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Introduction: Surgery is the mainstay treatment of breast cancer and has been improving in aesthetic outcomes even in locally advanced disease. Radical mastectomies are being replaced by less aggressive surgeries with immediate breast reconstruction. Nipple-Sparing Mastectomy (NSM) preserves Nipple Areolar Complex (NAC) along with the entire breast skin envelope and is associated with better aesthetic results and quality of life, improving patient satisfaction. Since NSM is a relatively recent technique and few studies have shown the feasibility of NSM after neoadjuvant chemotherapy (NACT), there are some concerns and controversies about its oncological safety, especially, with regard to the NAC recurrence due to the remaining tissue in the retroareolar area. This study compares the long-term oncological outcomes and correlated factors of NSM and skin-sparing mastectomy (SSM) after NACT. Methods: After approval by the institution’s ethics committee a retrospective review was conducted to identify all patients who underwent NSM and SSM with immediate breast reconstruction after NACT between January 2011 and December 2018 at Centro de Doenças da Mama- Breast Unit Hospital Nossa Senhora das Graças. Metastatic disease, recurrent breast cancer, and other types of mastectomies were excluded. Clinicopathological and survival data, as well as recurrence events were collected from the electronic medical records. NSM was offered to the patients without involvement of the NAC and skin clinically and on imaging. All patients underwent ultrasonography, mammography and breast MRI in the preoperative period, as well as breast MRI after NACT. The decision to undergo adjuvant radiotherapy was determined by the treating radiation oncologists according to NCCN and ASTRO recommendation. A propensity score match was used to reduce the effect of selection bias on type of surgery and create well-balanced groups. The covariates included for matching were: anatomical stage, radiotherapy and molecular subtype. Results: A total of 188 patients underwent mastectomy in this period, 134 NSM and 54 SSM. After propensity score matching, 92 patients in the NSM group were matched to the 54 patients in the SSM group. The median follow up time was 44,7 months to NSM and 40,3 months to NSM. The characteristics of patients included in both groups after propensity score matching are described in Table 1. NAC recurrence was observed in 5 (5.4%) of 92 NSM patients, and median time of recurrence was 24.2 (11.7- 40.1) months. Ki67 showed a significant relationship with relapse in the NAC. The distance from the tumor to the NAC and other clinicopathologic variables were not correlated with NAC recurrence (Table 2). There were no significative differences between the groups in locorregional recurrence (p=0.102), distant metastasis (p=0.223) and death (p=0.610) (Table 3). Conclusion: In this matched control study, there was no difference in oncological outcomes in patients submitted to NSM and SSM after NACT, suggesting NSM with immediate breast reconstruction is a feasible option in this setting. Table 1. Characteristics of patients treated with NSM and SSM after propensity score matching *Student’s t test for independent samples or non-parametric Mann-Whitney test (quantitative variables); Fisher’s exact test or chi square test (categorical variables); p Table 2. Correlation between clinicopathologic variables and recurrence in the NAC *Fine and Gray Regression Model including death as a competitive risk and Wald test, p Table 3. Oncological outcomes in NSM and SSM groups *Model Fine & Gray and Wald test, p Citation Format: IRIS RABINOVICH, Leonardo P. Nissen, Isabela C. Soares, Alessandra C. Fornazari, Cleverton C. Spautz, Ana Paula M. Sebastião, CICERO A. URBAN, Karina F. Anselmi, Eduardo Schunemann, Flavia Kuroda, Maira T. Doria, Ana Clea S. Andrade, Rubens S. Lima. Oncological Outcomes of Nipple-Sparing Mastectomy after Neoadjuvant Chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-14-18.

Postnatal 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.

Astrocytes are non-neural cells, restricted to the brain and spinal cord, whose functions and morphology depend on their location. Astrocyte-astrocyte and astrocyte-neuron interactions occur through cytoplasmic Ca2+ levels changes, that are assessed to determine cell function and response (i.e. drug testing). Evaluation of changes in intracellular Ca2+ levels is mostly centered on fluorescence imaging approaches, performed through video recording of cells incubated with Ca2+-sensitive dyes. By observing ion concentration shifts over time in a delimited region of interest (ROI) comprising a single cell, it is possible to attain conclusions on cell responses to specific stimuli. Our work describes a tool named SIGAA – SIGnaling Automated Analysis, for astrocyte ROI-based fluorescent imaging, which is tailored for two wavelengths dyes by using two inputs of Ca2+ signaling recorded frames/videos and outputs a set of features relevant to the experiment’s conclusions and cell characterization. SIGAA performs automatic drift correction for the two recorded videos with a template matching algorithm, followed by astrocyte identification (ROI) using morphological reconstruction techniques. SIGAA then extracts intracellular Ca2+ evolution functions for all identified ROIs, detects function transients, and estimates a set of features for each signal, which are very similar to the ones obtained by the traditional methods and software used so far. SIGAA is a new fully automated tool, which can speed up hour-long studies and analysis to a few minutes, showing reliable results as the validity tests indicate.

Neural stem cells (NSCs), crucial for memory in the adult brain, are also pivotal to buffer depressive behavior. However, the mechanisms underlying the boost in NSC activity throughout life are still largely undiscovered. Here, we aimed to explore the role of deacetylase Sirtuin 3 (SIRT3), a central player in mitochondrial metabolism and oxidative protection, in the fate of NSC under aging and depression-like contexts. We showed that chronic treatment with tert-butyl hydroperoxide induces NSC aging, markedly reducing SIRT3 protein. SIRT3 overexpression, in turn, restored mitochondrial oxidative stress and the differentiation potential of aged NSCs. Notably, SIRT3 was also shown to physically interact with the long chain acyl-CoA dehydrogenase (LCAD) in NSCs and to require its activation to prevent age-impaired neurogenesis. Finally, the SIRT3 regulatory network was investigated in vivo using the unpredictable chronic mild stress (uCMS) paradigm to mimic depressive-like behavior in mice. Interestingly, uCMS mice presented lower levels of neurogenesis and LCAD expression in the same neurogenic niches, being significantly rescued by physical exercise, a well-known upregulator of SIRT3 and lipid metabolism. Our results suggest that targeting NSC metabolism, namely through SIRT3, might be a suitable promising strategy to delay NSC aging and confer stress resilience.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease commonly diagnosed among the elderly population. AD is characterized by the loss of synaptic connections, neuronal death, and progressive cognitive impairment, attributed to the extracellular accumulation of senile plaques, composed by insoluble aggregates of amyloid-β (Aβ) peptides, and to the intraneuronal formation of neurofibrillary tangles shaped by hyperphosphorylated filaments of the microtubule-associated protein tau. However, evidence showed that chronic inflammatory responses, with long-lasting exacerbated release of proinflammatory cytokines by reactive glial cells, contribute to the pathophysiology of the disease. NLRP3 inflammasome (NLRP3), a cytosolic multiprotein complex sensor of a wide range of stimuli, was implicated in multiple neurological diseases, including AD. Herein, we review the most recent findings regarding the involvement of NLRP3 in the pathogenesis of AD. We address the mechanisms of NLRP3 priming and activation in glial cells by Aβ species and the potential role of neurofibrillary tangles and extracellular vesicles in disease progression. Neuronal death by NLRP3-mediated pyroptosis, driven by the interneuronal tau propagation, is also discussed. We present considerable evidence to claim that NLRP3 inhibition, is undoubtfully a potential therapeutic strategy for AD.

Background: The use of Alzheimer’s disease (AD) models obtained by intracerebral infusion of amyloid-β (Aβ) has been increasingly reported in recent years. Nonetheless, these models may present important challenges. Objective: We have focused on canonical mechanisms of hippocampal-related neural plasticity to characterize a rat model obtained by an intracerebroventricular (icv) injection of soluble amyloid-β42 (Aβ42). Methods: Animal behavior was evaluated in the elevated plus maze, Y-Maze spontaneous or forced alternation, Morris water maze, and open field, starting 2 weeks post-Aβ42 infusion. Hippocampal neurogenesis was assessed 3 weeks after Aβ42 injection. Aβ deposition, tropomyosin receptor kinase B levels, and neuroinflammation were appraised at 3 and 14 days post-Aβ42 administration. Results: We found that immature neuronal dendritic morphology was abnormally enhanced, but proliferation and neuronal differentiation in the dentate gyrus was conserved one month after Aβ42 injection. Surprisingly, animal behavior did not reveal changes in cognitive performance nor in locomotor and anxious-related activity. Brain-derived neurotrophic factor related-signaling was also unchanged at 3 and 14 days post-Aβ icv injection. Likewise, astrocytic and microglial markers of neuroinflammation in the hippocampus were unaltered in these time points. Conclusion: Taken together, our data emphasize a high variability and lack of behavioral reproducibility associated with these Aβ injection-based models, as well as the need for its further optimization, aiming at addressing the gap between preclinical AD models and the human disorder.

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.

Supporting mammalian cells against reactive oxygen species such as hydrogen peroxide (H2O2) is essential. Bottom-up synthetic biology aims to integrate designed artificial units with mammalian cells. Here, we used manganese dioxide nanosheets (MnO2-NSs) as catalytically active entities that have superoxide dismutase-like and catalase-like activities. The integration of these MnO2-NSs into 7 μm reactors was able to assist SH-SY5Y neuroblastoma cells when stressed with H2O2. Complementary, Janus-shaped 800 nm reactors with one hemisphere coated with MnO2-NSs showed directed locomotion in cell media with top speeds up to 50 μm s−1 when exposed to 300 mM H2O2 as a fuel, while reactors homogeneously coated with MnO2-NSs were not able to outperform Brownian motion. These Janus-shaped reactors were able to remove H2O2 from the media, protecting cells cultured in the proximity. This effort advanced the use of bottom-up synthetic biology concepts in neuroscience.

Studies have correlated excessive S100B, a small inflammatory molecule, with demyelination and associated inflammatory processes occurring in multiple sclerosis. The relevance of S100B in multiple sclerosis pathology brought an emerging curiosity highlighting its use as a potential therapeutic target to reduce damage during the multiple sclerosis course, namely during inflammatory relapses. We examined the relevance of S100B and further investigated the potential of S100B-neutralizing small-molecule pentamidine in chronic experimental autoimmune encephalomyelitis. S100B depletion had beneficial pathological outcomes and based on promising results of a variety of S100B blockade strategies in an ex vivo demyelinating model, we choose pentamidine to assay its role in the in vivo experimental autoimmune encephalomyelitis. We report that pentamidine prevents more aggressive clinical symptoms and improves recovery of chronic experimental autoimmune encephalomyelitis. Blockade of S100B by pentamidine protects against oligodendrogenesis impairment and neuroinflammation by reducing astrocyte reactivity and microglia pro-inflammatory phenotype. Pentamidine also increased regulatory T cell density in the spinal cord suggesting an additional immunomodulatory action. These results showed the relevance of S100B as a main driver of neuroinflammation in experimental autoimmune encephalomyelitis and identified an uncharacterized mode of action of pentamidine, strengthening the possibility to use this drug as an anti-inflammatory and remyelinating therapy for progressive multiple sclerosis.

Introduction Nanoparticles (NPs), as drug delivery systems, appear to be a promising tool for prolonged therapeutic strategies as they allow a controlled drug release over time. However, most of the studies found in the literature simply contemplate the use of a single or low number of dosages with low NPs concentrations. In the context of chronic diseases, like Alzheimer's disease, cancer or human immunodeficiency virus (HIV), where the therapeutic scheme is also chronic, studies with numerous repeated dosages are often neglected. Methods We screened different NPs, polymeric and lipid-based, in a repeated-dose toxicity study, to evaluate the safety and tissue distribution of promising nanocarriers to be used in the treatment of long-lasting diseases. Results After administrating 24 high concentrated doses of the selected NPs intraperitoneally (i.p.) (3 times a week for 2 months), animals have presented NPs accumulation in different tissues. However, neither toxicity, bodyweight changes nor clinical signs of disease were observed. Discussion This work demonstrates no general adverse effects upon the studied NPs repeated-dose exposure, indicating the most promising NPs to be used in the different therapeutic circumstances, which may be useful in chronic diseases treatment.

The development of new therapies for psychiatric disorders is of utmost importance, given the enormous toll these disorders pose to society nowadays. This should be based on the identification of neural substrates and mechanisms that underlie disease etiopathophysiology. Adult neural stem cells (NSCs) have been emerging as a promising platform to counteract brain damage. In this perspective article, we put forth a detailed view of how NSCs operate in the adult brain and influence brain homeostasis, having profound implications at both behavioral and functional levels. We appraise evidence suggesting that adult NSCs play important roles in regulating several forms of brain plasticity, particularly emotional and cognitive flexibility, and that NSC dynamics are altered upon brain pathology. Furthermore, we discuss the potential therapeutic value of utilizing adult endogenous NSCs as vessels for regeneration, highlighting their importance as targets for the treatment of multiple mental illnesses, such as affective disorders, schizophrenia, and addiction. Finally, we speculate on strategies to surpass current challenges in neuropsychiatric disease modeling and brain repair.

© The Author(s), under exclusive licence to Springer Nature B.V. 2022, A recent work elegantly demonstrated a role of adenosine, through the high afnity Gs-coupled A2A receptors (A2AR), on stabilization of GABAergic synapses during a critical post-natal period, which is discussed here within the context of the most recent advances on our understanding of the neurodevelopmental actions of adenosine. Neuronal activity requires a proper balance between excitatory and inhibitory inputs, GABA being the predominant inhibitory neurotransmitter in the brain. Dysfunction of GABAergic signalling, in particular in the maturation of GABAergic synapses, leads to severe neurodevelopmental diseases, which may express as drug-resistant forms of epilepsy, cognitive impairment and, often, mental retardation or even premature death., The author is in receipt of funding from EU H2020 (EpiEpiNet Twinning Project, GA 952455) and Fundação para a Ciência e Tecnologia, Portugal (PTDC/MED-FAR/30933/2017 and PTDC/MED-FAR/4834/2021).

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease that involves the progressive degeneration of the motor neurons of the spinal cord and motor cortex. Severe muscle wasting...

The processes of neural genesis, namely neurogenesis, astrogliogenesis, and oligodendrogenesis, that is, the formation of new neurons, astrocytes, and oligodendrocytes start with embryonic developm...

Copyright © 2021, © SAGE Publications, 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., The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: 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 Fundação para a Ciência e Tecnologia (FCT), by European Union’s Horizon 2020 research and innovation programme under grant agreement No 692340 (SynaNet) and No 952455 (EpiEpinet) and GAPIC (20180014). SRT was in receipt of SFRH/BD/128091/2016 FCT fellowship.

Patients under cannabis-based therapies are usually chronically exposed to cannabinoids. Chronic treatment with a cannabinoid receptor agonist, WIN 55,212–2, affects brain metabolism and modifies functional connectivity between brain areas responsible for memory and learning. Therefore, it is of uttermost importance to discover strategies to mitigate the negative side-effects of cannabinoid-based therapies. Previously, we showed that a single treatment with the synthetic cannabinoid WIN 55,212-2 disrupts recognition memory, an effect mediated by cannabinoid receptor 1 (CB1R) and cancelled by concomitant administration of adenosine A2A receptor (A2AR) antagonists. We herein evaluate if memory deficits induced by chronic exposure to WIN 55,212–2 can also be reverted by A2AR antagonism, and assessed the synaptic mechanisms that could be involved in that reversal. We show that chronic administration of KW-6002 (istradefylline) (3 mg/kg/28days) reverts memory deficits (evaluated through the Novel Object Recognition Test) induced by chronic cannabinoid exposure (WIN 55,212–2, 1 mg/kg/28 days). Long Term Potentiation (LTP) of synaptic potentials recorded from the CA1 area of the hippocampus was impaired by WIN 55,212–2 (300 nM), an effect partially rescued by the A2AR antagonist, SCH 58261 (100 nM). Chronic administration of KW-6002 or WIN 55,212-2 did not affect A2AR or CB1R binding in the hippocampus and in the prefrontal cortex. These results, showing that A2AR antagonism can still revert memory deficits after chronic administration of a cannabinoid, an effect that involves mitigation of synaptic plasticity impairment, strongly indicate that adenosine A2ARs are appropriate targets to tackle side-effects of putative therapies involving the activation of cannabinoid receptors.

Niemann Pick type C is an inborn error of metabolism (IEM), classified as a lysosomal storage disease (LSD) caused by a dysfunction in NPC transport protein, that leads to intracellular accumulation of non-esterified cholesterol and other lipids. Clinical manifestations are ample, with visceral and neurological symptoms. Miglustat, a molecule that reversibly inhibits glucosylceramide synthase is used as treatment for this disorder. Studies demonstrated the influence of oxidative stress and inflammation in IEM, as well in animal model of NP-C disease. Nonetheless, literature lacks data on patients, so our work aimed to investigate if there is influence of chronic inflammation in the pathophysiology of NP-C disease, and the effect of miglustat, N-acetylcysteine (NAC) and Coenzyme Q10 (CoQ10). We evaluated the plasmatic cytokines in NPC patients at diagnosis and during the treatment with miglustat. Additionally, we performed an in vitro study with antioxidants NAC (1 mM and 2.5 mM) and CoQ10 (5 μM and 10 μM), where we could verify its effect on inflammatory parameters, as well as in cholesterol accumulation. Our results showed that NP-C patients have higher plasmatic levels of pro and anti-inflammatory cytokines (IL-6, IL-8, and IL-10) at diagnosis and the treatment with miglustat was able to restore it. In vitro study showed that treatment with antioxidants in higher concentrations significantly decrease cholesterol accumulation, and NAC at 2.5 mM normalized the level of pro-inflammatory cytokines. Although the mechanism is not completely clear, it can be related to restoration in lipid traffic and decrease in oxidative stress caused by antioxidants.

Adenosine A

21 páginas, 7 figuras., Adenosine A2A receptor (A2A R) activation modulates several brain processes, ranging from neuronal maturation to synaptic plasticity. Most of these actions occur through the modulation of the actions of the neurotrophin brain-derived neurotrophic factor (BDNF). In this work, we studied the role of A2A Rs in regulating postnatal and adult neurogenesis in the rat hippocampal dentate gyrus (DG). Here, we show that A2A R activation with CGS 21680 promoted neural stem cell self-renewal, protected committed neuronal cells from cell death and contributed to a higher density of immature and mature neuronal cells, particularly glutamatergic neurons. Moreover, A2A R endogenous activation was found to be essential for BDNF-mediated increase in cell proliferation and neuronal differentiation. Our findings contribute to further understand the role of adenosinergic signaling in the brain and may have an impact in the development of strategies for brain repair under pathological conditions., Fundaç~ao para a Ciência e a Tecnologia, Grant/Award Numbers: IF/01227/2015, SFRH/BD/74662/2010, IMM/CT/35-2018, SFRH/BD/128280/2017, SFRH/BD/129710/2017; H2020-WIDESPREAD-05-2020-Twinning (EpiEpinet) under grant agreement No, Grant/Award Number: 9524; Ministerio de Ciencia e Innovaci on, Grant/Award Number: PID2019-111225RB-I00; Spanish MICIU, Grant/Award Number: SAF2015-70433-R; Generalitat Valenciana, Grant/Award Number: PROMETEO/2018/055; COST Action, Grant/Award Number: BM1402

© 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.

Stroke is a complex disorder that includes several different pathophysiological entities, and therefore requires a personalized approach to therapy. Pharmacological acute stroke therapy includes fibrinolytics such as alteplase (rt-PA) or tenecteplase which has a more favorable pharmacokinetic profile. Hemorrhagic adverse events are the main risks of these therapies, and clinical, laboratorial, and genetic factors may influence the risk of severe bleeding. After the acute time window, prevention is paramount in order to avoid recurrent stroke. Antiplatelet drugs (aspirin, clopidogrel, dipyridamole) are the default secondary prevention therapies. In some patients, such as those with cardioembolic stroke, anticoagulants are the preferred preventive treatment. These include vitamin K antagonists (warfarin) and direct oral anticoagulants (dabigatran, rivaroxaban, apixaban, edoxaban). With some drugs, such as clopidogrel and warfarin, significant heterogeneity of the magnitude of effect and side effect profile exists between subjects. In addition to environmental factors and drug-drug interactions, genetic polymorphisms contribute to intersubject variability, whether through drug receptor or metabolic pathway variations. Knowledge of the precise pharmacodynamic, pharmacokinetic, and pharmacogenetic details of drugs used in stroke care enables the selection of the most appropriate option for each individual patient. Precision medicine and pharmacogenetic-guided drug algorithms may become the standard of care in the future.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder, characterized by the degeneration of upper and lower motor neurons of the motor cortex, brainstem, and ventral horn of the spinal cord. The role of glial cells in the onset and progression of ALS is increasingly being recognized. Dysfunctional astrocytes, with an atypical and neurotoxic phenotype, in the cerebral cortex and the spinal cord promote neuroinflammation and motor neuron degeneration. Indeed, cortical and spinal cord astrocytes from SOD1G93A (mSOD1) mice are neurotoxic, develop early deficits, and lose their neuro-supportive properties before disease onset. This chapter discusses the contribution of dysfunctional cortical and spinal cord astrocytes in the development and progression of ALS. Differences in astrocyte heterogeneity and reactivity, calcium signaling, neurotransmitters, and in paracrine signaling mechanisms along with implications for novel therapies in ALS are addressed.

© 2021 IBRO. Published by Elsevier Ltd. All rights reserved., Glioblastoma (GBM) is the most common brain primary tumour. Hypoxic regions in GBM are associated to tumour growth. Adenosine accumulates in hypoxic regions and can affect cell proliferation and survival. However, how proliferating GBM cells respond/adapt to increased adenosine levels compared to human astrocytes (HA) is not clarified and was addressed in the present work. GBM cell lines and HA were treated for 3 days with test drugs. Thirty Adenosine (30 µM) caused a 43% ± 5% (P < 0.05) reduction of cell proliferation/viability in HA, through an adenosine receptor-independent mechanism, but had no effect in GBM cell lines U87MG, U373MG and SNB19. Contrastingly, inhibition of adenosine phosphorylation (using the adenosine kinase (ADK) inhibitor 5-iodotubercidin (ITU) (25 µM)), produced a strong and similar decrease on cell proliferation in both HA and GBM cells. The effect of adenosine on HA proliferation/viability was potentiated by 100 µM-homocysteine. Combined application of 30 µM-adenosine and 100 µM-homocysteine reduced the cell proliferation/viability in all three GBM cell lines, but this reduction was much lower than that observed in HA. Adenosine alone did not induce cell death, assessed by lactate dehydrogenase (LDH) release, both in HA and GBM cells, but potentiated the cytotoxic effect of homocysteine in HA and in U87MG and U373MG cells. Results show a strong attenuation of adenosine anti-proliferative effect in GBM cells compared to HA, probably resulting from increased adenosine elimination by ADK, suggesting a proliferative-prone adaptation of tumour cells to increased adenosine levels., The present work was supported by CENTRO2020 and Lisboa 2020 (grant number POCI-01-0145-FEDER-016822) and FCT – Fundação para a Ciência e Tecnologia,I.P. (grant number PTDC/BIMONC/7121/2014). A.R. Costa received a scholarship from FCT (UI/BD/151025/2021)

© 2020 Springer Nature B.V., Caffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A1R and A2R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory post-synaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca2+ homeostasis. We found that caffeine (30-200 μM) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50 nM), thus likely mediated by blockade of inhibitory A1Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A2ARs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A2AR antagonist, SCH58261 (50 nM). Furthermore, caffeine increased the frequency of Ca2+ transients in neuronal cell culture, an action mimicked by the A1R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50 μM). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca2+ homeostasis., The authors in Portugal are members of an EU twinning action (Project grant 952455). Robertta Silva Martins was in receipt of a fellowship from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro–FAPERJ, Brasil, Bolsa de Doutorado Sanduíche Europa/Oriente (grant number E-26/201.599/2018). The work was supported by Fundação para a Ciência e Tecnologia, Portugal (project grant PTDC/MED-FAR/30933/2017).

© 2020 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 (http://creativecommons.org/licenses/by/4.0/)., Epilepsy is one of the most common brain diseases worldwide, having a huge burden in society. The main hallmark of epilepsy is the occurrence of spontaneous recurrent seizures, having a tremendous impact on the lives of the patients and of their relatives. Currently, the therapeutic strategies are mostly based on the use of antiepileptic drugs, and because several types of epilepsies are of unknown origin, a high percentage of patients are resistant to the available pharmacotherapy, continuing to experience seizures overtime. Therefore, the search for new drugs and therapeutic targets is highly important. One key aspect to be targeted is the aberrant adult hippocampal neurogenesis (AHN) derived from Neural Stem Cells (NSCs). Indeed, targeting seizure-induced AHN may reduce recurrent seizures and shed some light on the mechanisms of disease. The endocannabinoid system is a known modulator of AHN, and due to the known endogenous antiepileptic properties, it is an interesting candidate for the generation of new antiepileptic drugs. However, further studies and clinical trials are required to investigate the putative mechanisms by which cannabinoids can be used to treat epilepsy. In this manuscript, we will review how cannabinoid-induced modulation of NSCs may promote neural plasticity and whether these drugs can be used as putative antiepileptic treatment., This work was supported by IF/01227/2015 and UID/BIM/50005/2019, projeto financiado pela Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) através de Fundos do Orçamento de Estado. D.M.L. (PD/BD/141784/2018) and L.R.-R. (PD/BD/150344/2019) were in receipt of a fellowship from FCT. The authors are in hold of a H2020 Twinning Action from EU (EpiEpiNet).

Neural stem cells (NSCs) are found in discrete regions of the mammalian brain. During adulthood, NSCs can be a source of new neurons and oligodendrocytes in neurological disorders. However, these newborn cells are not sufficient to overcome the neurological deficits involved by neural loss. Therefore, the identification of novel mechanisms responsible for modulating NSC fate represent a major key issue for future brain repair strategies. Several studies suggest that mitochondria have an important role in regulating NSC differentiation and lineage determination. However, the molecular mechanisms involved in this regulation remain unknown. Hence, our work aims to dissect how mitochondria biogenesis and dynamics can modulate the NSC differentiation into neurons or oligodendrocytes. For this, NSCs were obtained by isolating subventricular zone (SVZ) and dentate gyrus (DG) cells from P1-3 mouse models. Seeding density, culture conditions and number of passages were determined. Moreover, the multipotency of SVZ/DG-derived NSPCs, obtained from different passages, was also accessed. Additionally, expression of proteins involved in mitochondrial biogenesis and fusion/fission appear altered during NSPC differentiation, while mitochondrial network revealed different morphologies in cells from different lineages. The results obtained will provide novel findings concerning the role of mitochondrial dynamics in NSC fate.

Late-onset sporadic Alzheimers disease (AD) is the most common neurodegenerative disorder worldwide, yet present treatment options are still very limited. Modulating adult hippocampal neurogenesis (AHN) as a potential therapeutic target for AD has been explored. However, how the process is regulated in AD remains unclear, as studies show controversial results, arising mainly from models of the familial form. Hence, in the present work, aiming at characterizing AHN in a model of the initial stages of sporadic AD, male Wistar rats were intracerebroventricularly injected with an Aβ1-42 peptide solution. Behaviour tests carried out two weeks after the injection unexpectedly revealed no impact on locomotor activity (open field), anxious-related behaviour (elevated plus maze) or learning and memory performance (novel object recognition, Y-maze, Morris water maze). Assessment of neurogenic markers one month after 5-bromo-2-deoxyuridine administration showed abnormally enhanced dendritic morphology of immature neurons in the dentate gyrus, while proliferation and neuronal differentiation were preserved. As brain-derived neurotrophic factor signalling has been shown to be compromised in the presence of Aβ due to the cleavage of TrkB full-length receptors, the levels of these receptors isoforms were evaluated. No evidence of cleavage was found at 3 or 14 days post-Aβ-infusion. Consistently, the levels of Aβ1-42 species were unaltered at these timepoints, as was the number of astrocytes and microglia. Overall, although this model has been previously described as a model of sporadic AD, the expected behavioural, cellular or molecular phenotypes were not observed, further highlighting the variability of results found in the literature.

Epilepsy is a common neurological disorder but resistance to pharmacotherapy makes it necessary the development of novel antiepileptic drugs (AEDs). An A1 adenosine receptor (A1R) agonist, MRS5474, possesses anticonvulsant activity in an animal model (Tosh et al.,2012-J.Med.Chem., 55,8075), without the cardiac side effects common to other A1R agonists, leading us to hypothesise that it could operate through a mechanism different from classical A1R agonists. We thus tested this hypothesis in an ex vivo model of epileptogenesis in rhinal cortex -hippocampus organotypic slices (Magalhães et al., 2018-J.Neuroinflam.15:203). MRS5474 (250nM) was incubated during 1h with slices under depolarizing ([K +] o=8.5mM) or non-depolarizing ([K +] o=3mM) conditions. Interestingly, MRS5474 decreased by 6615% (n=4, P+ but not under normal K +. Event frequency, amplitude and burst duration were not affected. The canonical A1R agonist, N 6-cyclopentyladenosine (30nM, n=4) prevented burst-like activity (number of bursts decreased to zero, spike amplitude markedly reduced) even under non-depolarizing conditions. Our results showing that MRS5474 prevents spontaneous neuronal firing only under depolarizing conditions, suggest that it may predominantly influence spontaneous synchronous firing of stressed neurons, sparing non-injured ones. This renders this compound with presumably less side effects than other A1R agonists and currently available AEDs.

Adult neural stem/progenitor cells (NSPC) are present in specialized niches of the mammalian brain and their proliferative and differentiative potential is modulated by a myriad of factors. Recent evidence sheds light on the interaction between cannabinoids and neurotrophic factors underlying a major regulative force of adult hippocampal neurogenesis, with important effects upon cognitive plasticity and mood flexibility. Herein, we aimed at evaluating the actions of cannabinoid type 2 receptor (CB2R) together with exercise upon hippocampal neurogenesis and whether this has significant behavioral implications. Our data suggests a participation of CB2Rs in fine-tuning the actions of physical exercise upon adult hippocampal neurogenesis. Specifically, CB2R ligands as well as exercise-regulated neurotrophic factors promote an acceleration in the differentiation of progenitor cells accompanied by an increase in the number of mature neurons in vitro. Moreover, preliminary results show that CB2Rs play an impactful role in controlling cognitive and depressive-like behavior. This is particularly important because brain physiology and mental health are known to be greatly affected by physical exercise, with adult neurogenesis playing a significant role in this process. Ultimately, this work will contribute to unravel the mechanisms behind the actions of cannabinoids and exercise in the brain and to develop strategies utilizing CB2Rs and physical exercise to boost neural stem cell capacity and treat several brain disorders. Acknowledgements: Supported by Fundação para a Ciência e a Tecnologia (FCT), projects SFRH/BD/129710/2017 and IF/01227/2015. No potential conflict of interest.

Under Multiple Sclerosis pathological conditions, oligodendrocyte precursor cells (OPCs) present in the brain parenchyma or derived from subventricular zone neural stem cells (SVZ-NSCs) can differentiate into oligodendrocytes (OLs), which migrate and partially remyelinate the lesioned areas. Previous data from our group demonstrated that activation of adenosine A2A receptors (A2AR) modulated SVZ-NSCs oligodendroglial differentiation, both in vitro and in vivo under physiological conditions. Hence, we aimed at understanding the role of A2AR in adult oligodendrogenesis derived from SVZ-NSCs in an in vivo mouse model of MS. For this, the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of MS was developed, and behavioural tests were performed to evaluate motor function. Cellular differentiation was assessed by immunohistochemistry assays for bromodeoxyuridine (BrdU) colocalization with oligodendrocytic markers in brain regions of interest. Western blot and ELISA assays were used for myelin protein levels and inflammatory cytokine quantification. Our results for EAE model characterization showed that motor impairment is proportional to the score of the disease and cellular and molecular data showed an increase in the levels of the pro-inflammatory cytokine TNFα (n=5, pin vivo modulation of A2AR and assessing its effect on EAE phenotype and adult oligodendrogenesis, ultimately unveiling the modulation of adult oligodendrogenesis derived from SVZ-NSCs by A2AR as a putative therapy for MS.

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1-3-day-old (P1-3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6-12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

© Mary Ann Liebert, Inc., Adenosine is an endogenous anticonvulsant and neuroprotectant of the brain. Seizure activity produces large quantities of adenosine, and it is this seizure-induced adenosine surge that normally stops a seizure. However, within the context of epilepsy, adenosine plays a wide spectrum of different roles. It not only controls seizures (ictogenesis), but also plays a major role in processes that turn a normal brain into an epileptic brain (epileptogenesis). It is involved in the control of abnormal synaptic plasticity and neurodegeneration and plays a major role in the expression of comorbid symptoms and complications of epilepsy, such as sudden unexpected death in epilepsy (SUDEP). Given the important role of adenosine in epilepsy, therapeutic strategies are in development with the goal to utilize adenosine augmentation not only for the suppression of seizures but also for disease modification and epilepsy prevention, as well as strategies to block adenosine A2A receptor overfunction associated with neurodegeneration. This review provides a comprehensive overview of the role of adenosine in epilepsy., The authors acknowledge grant support from the National Institutes of Health to D.B. (NINDS: NS103740, NS065957); support to A.M.S. by LISBOA-01-0145-FEDER-007391, project cofunded by Fundo Europeu Para o Desenvolvimento Regional through POR Lisboa 2020 (Programa Operacional Regional de Lisboa) from PORTUGAL 2020 and Fundação para a Ciência e Tecnologia (FCT), by an FCT project (PTDC/MED-FAR/30933/2017) and by Twinning action (SynaNet) from the EU H2020 programme (project number: 692340); and support to R.A.C. and A.R.T. by Fundacion LaCaixa (LCF/PR/HP17/52190001), Centro 2020 (CENTRO-01-0145-FEDER-000008: BrainHealth 2020 and CENTRO-01-0246-FEDER-000010) and FCT (POCI-01-0145-FEDER-03127).