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- IL-10 and Cdc42 modulate astrocyte-mediated microglia activation in methamphetamine-induced neuroinflammationPublication . Silva, Ana Isabel; Socodato, Renato; Pinto, Carolina; Terceiro, Ana Filipa; Canedo, Teresa; Relvas, JoĆ£o Bettencourt; Saraiva, Margarida; Summavielle, TeresaMethamphetamine (Meth) use is known to induce complex neuroinflammatory responses, particularly involving astrocytes and microglia. Building upon our previous research, which demonstrated that Meth stimulates astrocytes to release tumor necrosis factor (TNF) and glutamate, leading to microglial activation, this study investigates the role of the anti-inflammatory cytokine interleukin-10 (IL-10) in this process. Our findings reveal that the presence of recombinant IL-10 (rIL-10) counteracts Meth-induced excessive glutamate release in astrocyte cultures, which significantly reduces microglial activation. This reduction is associated with the modulation of astrocytic intracellular calcium (Ca2+) dynamics, particularly by restricting the release of Ca2+ from the endoplasmic reticulum to the cytoplasm. Furthermore, we identify the small Rho GTPase Cdc42 as a crucial intermediary in the astrocyte-to-microglia communication pathway under Meth exposure. By employing a transgenic mouse model that overexpresses IL-10 (pMT-10), we also demonstrate in vivo that IL-10 prevents Meth-induced neuroinflammation. These findings not only enhance our understanding of Meth-related neuroinflammatory mechanisms, but also suggest IL-10 and Cdc42 as putative therapeutic targets for treating Meth-induced neuroinflammation.
- Abnormal immunoreactivity to serotonin in cerebellar purkinje cells after neonatal cocaine exposurePublication . Summavielle, Teresa; Alves, CecĆlia J.; Monteiro, Pedro; Tavares, Maria AmĆ©liaNeonatal cocaine is known to affect the developing serotonergic system in many brain structures, including the cerebellum. Changes in the cerebellar Purkinje cells after drug exposure are well documented and result in impairment of movement and other cerebellar disorders such as ataxia. These cells have a major postnatal developmental pattern; therefore, neonatal exposure to cocaine is likely to affect them. In this work, male and female Wistar rats were injected with 15 mg of cocaine hydrochloride/kg body weight/day, subcutaneously, in two daily doses, from postnatal day 1 (PND1) to PND29. Controls were given 0.9% of saline. On PND14, PND21, and PND30, rats were transcardially perfused, and brains removed and cryoprotected. Coronal sections from the cerebellum were processed for immunocytochemistry of cells containing serotonin (5-hydroxytryptamine, or 5-HT). At the same postnatal age, rats from at least three different litters were sacrificed by decapitation, and brains were dissected for determination of 5-HT in the cerebellum by high-performance liquid chromatography with electrochemical detection. Upon the expected distribution of immunoreactivity to 5-HT, an abnormal immunoreactivity to 5-HT was observed in the Purkinje cells of six cocaineexposed animals, but not in control animals. Also, levels of cerebellar 5-HT in cocaine-exposed rats were significantly increased on PND21. These results, together with previously reported observations of altered patterns of motor behavior, indicate that neonatal cocaine exposure affects the serotonergic cerebellar system, altering the standard development of Purkinje cells and possibly compromising the motor function.
- Interlimb coordination during double support phase of gait in people with and without strokePublication . Couto, Ana G. B.; Vaz, MĆ”rio A. P.; Pinho, Liliana; FĆ©lix, JosĆ©; Moreira, Juliana; Pinho, Francisco; Mesquita, InĆŖs; Mesquita Montes, AntĆ³nio; Crasto, Carlos; Sousa, AndreiaThis study aims to identify differences between participants with and without stroke regarding the ipsilesional and contralesional lower limbs kinematics, kinetics, muscle activity and their variability during double support phase of gait. Eleven post-stroke and thirteen healthy participants performed 10 gait trials at a self-selected speed while being monitored by an optoelectronic motion capture system, two force plates and an electromyographic system. The following outcomes were evaluated during the double support: the time and the joint position; the external mechanical work on the centre of mass; and the relative electromyographic activity. Both, contralesional/ipsilesional and dominant/non-dominant of participants with and without stroke, respectively, were evaluated during double support phase of gait in trailing or leading positions. The average value of each parameter and the coefficient of variation of the 10 trials were analysed. Post-stroke participants present bilateral decreased mechanical work on the centre of mass and increased variability, decreased contralesional knee and ankle flexion in trailing position, increased ipsilesional knee flexion in leading position and increased variability. Increased relative muscle activity was observed in post-stroke participants with decreased variability. Mechanical work on the centre of mass seems to be the most relevant parameter to identify interlimb coordination impairments in post-stroke subjects.
- Microglial Rac1 is essential for experience-dependent brain plasticity and cognitive performancePublication . Socodato, Renato; Almeida, Tiago O.; Portugal, Camila C.; Santos, Evelyn C.S.; Tedim-Moreira, Joana; Ferreira, JoĆ£o GalvĆ£o; Canedo, Teresa; Baptista, Filipa I.; MagalhĆ£es, Ana; AmbrĆ³sio, AntĆ³nio F.; Brakebusch, Cord; Rubinstein, Boris; Moreira, Irina S.; Summavielle, Teresa; Pinto, InĆŖs Mendes; Relvas, JoĆ£o B.Microglia, the largest population of brain immune cells, continuously interact with synapses to maintain brain homeostasis. In this study, we use conditional cell-specific gene targeting in mice with multi-omics approaches and demonstrate that the RhoGTPase Rac1 is an essential requirement for microglia to sense and interpret the brain microenvironment. This is crucial for microglia-synapse crosstalk that drives experience-dependent plasticity, a fundamental brain property impaired in several neuropsychiatric disorders. Phosphoproteomics profiling detects a large modulation of RhoGTPase signaling, predominantly of Rac1, in microglia of mice exposed to an environmental enrichment protocol known to induce experience-dependent brain plasticity and cognitive performance. Ablation of microglial Rac1 affects pathways involved in microglia-synapse communication, disrupts experience-dependent synaptic remodeling, and blocks the gains in learning, memory, and sociability induced by environmental enrichment. Our results reveal microglial Rac1 as a central regulator of pathways involved in the microglia-synapse crosstalk required for experience-dependent synaptic plasticity and cognitive performance.
- Astrocyte-derived TNF and glutamate critically modulate microglia activation by methamphetaminePublication . Canedo, Teresa; Portugal, Camila Cabral; Socodato, Renato; Almeida, Tiago Oliveira; Terceiro, Ana Filipa; Bravo, Joana; Silva, Ana Isabel; MagalhĆ£es, JoĆ£o Duarte; Guerra-Gomes, SĆ³nia; Oliveira, JoĆ£o Filipe; Sousa, Nuno; MagalhĆ£es, Ana; Relvas, JoĆ£o Bettencourt; Summavielle, TeresaMethamphetamine (Meth) is a powerful illicit psychostimulant, widely used for recreational purposes. Besides disrupting the monoaminergic system and promoting oxidative brain damage, Meth also causes neuroinflammation, contributing to synaptic dysfunction and behavioral deficits. Aberrant activation of microglia, the largest myeloid cell population in the brain, is a common feature in neurological disorders triggered by neuroinflammation. In this study, we investigated the mechanisms underlying the aberrant activation of microglia elicited by Meth in the adult mouse brain. We found that binge Meth exposure caused microgliosis and disrupted risk assessment behavior (a feature that usually occurs in individuals who abuse Meth), both of which required astrocyte-to-microglia crosstalk. Mechanistically, Meth triggered a detrimental increase of glutamate exocytosis from astrocytes (in a process dependent on TNF production and calcium mobilization), promoting microglial expansion and reactivity. Ablating TNF production, or suppressing astrocytic calcium mobilization, prevented Meth-elicited microglia reactivity and re-established risk assessment behavior as tested by elevated plus maze (EPM). Overall, our data indicate that glial crosstalk is critical to relay alterations caused by acute Meth exposure.
- Profiling microglia in a mouse model of MachadoāJoseph diseasePublication . Campos, Ana Bela; Silva, Sara Duarte; Fernandes, Bruno; Neves, Sofia Pereira das; Marques, Fernanda; Castro, Andreia Teixeira; Carvalho, Andreia Neves; Fernandes, Daniela Monteiro; Portugal, Camila Cabral; Socodato, Renato; Summavielle, Teresa; AmbrĆ³sio, AntĆ³nio Francisco; Relvas, JoĆ£o Bettencourt; Maciel, PatrĆciaMicroglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in MachadoāJoseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder.
- Involving forensic students in integrative learningāa project proposalPublication . Teixeira, A.; Azevedo, A.; PĆ©rez-Mongiovi, D.; Caldas, I. M.; Costa-Rodrigues, JoĆ£oIn our experience, university students enrolling in health science and forensic science degrees show difficulty in retaining and integrating basic scientific knowledge learned in their first academic year. Furthermore, in the forensic sciences case, many students have oversimplified and unrealistic expectations as a result of the exposure to crime TV shows, internet blogs, and other social media platforms. Our pedagogical proposal is focused on second-year university students, aiming at promoting effective learning and the integration of scientific knowledge from previous courses, in this particular example, molecular and cell biology and biochemistry, with more advanced forensic courses, such as forensic anthropology and odontology. Teams composed of students and tutors from the teaching staff, with the help of dichotomous keys, are challenged to analyze a crime scene and choose the relevant evidence to further investigate, determine the scientific approach, execute the experimental work, interpret the results and, finally, resolve the case. To assess the pedagogical advantages and the receptivity of this project, a survey is to be carried out among students, and respective statistical analysis is also proposed. Finally, we hope this project outline may be adapted to other subjects, and, therefore, be used to address different pedagogical questions in forensic studies.
- Neuronāmicroglia contact-dependent mechanisms attenuate methamphetamine-induced microglia reactivity and enhance neuronal plasticityPublication . Bravo, Joana; Ribeiro, InĆŖs; Terceiro, Ana Filipa; Andrade, Elva B.; Portugal, Camila Cabral; Lopes, Igor M.; Azevedo, Maria M.; Sousa, Mafalda; Lopes, CĆ”tia D. F.; Lobo, Andrea C.; Canedo, Teresa; Relvas, JoĆ£o Bettencourt; Summavielle, TeresaExposure to methamphetamine (Meth) has been classically associated with damage to neuronal terminals. However, it is now becoming clear that addiction may also result from the interplay between glial cells and neurons. Recently, we demonstrated that binge Meth administration promotes microgliosis and microglia pro-inflammation via astrocytic glutamate release in a TNF/IP3R2-Ca2+-dependent manner. Here, we investigated the contribution of neuronal cells to this process. As the crosstalk between microglia and neurons may occur by contact-dependent and/or contact-independent mechanisms, we developed co-cultures of primary neurons and microglia in microfluidic devices to investigate how their interaction affects Meth-induced microglia activation. Our results show that neurons exposed to Meth do not activate microglia in a cell-autonomous way but require astrocyte mediation. Importantly, we found that neurons can partially prevent Meth-induced microglia activation via astrocytes, which seems to be achieved by increasing arginase 1 expression and strengthening the CD200/CD200r pathway. We also observed an increase in synaptic individual area, as determined by co-localization of pre- and post-synaptic markers. The present study provides evidence that contact-dependent mechanisms between neurons and microglia can attenuate pro-inflammatory events such as Meth-induced microglia activation.
- Bioactivity of ionic liquids based on valproate in SH-SY5Y human neuroblastoma cell linePublication . Dias, Ana Rita; Ferraz, Ricardo; Costa-Rodrigues, JoĆ£o; Santos, Andreia F. M.; Jacinto, Manuel L.; PrudĆŖncio, Cristina; Noronha, JoĆ£o Paulo; Branco, LuĆs C.; Petrovsk, ŽeljkoThe search for alternative and effective therapies to fight cancer is one of the main goals of the pharmaceutical industry. Recently, ionic liquids (ILs) have emerged as potential therapeutic agents with antitumor properties. The goal of this study was to synthesize and evaluate the bioactivity of different ILs coupled with the active pharmaceutical ingredient (API) valproate (VPA) as an antitumor agent. The toxicity of the prepared ionic liquids was evaluated by the MTT cell metabolic assay in human neuroblastoma SH-SY5Y and human primary Gingival Fibroblast (GF) cell lines, in which they showed inhibitory effects during the study period. In addition, low cytotoxicity against GF cell lines was observed, suggesting that these compounds are not toxic to human cell lines. [C2OHDMiM][VPA] demonstrated an outstanding antitumor activity against SH-SY5Y and lower activity against the non-neoplastic GF line. The herein assessed compounds played an important role in the modulation of the signaling pathways involved in the cellular behavior. This work also highlights the potential of these ILs-API as possible antitumor agents.
- Multimodal classification of anxiety based on physiological signalsPublication . Vaz, Mariana; Summavielle, Teresa; SebastiĆ£o, Raquel; Ribeiro, Rita P.Multiple studies show an association between anxiety disorders and dysregulation in the Autonomic Nervous System (ANS). Thus, understanding how informative the physiological signals are would contribute to effectively detecting anxiety. This study targets the classification of anxiety as an imbalanced binary classification problem using physiological signals collected from a sample of healthy subjects under a neutral condition. For this purpose, the Electrocardiogram (ECG), Electrodermal Activity (EDA), and Electromyogram (EMG) signals from the WESAD publicly available dataset were used. The neutral condition was collected for around 20 min on 15 participants, and anxiety scores were assessed through the shortened 6-item STAI. To achieve the described goal, the subsequent steps were followed: signal pre-processing; feature extraction, analysis, and selection; and classification of anxiety. The findings of this study allowed us to classify anxiety with discriminatory class features based on physiological signals. Moreover, feature selection revealed that ECG features play a relevant role in anxiety classification. Supervised feature selection and data balancing techniques, especially Borderline SMOTE 2, increased the performance of most classifiers. In particular, the combination of feature selection and Borderline SMOTE 2 achieved the best ROC-AUC with the Random Forest classifier.