functional response of SVZ-derived cells. We observed that CBDV-responsive cells displayed a two-phase calcium influx profile, being the initial phase dependent on TRPV1 activation. Taken together, this work unveiled a novel and untapped neurogenic potential of CBDV via TRPV1 modulation. These findings pave the way to future neural stem cell biological studies and repair strategies by repurposing this non-psychoactive cannabinoid as a valuable therapeutic target.
- Cholesterol redistribution triggered by CYP46A1 gene therapy improves major hallmarks of Niemann-Pick type C disease but is not sufficient to halt neurodegenerationPublication . Nunes, Maria João; Carvalho, Andreia N.; Reis, Joana; Costa, Daniela; Moutinho, Miguel; Mateus, Joana; Mendes de Almeida, Rita; Brito, Sara; Risso, Daniela; Nunes, Sofia; Castro-Caldas, Margarida; Gama, Maria João; Rodrigues, Cecília M. P.; Xapelli, Sara; Diógenes, Maria José; Cartier, Nathalie; Chali, Farah; Piguet, Françoise; Rodrigues, ElsaCholesterol 24-hydroxylase (CYP46A1) is an exclusively neuronal cytochrome P450 enzyme responsible for converting cholesterol into 24S-hydroxycholesterol, which serves as the primary pathway for eliminating cholesterol in the brain. We and others have shown that increased activity of CYP46A1 leads to reduced levels of cholesterol and has a positive effect on cognition. Therefore, we hypothesized that CYP46A1 could be a potential therapeutic target in Niemann-Pick type C (NPC) disease, a rare and fatal neurodegenerative disorder, characterized by cholesterol accumulation in endolysosomal compartments. Herein, we show that CYP46A1 ectopic expression, in cellular models of NPC and in Npc1tm(I1061T) mice by adeno-associated virus-mediated gene therapy improved NPC disease phenotype. Amelioration in functional, biochemical, molecular and neuropathological hallmarks of NPC disease were characterized. In vivo, CYP46A1 expression partially prevented weight loss and hepatomegaly, corrected the expression levels of genes involved in cholesterol homeostasis, and promoted a redistribution of brain cholesterol accumulated in late endosomes/lysosomes. Moreover, concomitant with the amelioration of cholesterol metabolism dysregulation, CYP46A1 attenuated microgliosis and lysosomal dysfunction in mouse cerebellum, favoring a pro-resolving phenotype. In vivo CYP46A1 ectopic expression improves important features of NPC disease and may represent a valid therapeutic approach to be used concomitantly with other drugs. However, promoting cholesterol redistribution does not appear to be enough to prevent Purkinje neuronal death in the cerebellum. This indicates that cholesterol buildup in neurons might not be the main cause of neurodegeneration in this human lipidosis.