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- IL-10 and Cdc42 as critical modulators in methamphtamine-induced neuroinflammationPublication . Silva, Ana Isabel; Socodato, Renato; Pinto, Carolina; Terceiro, Ana Filipa; Canedo, Teresa; Relvas, João Bettencourt; Saraiva, Margarida; Summavielle, Teresa; Summavielle, TeresaPsychoactive substances, such as Methamphetamine (Meth), can induce complex neuroinflammatory responses that modulate the neuron-glia cross talk and strongly affect behavioral responses. Recently we have reported that Meth stimulates astrocytes to release tumor necrosis factor (TNF) and glutamate, leading to microglial activation, microgliosis and loss of risk-assessment. Here, we started by investigating the anti-inflammatory power the cytokine interleukin-10 (IL-10), resorting to astrocyte and microglia primary transfected with different FRET probes and exposed to Meth (100µM), to elucidate the mechanisms involved. Then after, we confirmed these results in vivo, by employing a transgenic mouse model that overexpresses IL-10 (pMT-10), in time-controlled manner, and administering a binge Meth dosing (4 x 5mg/kg, with 2h intervals). In vitro, our findings reveal that the presence of recombinant IL-10 (rIL-10) counteracts Meth-induced excessive glutamate release in astrocytes, which significantly reduced microglial activation. This reduction was 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. In vivo, we observed that IL-10 overexpressing prevented Meth-induced neuroinflammation, microgliosis and Meth-induced behavioral changes. These findings enhance our understanding of Meth-related neuroinflammatory mechanisms, suggesting IL-10 and Cdc42 as putative therapeutic targets, and strengthen the view of a neuroimmune nature for addiction.
- The role of RAC1 in methamphtamine-induced neuroinflammationPublication . Terceiro, Ana Filipa; Canedo, Teresa; Silva, Ana Isabel; Magalhães, Ana; Relvas, João; Summavielle, Teresa; Summavielle, TeresaMethamphetamine (Meth), a powerful psychostimulant, induces detrimental neuroinflammatory responses, in the brain reward system that seem to contribute to maintenance of addictive behaviour. Yet, the mechanisms regulating these processes in microglial cells are not clear. We have previously shown that exposing WT mice to Meth (4x5 mg/kg, 2h intervals) induces microgliosis concomitant with decreased microglia cell volume and ramification. Furthermore, psychostimulants are known to induce structural plasticity mechanisms in neurons, and Rho GTPases, important regulators of the actin cytoskeleton, are involved in these responses. Here, we assessed Rho GTPases, specifically rhoA, rac1 and cdc42, activation in response to Meth in microglia. Exposing WT mice to the same pattern of Meth administration, we found an increase in the activation of rac1 in the striatum, 15 min following the last administration of Meth. To further explore these results, we then used a conditional mice model for ablation of rac1 in adult microglia (Rac1fl/fl:Cx3cr1CreER+) and exposed these mice to the same pattern of Meth administration. Rac1 ablation was sufficient to prevent Meth-induced morphological alterations in the striatum. Currently, we are assessing the role of rac1 in the behavioural response to Meth, using a locomotor sensitization test. Overall, we identified rac1 as a novel target of Meth in microglial cells. With these results, we expect to clarify if targeting Rho GTPases may contribute to improving the treatment of addictive disorders.
- Methamphetamine activates rac1 in striatal microgliaPublication . Terceiro, Ana Filipa; Canedo, Teresa; Silva, Ana Isabel; Magalhães, Ana; Relvas, João; Summavielle, Teresa; Summavielle, TeresaMethamphetamine (Meth), a powerful psychostimulant, induces profound synaptic and morphological alterations alongside with detrimental neuroinflammatory responses, in the brain reward system. Yet, the mechanisms regulating these processes in microglial cells are not clear. We have previously shown that exposing WT mice to Meth (4x5mg/kg, 2h intervals) induces microgliosis concomitant with decreased microglia cell volume and ramification. Furthermore, psychostimulants are known to induce structural plasticity mechanisms in neurons, and Rho GTPases, important regulators of the actin cytoskeleton, are involved in these responses. Here, we evaluate if Rho GTPases, specifically rhoA, rac1 and cdc42, are critical in the response to Meth in microglia. Exposing WT mice to the samepattern of Meth administration, we found an increase in the activation of rac1 in the striatum, 15 min following the last administration of Meth. To further explore these results, we then used a conditional mice model for ablation of rac1 in adult microglia (Rac1fl/fl:Cx3cr1CreER+) and exposed these mutants to the same pattern of Meth administration. We found that rac1 ablation is sufficient to prevent Meth-induced morphological alterations in the striatum. Currently, we are assessing whether ablation of rac1 is also sufficient to prevent the neuroinflammatory response induced by Meth. Overall, we identified rac1 as a novel target of Meth in microglial cells. With these results, we expect to clarifyif targeting Rho GTPases may contribute to improving the treatment of addictive disorders.
- Microglial Rac1 is essential for microglia-synapse crosstalk and cognitive performancePublication . Almeida, Tiago O.; Socodato, Renato; Portugal, Camila C.; Santos, Evelyn C. S.; Moreira, Joana Tedim; Ferreira, João Galvão; Canedo, Teresa; Magalhães, Ana; Summavielle, Teresa; Relvas, João B.; Summavielle, TeresaMicroglia, the main immune defenders of the brain, rapidly detect and react to stimuli due to constant extension andretraction of their processes. When engaged by external cues, that can be either inflammatory or products resulting from synaptic activity, microglia dramatically change their morphology and initiate a response to reestablish brain homeostasis. Additionally, microglia can also regulate and sustain synaptic activity by secreting a plethora of factors. While some of these factors are already described, there is still much to understand on how exactly microglia-secreted factors modulate synaptic function. Rac1, a well-known member of the Rho GTPase family, is a critical regulator of actin cytoskeleton dynamics and reorganization. Furthermore, Rac1 is a component of NADPH oxidase complex, a key element for phagocytic cup formation and it also regulates NF-κB pathway activation. In the central nervous system (CNS), Rac1 is involved in axon guidance and growth, but it is also linked with Alzheimer´s disease,since it regulates the expression of amyloid precursor protein in hippocampal neurons. Although extensively studied in other cell types in and outside of the CNS, there is a profound knowledge gap on how Rac1 regulates microglia function in homeostasis and in response to external stimuli. Combining cell-specific conditional gene ablation, RNAseq, flow cytometry, immunofluorescence, proteomics and phosphoproteomics, FRET live cell imaging and mouse behavior, we aimed at describing for the first time Rac1 roles in microglial function. We observed that microglia specific Rac1 ablation impaired the capacity of microglia to sense and respond to changes in their local environment. To promote changes on the brain environment, we performed a protocol of environmental enrichment (EE), mimicking currently used therapeutic approaches for enhancing brain plasticity in patients with brain disorders. EE had a profound impact on the microglial phosphoproteomic landscape, showing a strong effect on Rho GTPase signaling. Interestingly, we showed that Rac1 signaling was the most significantly altered pathway, followed by Cdc42 and RhoA signaling, allowing us to define a hierarchy between them. Besides, EE led to an overall improvement of cognitive performance. Strikingly, ablating Rac1 from microglia completely prevented this EE-dependent cognitive enhancement and disrupted microglia-synapse crosstalk, ultimately impacting the synaptic proteomic and phosphoproteomic profiles of these mice. Overall, this is a first step into understanding how Rac1 mediates microglial responses to their local environment. This places Rac1 as anessential target for further studies and reinforces the importance of Rho GTPases signaling for adult microglial function.
- Psychostimulants and neuroinflammation: finding critical players in the crostalk between glial cells and neuronsPublication . Bravo, Joana; Andrade, Elva Bonifácio; Vieira, Renato; Lorga, Inês; Azevedo, M.; Rodrigues, J.; Magalhães, Ana; Relvas, João B.; Summavielle, Teresa; Summavielle, Teresa; Bonifácio Andrade, Elva; Bravo, JoanaExposure to psychostimulants has been classically associated with damage to neuronal terminals. However, it is now accepted that interaction between neuronal and glial cells also contributes to the addictive behavior. We have recently shown that acute methamphetamine (Meth), a powerful psychostimulant, causes microgliosis and increases microglia activation through astrocytic-TNF release. We are now interested in clarifying the progression of neuroinflammation under chronic drug exposure and how different brain and immune cells contribute to this inflammatory process.To explore this, firstly, we performed a proteomic analysis, in different phases of the addictive process, in mice exposed to an escalating dosing of Meth for ten days (Meth10d). To validate the conditioning power of our model,mice were tested in a condition place preference (CPP) at 10d of Meth, and 2 or 10 days of withdrawal (WD). At all these time points, mice were seen to be strongly conditioned by Meth. Next, we conducted a proteomic analysis to compare the different time points (using the hippocampus, where we previously found robust microgliosis underMeth). We found a proteome profile that varied substantially with exposure (Meth10d) and after a short- (WD2d)and long-term withdrawal (WD10d) periods. Interestingly, the most altered pathways were neuro transmitter-related. However, we also identified significant differences in Wnt signaling, which was previously linked to regulation of microglia reactivity. As such, we evaluated the microglia profile after chronic Meth exposure and at withdrawal. In the hippocampus, the number of microglia cells was significantly increased at Meth10d and remained also increased at WD2d. Microglia presented a more ameboid-like shape at Meth10d, but its ramified morphology was recovered atWD2d. Importantly, our proteomic data also revealed that during Meth withdrawal, several microglial receptors were down regulated, suggesting that microglia was in a “primed” state. In addition, as the crosstalk between neurons and microglia seems to be relevant for the behavioral expression of Meth, we are dissecting the modulation of microglia by neurons under Meth exposure, to evaluate neuroimmune regulatory ligand-receptor pairs that seem to impact onthe neuron-microglia interaction. Of note, some these ligand-receptor pairs seem to be down regulated by chronic Meth and during abstinence, which may be associated with reduced neuronal ability to down regulate microglia reactivity, and lead to increased neuronal damage.We fore see that these receptors may prove to be interesting therapeutic targets for the treatment of addiction, and therefore we will manipulate them to confirm their value in reducing relapse rates and improve addiction treatments.
- Blockingmethamphetamine-induced microglia reactivity by targeting glutamate receptorsPublication . Summavielle, Teresa; Canedo, Teresa; Silva, Ana Isabel; Andrade, Elva Bonifácio; Almeida, Tiago O.; Bravo, Joana; Terceiro, Ana Filipa; Canedo, Teresa; Silva, Ana Isabel; Magalhães, Ana; Relvas, João B.; Bonifácio Andrade, Elva; Bravo, JoanaExposure to psychostimulants has been classically associated with damage to neuronal terminals. However, it is now accepted that interaction between neuronal and glial cells also contributes to the addictive behavior. We have recently shown that acute methamphetamine (Meth), a powerful psychostimulant, causes microgliosis and increases microglia activation through astrocytic-TNF release1. We are now interested in clarifying the progression of neuroinflammation under chronic drug exposure and how different brain and immune cells contribute to this inflammatory process.To explore this, firstly, we performed a proteomic analysis, in different phases of the addictive process, in mice exposed to an escalating dosing of Meth for ten days (Meth10d). To validate the conditioning power of our model, mice were tested in a condition place preference (CPP) at 10d of Meth, and 2 or 10 days of withdrawal (WD). At all these time points, mice were seen to be strongly conditioned by Meth. Next, we conducted a proteomic analysis to compare the different time points (using the hippocampus, where we previously found robust microgliosis underMeth1). We found a proteome profile that varied substantially with exposure (Meth10d) and after a short- (WD2d)and long-term withdrawal (WD10d) periods. Interestingly, the most altered pathways were neuro transmitter-related.However, we also identified significant differences in Wnt signaling, which was previously linked to regulation of microglia reactivity. As such, we evaluated the microglia profile after chronic Meth exposure and at withdrawal. In the hippocampus, the number of microglia cells was significantly increased at Meth10d and remained also increased at WD2d. Microglia presented a more ameboid-like shape at Meth10d, but its ramified morphology was recovered at WD2d. Importantly, our proteomic data also revealed that during Meth withdrawal, several microglial receptors were down regulated, suggesting that microglia was in a “primed” state. In addition, as the crosstalk between neurons and microglia seems to be relevant for the behavioral expression of Meth, we are dissecting the modulation of microgliaby neurons under Meth exposure, to evaluate neuroimmune regulatory ligand-receptor pairs that seem to impact on the neuron-microglia interaction. Of note, some these ligand-receptor pairs seem to be down regulated by chronic Meth and during abstinence, which may be associated with reduced neuronal ability to down regulate microglia reactivity, and lead to increased neuronal damage. We fore see that these receptors may prove to be interesting therapeutic targets for the treatment of addiction, and therefore we will manipulate them to confirm their value in reducing relapse rates and improve addiction treatments.
- Untangling sex differences in glia-to-neuron communication in chronic alcohol exposurePublication . Pacheco, Raquel; Canedo, Teresa; Rodrigues, Ana Margarida; Moreira, Joana; Relvas, João B.; Vieira, Cristina P.; Socodato, Renato; Sumavielle, Teresa; Summavielle, TeresaExcessive alcohol consumption is global health challenge with profound neurobiological consequences. Our laboratory has shown that alcohol exposure induces reactive astrocytic changes, affecting gene expression, activity, and proliferation, while also altering microglial morphology and immune responses [1]. This study aims to characterise sex-specific effects of chronic alcohol consumption on the prefrontal cortex (PFC), focusing on glial cell morphology, synaptic density, and behaviour. Adult mice underwent a voluntary drinking model using an intermittent ‘every-other-day’ (EOD) paradigm with 15% (v/v) ethanol for three weeks. Behavioural tests for anxiety, depression, and memory were conducted before sacrifice. Brains were processed for glial cell analysis using immunohistochemistry and confocal microscopy. Synaptosomes were isolated for proteomics and analysis used STRING and Panther. Preliminary data reveal sex-dependent glial and synaptic adaptations. Males showed increased astrocyte volume in the ventromedial PFC (vmPFC) and hyper-ramification in the ventrolateral PFC (vlPFC), while females showed reductions in astrocyte size and complexity. Microglia also displayed sex-specific changes: males had decreased microglial volume in the vlPFC, while females exhibited increased microglial size. Inhibitory synapse density was elevated in males, while females showed an increase in excitatory synapses. These changes correlated with behavioural differences—males displayed heightened anxiety, whereas females exhibited reduced anxiety. Proteomic analysis further supports sex-dependent molecular adaptations: male-enriched proteins were linked to myelination and glial development, while female-specific proteins were associated with mRNA processing, RNA transport, and axonogenesis. Chronic alcohol exposure induces sex-specific neuroimmune and synaptic alterations, potentially contributing to differential susceptibility to alcohol use disorders (AUD).
- Association between placental weight and birthweight, adjusted for parity, sex and PAPP-A levels: a retrospective cohort studyPublication . Santos, Inês; Moreira, Rui; Gonçalves, Inês Sarmento; Dias, Cláudia Camila; Ramalho, CarlaPlacental weight has been studied as an indicator of placental function, correlating with birthweight and perinatal outcomes. The fetal-to-placental ratio (FPR) reflects the adaptation to hypoxemia and fetal growth needs. This study aims to assess the association between placental weight and birthweight, adjusting for parity, fetal sex and PAPP-A. A retrospective cohort study was conducted at a tertiary university hospital. We included singleton pregnancies undergoing first-trimester screening and delivering at the same hospital between May 2013 and September 2024. Clinical data were gathered from electronic databases (SClínico®, ObsCare®, ASTRAIA®). Categorical data were analyzed using Chi-squared tests. Continuous variables were assessed through ANOVA or T test. Pearson or Spearman correlation was applied as appropriate. For significance, a p-value<0.05 was assumed. All data was processed with IBM SPSS v29.0. Results: Among 16492 singleton pregnancies, 54.9% women were nulliparous and 8.9% newborns were small for gestational age. Placental weight was positively correlated with birthweight (r=0.517,p<0.001), which increased by 1.77g per gram of placental weight, explaining 26.7% of the birthweight variation. Preterm deliveries (6.8%) had lower mean placental weight (493.1g versus 579.2g) and birthweight (900g difference, p<0.001) compared to term group. Mean male fetuses’ birthweight was 121.3g higher than female (p<0.001). Additionally, “female fetuses” and “nulliparity” were associated with decreased placental weight (p<0.001). On average, the FPR was 6:1, with male fetuses exhibiting greater FPR (p<0.001) as well as term deliveries (5.7) compared to preterm (4.8), p<0.001. FPR was also correlated with Apgar score at 5min (Apgar<7:5.0 vs. Apgar≥7:5.7, p<0.001) and PAPP-A levels (p<0.001). However, parity (p=0.121) showed no significant association with FPR. Placental weight correlates with birthweight. FPR reflects placental efficiency and perinatal outcome, influenced by fetal sex and gestational age.
- Breastfeeding and malocclusions: Characterization of a pediatric dentistry population enrolled in a birth cohort studyPublication . Marques, Sofia Cameron; Baptista, Manuel; Magalhães, Inês; Morais, Juliana; Ferreira, Ana Filipa; Pestana, Rebeca; Jerónimo, Maria Luís; Pinto, Carla; Sousa, Marta; Pires, Inês Falcão; Deuster, Denise; Zaura, Egija; Areias, Cristina; Maia, Benedita SampaioBreastfeeding plays an important role in the development of the craniofacial complex (1,2). However, the role of breastfeeding duration, non-nutritive sucking habits, and the timing of solid food introduction on the development of malocclusions is still in debate (25). Therefore, this study aimed to investigate these associations in a birth cohort. In the ongoing OralBioBorn birth cohort, occlusion of three-year-old children was assessed in maxiumum intercuspidation, examining the left and right sides, followed by a frontal assessment. In addition, the information on breastfeeding type and duration, solid food introduction, and non-nutritive sucking habits was collected through a parental questionnaire. Preliminary data from 39 children revealed a high prevalence of open bite (33.3%), crossbite (20.5%), and overbite (10.3%). Scissor bite, high/narrow palate, and crowding were observed in only one child each. The relationship between these occlusal characteristics and breastfeeding duration, non-nutritive sucking habits, and solid food introduction will be further explored. The preliminary findings suggest that malocclusions are frequent at an early age, reinforcing the need to investigate their potential early-life determinants. Future work will involve a more in-depth analysis of the associations between occlusal development and early feeding practices, considering additional factors such as genetic predisposition and environmental influences. Longitudinal follow-up of this cohort will provide valuable insights into the long-term impact of breastfeeding and oral habits on craniofacial growth, ultimately contributing to early preventive strategies.
- Association between maternal cardiometabolic risk factors and oral health of infantsPublication . Pestana, Rebeca Saad; Baptista, Manuel; Magalhães, Inês; Morais, Juliana; Ferreira, Ana Filipa; Marques, Sofia Cameron; Jerónimo, Maria Luís; Pinto, Carla; Sousa, Marta; Falcão-Pires, Inês; Duister, Denise; Zaura, Egija; Azevedo, Maria João; Maia, Benedita SampaioMaternal health conditions during pregnancy influence infant health1-3. Cardiometabolic risk factors (CRFs), such as obesity, hypertension, and gestational diabetes, increase maternal and neonatal complications due to chronic inflammation, which disrupts immune and endocrine adaptation4,5. While the effects of CRFs on infant development are well studied, their impact on oral health remains unclear. Therefore, this study aims to assess the association between maternal CRFs and oral health of infants at three years of age. The prospective OralBioBorn cohort follows pregnant women (healthy and with CRFs) and their children up to 36 months postpartum. At age three, oral hygiene habits, caries experience using ICDAS, and Quigley-Hein plaque index (PI) of children were assessed. PI was evaluated on buccal and lingual surfaces. Preliminary data from 36 children (26 from healthy mothers, 10 from mothers with CRFs) showed no cavitated, missing, or filled teeth due to caries, nor PUFA Index lesions, in either group. The PI did not differ significantly between children of healthy mothers and those of mothers with CRFs (0.17±0.32 vs. 0.49±0.72, p>0.05, Mann-Whitney U test). The lack of associations between maternal CRFs and child oral health may reflect multiple factors, including the small sample size, the young age of the children, and the possibility that maternal CRFs do not exert a strong early influence on oral health. Future work will focus on evaluating a larger cohort and considering additional determinants, such as oral hygiene and dietary habits, which may play a more prominent role in shaping early oral health outcomes. This will provide a deeper understanding of potential mediating factors influencing early oral health trajectories