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Abstract Background Neural progenitor cells (NPCs) can be cultivated from developing brains, reproducing many of the processes that occur during neural development. They can be isolated from a variety of animal models, such as transgenic mice carrying mutations in amyloid precursor protein (APP) and presenilin 1 and 2 (PSEN 1 and 2), characteristic of familial Alzheimer’s disease (fAD). Modulating the development of these cells with inflammation-related peptides, such as bradykinin (BK) and its antagonist HOE-140, enables the understanding of the impact of such molecules in a relevant AD model. Results We performed a global gene expression analysis on transgenic neurospheres treated with BK and HOE-140. To validate the microarray data, quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed on 8 important genes related to the immune response in AD such as CCL12, CCL5, CCL3, C3, CX3CR1, TLR2 and TNF alpha and Iba-1. Furthermore, comparative analysis of the transcriptional profiles was performed between treatments, including gene ontology and reactome enrichment, construction and analysis of protein-protein interaction networks and, finally, comparison of our data with human dataset from AD patients. The treatments affected the expression levels of genes mainly related to microglia-mediated neuroinflammatory responses, with BK promoting an increase in the expression of genes that enrich processes, biological pathways, and cellular components related to immune dysfunction, neurodegeneration and cell cycle. B2 receptor inhibition by HOE-140 resulted in the reduction of AD-related anomalies caused in this system. Conclusions BK is an important immunomodulatory agent and enhances the immunological changes identified in transgenic neurospheres carrying the genetic load of AD. Bradykinin treatments modulate the expression rates of genes related to microglia-mediated neuroinflammation. Inhibiting bradykinin activity in Alzheimer’s disease may slow disease progression.

Purinergic signaling has been implicated in many biological functions, including development. In this study, we investigate the functions of extracellular adenosine and adenosine receptors using a mouse embryonic stem cell (ESC) line and morula stages isolated from mouse embryos. Feeder-free mouse ESC was investigated in the absence and presence of the leukemia inhibitory factor (LIF), configuring undifferentiated cells and cells undergoing spontaneous differentiation. High alkaline phosphatase (ALPL) and low CD73 levels resulting in low adenosine (eADO) levels were characteristic for pluripotent cells in the presence of the LIF, while LIF deprivation resulted in augmented adenosine levels and reduced pluripotency marker expression, which indicated differentiation. Tracing ESC proliferation by BrdU labeling revealed that the inhibition of ALPL by levamisole resulted in a decrease in proliferation due to less eADO accumulation. Furthermore, caffeine and levamisole treatment, inhibiting adenosine receptor and eADO accumulation, respectively, reduced ESC migration, similar to that observed in the absence of the LIF. Pharmacological approaches of selective adenosine receptor subtype inhibition triggered specific adenosine receptor activities, thus triggering calcium or MAP kinase pathways leading to differentiation. In line with the in vitro data, mouse embryos at the morula stage were sensitive to treatments with A1 and A3 receptor antagonists, leading to the conclusion that A1 receptor and A3 receptor inhibition impairs proliferation and self-renewal and triggers inappropriate differentiation, respectively. The findings herein define the functions of eADO signaling in early development with implications for developmental disorders, in which adenosine receptors or ectonucleotidase dysfunctions are involved, and which could lead to malformations and miscarriages, due to exposure to caffeine.

Purinergic receptors (Rs) of the ATP/ADP, UTP/UDP (P2X, P2Y) and adenosine (A1, A2A)-sensitive classes broadly interfere with cognitive processes both under quasi normal and disease conditions. During neurodegenerative illnesses, high concentrations of ATP are released from the damaged neuronal and non-neuronal cells of the brain; then, this ATP is enzymatically degraded to adenosine. Thus, the primary injury in neurodegenerative diseases appears to be caused by various protein aggregates on which a superimposed damage mediated by especially P2X7 and A2AR activation develops; this can be efficiently prevented by small molecular antagonists in animal models of the above diseases, or are mitigated in the respective knockout mice. Dementia is a leading symptom in Alzheimer's disease (AD), and accompanies Parkinson's disease (PD) and Huntington's disease (HD), especially in the advanced states of these illnesses. Animal experimentation suggests that P2X7 and A2ARs are also involved in a number of psychiatric diseases, such as major depressive disorder (MDD), obsessive compulsive behavior, and attention deficit hyperactivity disorder. In conclusion, small molecular antagonists of purinergic receptors are expected to supply us in the future with pharmaceuticals which are able to combat in a range of neurological/psychiatric diseases the accompanying cognitive deterioration.

Abstract The aim of this study was to identify the bio‐efficacy of sodium butyrate (NaBu) on preventing the development of peritoneal fibrovascular tissue induced by implantation of a synthetic matrix in the abdominal cavity. Polyether–polyurethane sponge discs were implanted in the peritoneal cavity of mice, which were treated daily with oral administration of NaBu (100 mg/kg). Control animals received water (100 μl). After 7 days, the implants were removed for assessment of inflammatory, angiogenic and fibrogenic markers. Compared with control values, NaBu treatment decreased mast cell recruitment/activation, inflammatory enzyme activities, levels of pro‐inflammatory cytokines, and the proteins p65 and p50 of the nuclear factor‐κB pathway. Angiogenesis, as determined by haemoglobin content, vascular endothelial growth factor levels and the number of blood vessels in the implant, was reduced by the treatment. In NaBu‐treated animals, the predominant collagen present in the abdominal fibrovascular tissue was thin collagen, whereas in control implants it was thick collagen. Transforming growth factor‐β1 levels were also lower in implants of treated animals. Sodium butyrate downregulated the inflammatory, angiogenesis and fibrogenesis axes of the fibroproliferative tissue induced by the intraperitoneal synthetic matrix. This compound has potential to control/regulate chronic inflammation and adverse healing processes in the abdominal cavity.

Abstract Important functions of the prefrontal cortex (PFC) are established during early life, when neurons exhibit enhanced synaptic plasticity and synaptogenesis. This developmental stage drives the organization of cortical connectivity, responsible for establishing behavioral patterns. Serotonin (5-HT) emerges among the most significant factors that modulate brain activity during postnatal development. In the PFC, activated 5-HT receptors modify neuronal excitability and interact with intracellular signaling involved in synaptic modifications, thus suggesting that 5-HT might participate in early postnatal plasticity. To test this hypothesis, we employed intracellular electrophysiological recordings of PFC layer 5 neurons to study the modulatory effects of 5-HT on plasticity induced by theta-burst stimulation (TBS) in two postnatal periods of rats. Our results indicate that 5-HT is essential for TBS to result in synaptic changes during the third postnatal week, but not later. TBS coupled with 5-HT2A or 5-HT1A and 5-HT7 receptors stimulation leads to long-term depression (LTD). On the other hand, TBS and synergic activation of 5-HT1A, 5-HT2A, and 5-HT7 receptors lead to long-term potentiation (LTP). Finally, we also show that 5-HT dependent synaptic plasticity of the PFC is impaired in animals that are exposed to early-life chronic stress.

Serotonergic neurons constitute one of the main systems of neuromodulators, whose diffuse projections regulate the functions of the cerebral cortex. Serotonin (5-HT) is known to play a crucial role in the differential modulation of cortical activity related to behavioral contexts. Some features of the 5-HT signaling organization suggest its possible participation as a modulator of activity-dependent synaptic changes during the critical period of the primary visual cortex (V1). Cells of the serotonergic system are among the first neurons to differentiate and operate. During postnatal development, ramifications from raphe nuclei become massively distributed in the visual cortical area, remarkably increasing the availability of 5-HT for the regulation of excitatory and inhibitory synaptic activity. A substantial amount of evidence has demonstrated that synaptic plasticity at pyramidal neurons of the superficial layers of V1 critically depends on a fine regulation of the balance between excitation and inhibition (E/I). 5-HT could therefore play an important role in controlling this balance, providing the appropriate excitability conditions that favor synaptic modifications. In order to explore this possibility, the present work used in vitro intracellular electrophysiological recording techniques to study the effects of 5-HT on the E/I balance of V1 layer 2/3 neurons, during the critical period. Serotonergic action on the E/I balance has been analyzed on spontaneous activity, evoked synaptic responses, and long-term depression (LTD). Our results pointed out that the predominant action of 5-HT implies a reduction in the E/I balance. 5-HT promoted LTD at excitatory synapses while blocking it at inhibitory synaptic sites, thus shifting the Hebbian alterations of synaptic strength towards lower levels of E/I balance.

Abstract Oligogenic inheritance of autism spectrum disorder (ASD) has been supported by several studies. However, little is known about how the risk variants interact and converge on causative neurobiological pathways. We identified in an ASD proband deleterious compound heterozygous missense variants in the Reelin (RELN) gene, and a de novo splicing variant in the Cav3.2 calcium channel (CACNA1H) gene. Here, by using iPSC-derived neural progenitor cells (NPCs) and a heterologous expression system, we show that the variant in Cav3.2 leads to increased calcium influx into cells, which overactivates mTORC1 pathway and, consequently, further exacerbates the impairment of Reelin signaling. Also, we show that Cav3.2/mTORC1 overactivation induces proliferation of NPCs and that both mutant Cav3.2 and Reelin cause abnormal migration of these cells. Finally, analysis of the sequencing data from two ASD cohorts—a Brazilian cohort of 861 samples, 291 with ASD; the MSSNG cohort of 11,181 samples, 5,102 with ASD—revealed that the co-occurrence of risk variants in both alleles of Reelin pathway genes and in one allele of calcium channel genes confer significant liability for ASD. Our results support the notion that genes with co-occurring deleterious variants tend to have interconnected pathways underlying oligogenic forms of ASD.

IntroductionPhysical exercise has beneficial effects by providing neuroprotective and anti-inflammatory responses to AD. Most studies, however, have been conducted with aerobic exercises, and few have investigated the effects of other modalities that also show positive effects on AD, such as resistance exercise (RE). In addition to its benefits in developing muscle strength, balance and muscular endurance favoring improvements in the quality of life of the elderly, RE reduces amyloid load and local inflammation, promotes memory and cognitive improvements, and protects the cortex and hippocampus from the degeneration that occurs in AD. Similar to AD patients, double-transgenic APPswe/PS1dE9 (APP/PS1) mice exhibit Αβ plaques in the cortex and hippocampus, hyperlocomotion, memory deficits, and exacerbated inflammatory response. Therefore, the aim of this study was to investigate the effects of 4 weeks of RE intermittent training on the prevention and recovery from these AD-related neuropathological conditions in APP/PS1 mice.MethodsFor this purpose, 6-7-month-old male APP/PS1 transgenic mice and their littermates, negative for the mutations (CTRL), were distributed into three groups: CTRL, APP/PS1, APP/PS1+RE. RE training lasted four weeks and, at the end of the program, the animals were tested in the open field test for locomotor activity and in the object recognition test for recognition memory evaluation. The brains were collected for immunohistochemical analysis of Aβ plaques and microglia, and blood was collected for plasma corticosterone by ELISA assay.ResultsAPP/PS1 transgenic sedentary mice showed increased hippocampal Aβ plaques and higher plasma corticosterone levels, as well as hyperlocomotion and reduced central crossings in the open field test, compared to APP/PS1 exercised and control animals. The intermittent program of RE was able to recover the behavioral, corticosterone and Aβ alterations to the CTRL levels. In addition, the RE protocol increased the number of microglial cells in the hippocampus of APP/PS1 mice. Despite these alterations, no memory impairment was observed in APP/PS1 mice in the novel object recognition test.DiscussionAltogether, the present results suggest that RE plays a role in alleviating AD symptoms, and highlight the beneficial effects of RE training as a complementary treatment for AD.

Abstract Background Fragile X syndrome, the major cause of inherited intellectual disability among men, is due to deficiency of the synaptic functional regulator FMR1 protein (FMRP), encoded by the FMRP translational regulator 1 (FMR1) gene. FMR1 alternative splicing produces distinct transcripts that may consequently impact FMRP functional roles. In transcripts without exon 14 the translational reading frame is shifted. For deepening current knowledge of the differential expression of Fmr1 exon 14 along the rat nervous system development, we conducted a descriptive study employing quantitative RT-PCR and BLAST of RNA-Seq datasets. Results We observed in the rat forebrain progressive decline of total Fmr1 mRNA from E11 to P112 albeit an elevation on P3; and exon-14 skipping in E17–E20 with downregulation of the resulting mRNA. We tested if the reduced detection of messages without exon 14 could be explained by nonsense-mediated mRNA decay (NMD) vulnerability, but knocking down UPF1, a major component of this pathway, did not increase their quantities. Conversely, it significantly decreased FMR1 mRNA having exon 13 joined with either exon 14 or exon 15 site A. Conclusions The forebrain in the third embryonic week of the rat development is a period with significant skipping of Fmr1 exon 14. This alternative splicing event chronologically precedes a reduction of total Fmr1 mRNA, suggesting that it may be part of combinatorial mechanisms downregulating the gene’s expression in the late embryonic period. The decay of FMR1 mRNA without exon 14 should be mediated by a pathway different from NMD. Finally, we provide evidence of FMR1 mRNA stabilization by UPF1, likely depending on FMRP.

Prostate cancer (PC) is the most diagnosed tumor in males and ranks as the second leading cause of male mortality in the western world. The CD39 and CD73 enzymes play a crucial role in cancer regulation by degrading nucleotides and forming nucleosides. This study aimed to investigate the expression of the CD39 and CD73 enzymes as potential therapeutic targets for PC. The initial part of this study retrospectively analyzed tissue samples from 23 PC patients. Using the TissueFAXSTM cytometry platform, we found significantly higher levels of CD39—labeling its intensity compared to CD73. Additionally, we observed a correlation between the Gleason score and the intensity of CD39 expression. In the prospective arm, blood samples were collected from 25 patients at the time of diagnosis and after six months of treatment to determine the expression of CD39 and CD73 in the serum extracellular vesicles (EVs) and to analyze nucleotide hydrolysis. Notably, the expression of CD39 in the EVs was significantly increased compared to the CD73 and/or combined CD39/CD73 expression levels at initial collection. Furthermore, our results demonstrated positive correlations between ADP hydrolysis and the transurethral resection and Gleason score. Understanding the role of ectonucleotidases is crucial for identifying new biomarkers in PC.

Aim: Excitotoxicity results from unusually increased activation of excitatory amino acid receptors leading to neuronal death. Since glutamate is the main excitatory neurotransmitter in the central nervous system, it is also the most common excitotoxicity trigger. This uncontrolled neuronal response participates in various neurodegenerative diseases, such as ischemia, hypoglycemia, Huntington’s, Parkinson’s and Alzheimer’s disease. Thus, the investigation in the field expanded a lot in the past decade, leading to in vitro modelling adaptations. However, much performed work on glutamate-induced excitotoxicity is methodologically inconsistent in the literature. The field lacks reproducibility, which is one of the main fundaments of empirical science. In this regard, the literature was summarized and the main methodological features were critically evaluated, aiming to guide the researchers that are starting in the field. Methods: Published data since 1985 from PUBMED were collected and analyzed to observe which in vitro experimental conditions of excitotoxicity were reproducible. The suggested methods were based on the characteristics of excitotoxicity, such as abnormal intracellular calcium mediated signaling, mitochondria impairment, reactive oxygen species accumulation and cell death. Various conditions and comparative controls were used to design the standard investigation of excitotoxicity, such as culture medium content (presence of glutamate and aspartate), time interval of induction and the concentration of the inductor, based on the most reproducible published ones. Results: Our results and critical analysis point to some experimental conditions to consider, such as primary cultured neurons are more sensitive to glutamate and the response obtained is more robust than in other models; excitotoxicity mediated effects are better observed one hour following the stimulus; the culture medium should contain low levels of glutamate or aspartate or glycine. Online available phosphoproteomic data on excitotoxicity using the primary cortical neurons in vitro model supported the same conditions proposed by us. Conclusions: This manuscript will facilitate the design of any research for excitotoxic or neuroprotective compounds in physiological and pathophysiological conditions by standardizing and improving experimental conditions.

In this work, citric acid molecules mediated simultaneously the crosslinking of xanthan gum (XG) chains and the grafting of glutamic acid (Glu), cysteine (Cys), histidine (His) or tryptophan (Trp) to XG chains, as evidenced by Fourier-transform infrared spectroscopy, elemental and thermogravimetric analyses, and high-resolution X-ray photoelectron spectroscopy. XG coatings (∼60 nm thick) presented total surface energy (γS) of 65 mJ/m². The attachment of amino acids to the XG chains decreased γS values to ∼ 48 mJ/m² because the surface energy polar (γpS) component decreased. Circular dichroism (CD) spectra revealed that the helix conformation of XG chains was retained in XG and XG-Glu-coatings, but it was lost in the XG-His, XG-Cys-and XG-Trp-coatings due to intermolecular interactions between the amino acid side groups (thiol, imidazole, and indole). XG-based coatings presented no cytotoxicity. After 3 h of incubation, the adhesion of SH-5YSY neural cells in comparison to the control (100%) was XG-His (18%), XG-Cys (21.4%), XG (29%), XG-Trp (28.6%) and XG-Glu (46.4%). The decrease of% cell adhesion tended to be favored by the decrease of γpS, except for XG-Glu-and XG-Trp, and by the loss of helix molecular conformation of chains in the coatings.

Ectonucleotidases modulate inflammatory responses by balancing extracellular ATP and adenosine (ADO) and might be involved in COVID-19 immunopathogenesis. Here, we explored the contribution of extracellular nucleotide metabolism to COVID-19 severity in mild and severe cases of the disease. We verified that the gene expression of ectonucleotidases is reduced in the whole blood of patients with COVID-19 and is negatively correlated to levels of CRP, an inflammatory marker of disease severity. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of ADO when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into ADP and ADO. Furthermore, impaired expression of ADO receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of ADO in vitro, however, suppressed inflammatory responses triggered in patients’ cells. In summary, our findings support the idea that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity, and suggest that ADO may be a therapeutic approach for the disease.

Neurodegenerative disorders are characterized by a progressive process of degeneration and neuronal death, where oxidative stress and neuroinflammation are key factors that contribute to the progression of these diseases. Therefore, two major pathways involved in these pathologies have been proposed as relevant therapeutic targets: The nuclear transcription factor erythroid 2 (Nrf2), which responds to oxidative stress with cytoprotecting activity; and the nuclear factor NF-κB pathway, which is highly related to the neuroinflammatory process by promoting cytokine expression. Caffeic acid phenethyl ester (CAPE) is a phenylpropanoid naturally found in propolis that shows important biological activities, including neuroprotective activity by modulating the Nrf2 and NF-κB pathways, promoting antioxidant enzyme expression and inhibition of proinflammatory cytokine expression. Its simple chemical structure has inspired the synthesis of many derivatives, with aliphatic and/or aromatic moieties, some of which have improved the biological properties. Moreover, new drug delivery systems increase the bioavailability of these compounds in vivo, allowing its transcytosis through the blood-brain barrier, thus protecting brain cells from the increased inflammatory status associated to neurodegenerative and psychiatric disorders. This review summarizes the biosynthesis and chemical synthesis of CAPE derivatives, their miscellaneous activities, and relevant studies (from 2010 to 2023), addressing their neuroprotective activity in vitro and in vivo.

Drug resistance is a major challenge for all oncological treatments that involve the use of cytotoxic agents. Recent therapeutic alternatives cannot circumvent the ability of cancer cells to adapt or alter the natural selection of resistant cells, so the problem persists. In neuroblastoma, recurrence can occur in up to 50% of high-risk patients. Therefore, the identification of novel therapeutic targets capable of modulating survival or death following classical antitumor interventions is crucial to address this problem. In this study, we investigated the role of the P2X7 receptor in chemoresistance. Here, we elucidated the contributions of P2X7 receptor A and B isoforms to neuroblastoma chemoresistance, demonstrating that the B isoform favors resistance through a combination of mechanisms involving drug efflux via MRP-type transporters, resistance to retinoids, retaining cells in a stem-like phenotype, suppression of autophagy, and EMT induction, while the A isoform has opposite and complementary roles.

Multiple sclerosis (MS) is a highly disabling, progressive neurodegenerative disease with no curative treatment available. Although significant progress has been made in understanding how MS develops, there remain aspects of disease pathogenesis that are yet to be fully elucidated. In this regard, studies have shown that dysfunctional adenosinergic signaling plays a pivotal role, as patients with MS have altered levels adenosine (ADO), adenosine receptors and proteins involved in the generation and termination of ADO signaling, such as CD39 and adenosine deaminase (ADA). We have therefore performed a literature review regarding the involvement of the adenosinergic system in the development of MS and propose mechanisms by which the modulation of this system can support drug development and repurposing.

Glioblastoma (GB) is the most aggressive primary tumor of central nervous system, where no efficient therapy has been found so far. This is due to high intra- and inter- tumor heterogeneity, fast invasion and the therapy-resistant subpopulation of GB stem cells and their plasticity. Tumor heterogeneity involves interactions among cancer cells and stromal cells, such as mesenchymal stem cells (MSCs), that are recruited into the tumor microenvironment and migrate to the tumor site. Here, we characterized the impact of kinin receptors on the interaction of MSCs with GB cells which is further enhanced by kinin receptor agonists. Interactions between GB cells and MSCs were studied in two- and three-dimensional co-cultures. Kinin receptor activity was modulated by selective agonists and antagonists to evaluate their influence on cell viability, cell-cell interactions, GB U87 cell invasiveness, and phenotype alterations. Tumor cell invasion was enhanced by the kinin-B2 receptor agonist bradykinin, while it was blocked by B2 receptor antagonists applied in U87 cells monoculture and in co-culture with MSCs. Kinin receptor inhibition correlated with enhanced direct U87/MSC interactions, such as heterotypic fusion, vesicle transfer and entosis. Furthermore, kinin receptor modulation influenced expression of F-actin expression and genes associated with epithelial-to-mesenchymal transition in U87 cells upon U87/MSCs co-cultures. Our data support ongoing investigations of kinin receptor inhibition as an adjuvant approach in GB therapy. MSC impacts on cancer progression need further investigation, as they may have a synergistic effect with kininergic receptor activation.

Abstract Purines and their derivatives, most notably adenosine and ATP, are the key molecules controlling intracellular energy homoeostasis and nucleotide synthesis. Besides, these purines support, as chemical messengers, purinergic transmission throughout tissues and species. Purines act as endogenous ligands that bind to and activate plasmalemmal purinoceptors, which mediate extracellular communication referred to as “purinergic signalling”. Purinergic signalling is cross-linked with other transmitter networks to coordinate numerous aspects of cell behaviour such as proliferation, differentiation, migration, apoptosis and other physiological processes critical for the proper function of organisms. Pathological deregulation of purinergic signalling contributes to various diseases including neurodegeneration, rheumatic immune diseases, inflammation, and cancer. Particularly, gout is one of the most prevalent purine-related disease caused by purine metabolism disorder and consequent hyperuricemia. Compelling evidence indicates that purinoceptors are potential therapeutic targets, with specific purinergic agonists and antagonists demonstrating prominent therapeutic potential. Furthermore, dietary and herbal interventions help to restore and balance purine metabolism, thus addressing the importance of a healthy lifestyle in the prevention and relief of human disorders. Profound understanding of molecular mechanisms of purinergic signalling provides new and exciting insights into the treatment of human diseases.

Epothilone is a natural 16-membered macrolide cytotoxic compound produced by the metabolism of the cellulose-degrading myxobacterium Sorangium cellulosum. This review summarizes results in the study of epothilones against cancer with preclinical results and clinical studies from 2010–2022. Epothilone have mechanisms of action similar to paclitaxel by inducing tubulin polymerization and apoptosis with low susceptibility to tumor resistance mechanisms. It is active against refractory tumors, being superior to paclitaxel in many respects. Since the discovery of epothilones, several derivatives have been synthesized, and most of them have failed in Phases II and III in clinical trials; however, ixabepilone and utidelone are currently used in clinical practice. There is robust evidence that triple-negative breast cancer (TNBC) treatment improves using ixabepilone plus capecitabine or utidelone in combination with capecitabine. In recent years innovative synthetic strategies resulted in the synthesis of new epothilone derivatives with improved activity against refractory tumors with better activities when compared to ixabepilone or taxol. These compounds together with specific delivery mechanisms could be developed in anti-cancer drugs.

Caffeine (CAF) is a psychostimulant present in many beverages and with rapid bioabsorption. For this reason, matrices that effectuate the sustained release of a low amount of CAF would help reduce the intake frequency and side effects caused by high doses of this stimulant. Thus, in this study, CAF was loaded into magnetic gelatin/alginate (Gel/Alg/MNP) hydrogels at 18.5 mg/ghydrogel. The in vitro release of CAF was evaluated in the absence and presence of an external magnetic field (EMF) and Ca2+. In all cases, the presence of Ca2+ (0.002 M) retarded the release of CAF due to favorable interactions between them. Remarkably, the release of CAF from Gel/Alg/MNP in PBS/CaCl2 (0.002 M) at 37 °C under an EMF was more sustained due to synergic effects. In PBS/CaCl2 (0.002 M) and at 37 °C, the amounts of CAF released after 45 min from Gel/Alg and Gel/Alg/MNP/EMF were 8.3 ± 0.2 mg/ghydrogel and 6.1 ± 0.8 mg/ghydrogel, respectively. The concentration of CAF released from Gel/Alg and Gel/Alg/MNP hydrogels amounted to ~0.35 mM, thereby promoting an increase in cell viability for 48 h. Gel/Alg and Gel/Alg/MNP hydrogels can be applied as reservoirs to release CAF at suitable concentrations, thus forestalling possible side effects and improving the viability of SH-SY5Y cells.

Abstract Duplication or deficiency of the X‐linked MECP2 gene reliably produces profound neurodevelopmental impairment. MECP2 mutations are almost universally responsible for Rett syndrome (RTT), and particular mutations and cellular mosaicism of MECP2 may underlie the spectrum of RTT symptomatic severity. No clinically approved treatments for RTT are currently available, but human pluripotent stem cell technology offers a platform to identify neuropathology and test candidate therapeutics. Using a strategic series of increasingly complex human stem cell‐derived technologies, including human neurons, MECP2‐mosaic neurospheres to model RTT female brain mosaicism, and cortical organoids, we identified synaptic dysregulation downstream from knockout of MECP2 and screened select pharmacological compounds for their ability to treat this dysfunction. Two lead compounds, Nefiracetam and PHA 543613, specifically reversed MECP2‐knockout cytologic neuropathology. The capacity of these compounds to reverse neuropathologic phenotypes and networks in human models supports clinical studies for neurodevelopmental disorders in which MeCP2 deficiency is the predominant etiology.

Background: The blood–brain barrier (BBB) describes the brain’s highly specialized capillaries, which form a dynamic interface that maintains central nervous system (CNS) homeostasis. The BBB supports the CNS, in part, by preventing the entry of potentially harmful circulating molecules into the brain. However, this specialized function is challenging for the development of CNS therapeutics. Several strategies to facilitate drug delivery into the brain parenchyma via disruption of the BBB have been proposed. Bradykinin has proven effective in disrupting mechanisms across the blood–tumor barrier. Unfortunately, bradykinin has limited therapeutic value because of its short half-life and the undesirable biological activity elicited by its active metabolites.Objective: To evaluate NG291, a stable bradykinin analog, with selective agonist activity on the bradykinin-B2 receptor and its ability to disrupt the BBB transiently.Methods: Sprague Dawley rats and CD-1 mice were subjected to NG291 treatment (either 50 or 100 μg/kg, intravenously). Time and dose-dependent BBB disruption were evaluated by histological analysis of Evans blue (EB) extravasation. Transcellular and paracellular BBB leakage were assessed by infiltration of 99mTc-albumin (66.5 KDa) and 14C-sucrose (340 Da) radiolabeled probes into the brains of CD-1 mice treated with NG291. NG291 influence on P-glycoprotein (P-gp) efflux pump activity was evaluated by quantifying the brain accumulation of 3H-verapamil, a known P-gp substrate, in CD-1 mice.Results: NG291-mediated BBB disruption was localized, dose-dependent, and reversible as measured by EB extravasation. 99mTc-albumin leakage was significantly increased by 50 μg/kg of NG291, whereas 100 μg/kg of NG291 significantly augmented both 14C-sucrose and 99mTc-albumin leakage. NG291 enhanced P-gp efflux transporter activity and was unable to increase brain uptake of the P-gp substrate pralidoxime. NG291 did not evoke significant short-term neurotoxicity, as it did not increase brain water content, the number of Fluoro-Jade C positive cells, or astrocyte activation.Conclusion: Our findings strongly suggest that NG291 increases BBB permeability by two different mechanisms in a dose-dependent manner and increases P-gp efflux transport. This increased permeability may facilitate the penetration into the brain of therapeutic candidates that are not P-gp substrates.

Cannabis sativa is one of the first medicinal plants used by humans. Its medical use remains controversial because it is a psychotropic drug whose use has been banned. Recently, however, some countries have approved its use, including for recreational and medical purposes, and have allowed the scientific study of its compounds. Cannabis is characterized by the production of special types of natural products called phytocannabinoids that are synthesized exclusively by this genus. Phytocannabinoids and endocannabinoids are chemically different, but both pharmacologically modulate CB1, CB2, GRP55, GRP119 and TRPV1 receptor activities, involving activities such as memory, sleep, mood, appetite and motor regulation, pain sensation, neuroinflammation, neurogenesis and apoptosis. Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are phytocannabinoids with greater pharmacological potential, including anti-inflammatory, neuroprotective and anticonvulsant activities. Cannabidiol is showing promising results for the treatment of COVID-19, due to its capability of acting on the unleashed cytokine storm, on the proteins necessary for both virus entry and replication and on the neurological consequences of patients who have been infected by the virus. Here, we summarize the latest knowledge regarding the advantages of using cannabinoids in the treatment of COVID-19.

Summary: Extracellular agonists linked to inositol-1,4,5-trisphosphate (IP3) formation elicit cytosolic Ca2+ oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca2+ spike patterns are observed. Using qPCR, we show that rat hepatocytes express multiple purinergic P2Y and P2X receptors (R). ADP acting through P2Y1R elicits narrow Ca2+ oscillations, whereas UTP acting through P2Y2R elicits broad Ca2+ oscillations, with composite patterns observed for ATP. P2XRs do not play a role at physiological agonist levels. The discrete Ca2+ signatures reflect differential effects of protein kinase C (PKC), which selectively modifies the falling phase of the Ca2+ spikes. Negative feedback by PKC limits the duration of P2Y1R-induced Ca2+ spikes in a manner that requires extracellular Ca2+. By contrast, P2Y2R is resistant to PKC negative feedback. Thus, the PKC leg of the bifurcated IP3 signaling pathway shapes unique Ca2+ oscillation patterns that allows for distinct cellular responses to different agonists.

Recent studies have highlighted the mechanisms controlling the formation of cerebral cholesterol, which is synthesized in situ primarily by astrocytes, where it is loaded onto apolipoproteins and delivered to neurons and oligodendrocytes through interactions with specific lipoprotein receptors. The “cholesterol shuttle” is influenced by numerous proteins or carbohydrates, which mainly modulate the lipoprotein receptor activity, function and signaling. These molecules, provided with enzymatic/proteolytic activity leading to the formation of peptide fragments of different sizes and specific sequences, could be also responsible for machinery malfunctions, which are associated with neurological, neurodegenerative and neurodevelopmental disorders. In this context, we have pointed out that purines, ancestral molecules acting as signal molecules and neuromodulators at the central nervous system, can influence the homeostatic machinery of the cerebral cholesterol turnover and vice versa. Evidence gathered so far indicates that purine receptors, mainly the subtypes P2Y2, P2X7 and A2A, are involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer’s and Niemann–Pick C diseases, by controlling the brain cholesterol homeostasis; in addition, alterations in cholesterol turnover can hinder the purine receptor function. Although the precise mechanisms of these interactions are currently poorly understood, the results here collected on cholesterol–purine reciprocal control could hopefully promote further research.

P2X7 receptors are ion-gated channels activated by ATP. Under pathological conditions, the extensive release of ATP induces sustained P2X7 receptor activation, culminating in induction of proinflammatory pathways with inflammasome assembly and cytokine release. These inflammatory conditions, whether occurring peripherally or in the central nervous system (CNS), increase blood-brain-barrier (BBB) permeability. Besides its well-known involvement in neurodegeneration and neuroinflammation, the P2X7 receptor may induce BBB disruption and chemotaxis of peripheral immune cells to the CNS, resulting in brain parenchyma infiltration. For instance, despite common effects on cytokine release, P2X7 receptor signaling is also associated with metalloproteinase secretion and activation, as well as migration and differentiation of T lymphocytes, monocytes and dendritic cells. Here we highlight that peripheral immune cells mediate the pathogenesis of Multiple Sclerosis and Parkinson’s and Alzheimer’s disease, mainly through T lymphocyte, neutrophil and monocyte infiltration. We propose that P2X7 receptor activation contributes to neurodegenerative disease progression beyond its known effects on the CNS. This review discusses how P2X7 receptor activation mediates responses of peripheral immune cells within the inflamed CNS, as occurring in the aforementioned diseases.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) are constantly threatening global public health. With no end date, the pandemic persists with the emergence of novel variants that threaten the effectiveness of diagnostic tests and vaccines. Mutations in the Spike surface protein of the virus are regularly observed in the new variants, potentializing the emergence of novel viruses with different tropism from the current ones, which may change the severity and symptoms of the disease. Growing evidence has shown that mutations are being selected in favor of variants that are more capable of evading the action of neutralizing antibodies. In this context, the most important factor guiding the evolution of SARS-CoV-2 is its interaction with the host’s immune system. Thus, as current vaccines cannot block the transmission of the virus, measures complementary to vaccination, such as the use of masks, hand hygiene, and keeping environments ventilated remain essential to delay the emergence of new variants. Importantly, in addition to the involvement of the immune system in the evolution of the virus, we highlight several chemical parameters that influence the molecular interactions between viruses and host cells during invasion and are also critical tools making novel variants more transmissible. In this review, we dissect the impacts of the Spike mutations on biological parameters such as (1) the increase in Spike binding affinity to hACE2; (2) bound time for the receptor to be cleaved by the proteases; (3) how mutations associate with the increase in RBD up-conformation state in the Spike ectodomain; (4) expansion of uncleaved Spike protein in the virion particles; (5) increment in Spike concentration per virion particles; and (6) evasion of the immune system. These factors play key roles in the fast spreading of SARS-CoV-2 variants of concern, including the Omicron.

The P2X7 receptor is a cation channel activated by high concentrations of adenosine triphosphate (ATP). Upon long-term activation, it complexes with membrane proteins forming a wide pore that leads to cell death and increased release of ATP into the extracellular milieu. The P2X7 receptor is widely expressed in the CNS, such as frontal cortex, hippocampus, amygdala and striatum, regions involved in neurodegenerative diseases and psychiatric disorders. Despite P2X7 receptor functions in glial cells have been extensively studied, the existence and roles of this receptor in neurons are still controversially discussed. Regardless, P2X7 receptors mediate several processes observed in neuropsychiatric disorders and brain tumors, such as activation of neuroinflammatory response, stimulation of glutamate release and neuroplasticity impairment. Moreover, P2X7 receptor gene polymorphisms have been associated to depression, and isoforms of P2X7 receptors are implicated in neuropsychiatric diseases. In view of that, the P2X7 receptor has been proposed to be a potential target for therapeutic intervention in brain diseases. This review discusses the molecular mechanisms underlying P2X7 receptor-mediated signaling in neurodegenerative diseases, psychiatric disorders, and brain tumors. In addition, it highlights the recent advances in the development of P2X7 receptor antagonists that are able of penetrating the central nervous system.

Glioblastomas (GBMs) are tumors that have a high ability to migrate, invade and proliferate in the healthy tissue, what greatly impairs their treatment. These characteristics are associated with the complex microenvironment, formed by the perivascular niche, which is also composed of several stromal cells including astrocytes, microglia, fibroblasts, pericytes and endothelial cells, supporting tumor progression. Further microglia and macrophages associated with GBMs infiltrate the tumor. These innate immune cells are meant to participate in tumor surveillance and eradication, but they become compromised by GBM cells and exploited in the process. In this review we discuss the context of the GBM microenvironment together with the actions of flavonoids, which have attracted scientific attention due to their pharmacological properties as possible anti-tumor agents. Flavonoids act on a variety of signaling pathways, counteracting the invasion process. Luteolin and rutin inhibit NFκB activation, reducing IL-6 production. Fisetin promotes tumor apoptosis, while inhibiting ADAM expression, reducing invasion. Naringenin reduces tumor invasion by down-regulating metalloproteinases expression. Apigenin and rutin induce apoptosis in C6 cells increasing TNFα, while decreasing IL-10 production, denoting a shift from the immunosuppressive Th2 to the Th1 profile. Overall, flavonoids should be further exploited for glioma therapy.

Signaling mediated by purines is a widespread mechanism of cell–cell communication related to vasomotor responses and the control of platelet function in the vascular system. However, little is known about the involvement of this signaling as well as the role of reactive oxygen species (ROS) in the development of hypothyroidism. Therefore, the present study investigates changes in the purinergic system, including enzyme activities and expression in platelets, and oxidative profiles in patients with post-thyroidectomy hypothyroidism. The nucleoside triphosphate diphosphohydrolase 1 (NTPDase/CD39) expression in patients increased by 40%, and the adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolyzing activity increased by 82% and 70%, respectively. The activities of ecto-5´-nucleotidase and adenosine deaminase (ADA) also significantly enhanced (39% and 52%, respectively), which correlates with a 45% decrease in adenosine concentration. Furthermore, these patients demonstrated an increased production of ROS (42%), thiobarbituric acid reactive substances (TBARS) (115%), carbonyl protein (30%) and a decreased glutathione S-transferase (GST) activity (20%). This study demonstrates that hypothyroidism interferes with adenine nucleoside and nucleotide hydrolysis and this is correlated with oxidative stress, which might be responsible for the increase in ADA activity. This increase causes rapid adenosine deamination, which can generate a decrease in their concentration in the systemic circulation, which can be associated with the development of vascular complications.

Breast cancer remains a clinical problem despite advancements in the field. Cancer stem cells (CSCs) within the breast cancer population are implicated in cancer relapse. The dormant CSCs generally resist available treatment, thus challenging the current treatment paradigm. Radiation is an aggressive form of treatment typically used to reduce tumor mass in breast cancer patients. Several clinical and research-based studies have shown that radiation treatment cannot target all cancer cells, leaving behind radioresistant cells. The radioresistant cells have the potential to acquire stem cell-like features that render them untargetable with respect to the current technology. This review elaborates on cancer cells acquiring stem cell phenotype. In addition, we discuss the phenotype and function of cancer cells that are derived from radioresistant cells as well as indirect changes as a consequence to bystander effect. In addition, the epigenetic profile of the radioresistant cells plays a crucial role in the acquisition of cycling quiescence and stem cell-like phenotype and is detailed in this review.

Studies have confirmed that bone marrow-derived mesenchymal stem cells (MSCs) can be used for treatment of several nervous system diseases. However, isolation of bone marrow-derived MSCs (BMSCs) is an invasive and painful process and the yield is very low. Therefore, there is a need to search for other alterative stem cell sources. Adipose-derived MSCs (ADSCs) have phenotypic and gene expression profiles similar to those of BMSCs. The production of ADSCs is greater than that of BMSCs, and ADSCs proliferate faster than BMSCs. To compare the effects of venous grafts containing BMSCs or ADSCs on sciatic nerve injury, in this study, rats were randomly divided into four groups: sham (only sciatic nerve exposed), Matrigel (MG; sciatic nerve injury + intravenous transplantation of MG vehicle), ADSCs (sciatic nerve injury + intravenous MG containing ADSCs), and BMSCs (sciatic nerve injury + intravenous MG containing BMSCs) groups. Sciatic functional index was calculated to evaluate the function of injured sciatic nerve. Morphologic characteristics of nerves distal to the lesion were observed by toluidine blue staining. Spinal motor neurons labeled with Fluoro-Gold were quantitatively assessed. Compared with sham-operated rats, sciatic functional index was lower, the density of small-diameter fibers was significantly increased, and the number of motor neurons significantly decreased in rats with sciatic nerve injury. Neither ADSCs nor BMSCs significantly improved the sciatic nerve function of rats with sciatic nerve injury, increased fiber density, fiber diameters, axonal diameters, myelin sheath thickness, and G ratios (axonal diameter/fiber diameter ratios) in the sciatic nerve distal to the lesion site. There was no significant difference in the number of spinal motor neurons among ADSCs, BMSCs and MG groups. These results suggest that neither BMSCs nor ADSCs provide satisfactory results for peripheral nerve repair when using MG as the conductor for engraftment.

The heterogeneity of tumor cell mass and the plasticity of cancer cell phenotypes in solid tumors allow for the insurgence of resistant and metastatic cells, responsible for cancer patients’ clinical management’s main challenges. Among several factors that are responsible for increased cancer aggression, metabolic reprogramming is recently emerging as an ultimate cancer hallmark, as it is central for cancer cell survival and self-renewal, metastasis and chemoresistance. The P2X7 receptor, whose expression is upregulated in many solid and hematological malignancies, is also emerging as a good candidate in cancer metabolic reprogramming and the regulation of stem cell proliferation and differentiation. Metabostemness refers to the metabolic reprogramming of cancer cells toward less differentiated (CSCs) cellular states, and we believe that there is a strong correlation between metabostemness and P2X7 receptor functions in oncogenic processes. Here, we summarize important aspects of P2X7 receptor functions in normal and tumor tissues as well as essential aspects of its structure, regulation, pharmacology and its clinical use. Finally, we review current knowledge implicating P2X7 receptor functions in cancer-related molecular pathways, in metabolic reprogramming and in metabostemness.

Huntington’s disease (HD) is a multi-system disorder that is caused by expanded CAG repeats within the exon-1 of the huntingtin (HTT) gene that translate to the polyglutamine stretch in the HTT protein. HTT interacts with the proteins involved in gene transcription, endocytosis, and metabolism. HTT may also directly or indirectly affect purine metabolism and signaling. We aimed to review existing data and discuss the modulation of the purinergic system as a new therapeutic target in HD. Impaired intracellular nucleotide metabolism in the HD affected system (CNS, skeletal muscle and heart) may lead to extracellular accumulation of purine metabolites, its unusual catabolism, and modulation of purinergic signaling. The mechanisms of observed changes might be different in affected systems. Based on collected findings, compounds leading to purine and ATP pool reconstruction as well as purinergic receptor activity modulators, i.e., P2X7 receptor antagonists, may be applied for HD treatment.

Aptamers are single-stranded DNA or RNA molecules which are submitted to a process denominated SELEX. SELEX uses reiterative screening of a random oligonucleotide library to identify high-affinity binders to a chosen target, which may be a peptide, protein, or entire cells or viral particles. Aptamers can rival antibodies in target recognition, and benefit from their non-proteic nature, ease of modification, increased stability, and pharmacokinetic properties. This turns them into ideal candidates for diagnostic as well as therapeutic applications. Here, we review the recent accomplishments in the development of aptamers targeting emerging viral diseases, with emphasis on recent findings of aptamers binding to coronaviruses. We focus on aptamer development for diagnosis, including biosensors, in addition to aptamer modifications for stabilization in body fluids and tissue penetration. Such aptamers are aimed at in vivo diagnosis and treatment, such as quantification of viral load and blocking host cell invasion, virus assembly, or replication, respectively. Although there are currently no in vivo applications of aptamers in combating viral diseases, such strategies are promising for therapy development in the future.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by decreased dopamine bioavailability in the substantia nigra and the striatum. Taking into account that adenosine-5’-triphosphate (ATP) and its metabolites are intensely released in the 6-hydroxydopamine (6-OHDA) animal model of PD, screening of purinergic receptor gene expression was performed. Effects of pharmacological P2Y6 or P2X7 receptor antagonism were studied in preventing or reversing hemiparkinsonian behavior and dopaminergic deficits in this animal model. P2X7 receptor antagonism with Brilliant Blue G (BBG) at a dose of 75 mg/kg re-established the dopaminergic nigrostriatal pathway in rats injured with 6-OHDA. Selective P2Y6 receptor antagonism by MRS2578 prevented dopaminergic neuron death in SH-SY5Y cells in vitro and in vivo in the substantia nigra of rats injured with 6-OHDA. Moreover, in vivo analysis showed that both treatments were accompanied by a reduction of microglial activation in the substantia nigra. Altogether, these data provide evidence that antagonism of P2X7 or P2Y6 receptors results in neuroregenerative or neuroprotective effects, respectively, possibly through modulation of neuroinflammatory responses.

Despite recent advances unraveling mechanisms of hostâpathogen interactions in innate immunity, the participation of purinergic signaling in infection-driven inflammation remains an emerging research field with many unanswered questions. As one of the most-studied oral pathogens, Porphyromonas gingivalis is considered as a keystone pathogen with a central role in development of periodontal disease. This pathogen needs to evade immune-mediated defense mechanisms and tolerate inflammation in order to survive in the host. In this review, we summarize evidence showing that purinergic signaling modulates P. gingivalis survival and cellular immune responses, and discuss the role played by inflammasome activation and cell death during P. gingivalis infection. Keywords: Purinergic receptors, Innate immunity, Porphyromonas gingivalis, P2X7 receptor, Oral microbes, Inflammasome

Microglial cells, the resident macrophages of the central nervous system (CNS), exist in a process-bearing, ramified/surveying phenotype under resting conditions. Upon activation by cell-damaging factors, they get transformed into an amoeboid phenotype releasing various cell products including pro-inflammatory cytokines, chemokines, proteases, reactive oxygen/nitrogen species, and the excytotoxic ATP and glutamate. In addition, they engulf pathogenic bacteria or cell debris and phagocytose them. However, already resting/surveying microglia have a number of important physiological functions in the CNS; for example, they shield small disruptions of the blood–brain barrier by their processes, dynamically interact with synaptic structures, and clear surplus synapses during development. In neurodegenerative illnesses, they aggravate the original disease by a microglia-based compulsory neuroinflammatory reaction. Therefore, the blockade of this reaction improves the outcome of Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, amyotrophic lateral sclerosis, etc. The function of microglia is regulated by a whole array of purinergic receptors classified as P2Y12, P2Y6, P2Y4, P2X4, P2X7, A2A, and A3, as targets of endogenous ATP, ADP, or adenosine. ATP is sequentially degraded by the ecto-nucleotidases and 5′-nucleotidase enzymes to the almost inactive inosine as an end product. The appropriate selective agonists/antagonists for purinergic receptors as well as the respective enzyme inhibitors may profoundly interfere with microglial functions and reconstitute the homeostasis of the CNS disturbed by neuroinflammation.

Bone marrow metastasis occurs in approximately 350,000 patients that annually die in the U.S. alone. In view of the importance of tumor cell migration into the bone marrow, we have here investigated effects of various concentrations of stromal cell-derived factor-1 (SDF-1), bradykinin- and ATP on bone marrow metastasis. We show for first time that bradykinin augmented chemotactic responsiveness of neuroblastoma cells to SDF-1 and ATP concentrations, encountered under physiological conditions. Bradykinin upregulated VEGF expression, increased metalloproteinase activity and induced adhesion of neuroblastoma cells. Bradykinin augmented SDF-1-induced intracellular Ca2+ mobilization as well as resensitization and expression of ATP-sensing P2X7 receptors. Bradykinin treatment resulted in higher gene expression levels of the truncated P2X7B receptor compared to those of the P2X7A full-length isoform. Bradykinin as pro-metastatic factor induced tumor proliferation that was significantly decreased by P2X7 receptor antagonists; however, the peptide did not enhance cell death nor P2X7A receptor-related pore activity, promoting neuroblastoma growth. Furthermore, immunodeficient nude/nude mice transplanted with bradykinin-pretreated neuroblastoma cells revealed significantly higher metastasis rates compared to animals injected with untreated cells. In contrast, animals receiving Brilliant Blue G, a P2X7 receptor antagonist, did not show any specific dissemination of neuroblastoma cells to the bone marrow and liver, and metastasis rates were drastically reduced. Our data suggests correlated actions of kinins and purines in neuroblastoma dissemination, providing novel avenues for clinic research in preventing metastasis.

Since proving adenosine triphosphate (ATP) functions as a neurotransmitter in neuron/glia interactions, the purinergic system has been more intensely studied within the scope of the central nervous system. In neurological disorders with associated motor symptoms, including Parkinson's disease (PD), motor neuron diseases (MND), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Huntington's Disease (HD), restless leg syndrome (RLS), and ataxias, alterations in purinergic receptor expression and activity have been noted, indicating a potential role for this system in disease etiology and progression. In neurodegenerative conditions, neural cell death provokes extensive ATP release and alters calcium signaling through purinergic receptor modulation. Consequently, neuroinflammatory responses, excitotoxicity and apoptosis are directly or indirectly induced. This review analyzes currently available data, which suggests involvement of the purinergic system in neuro-associated motor dysfunctions and underlying mechanisms. Possible targets for pharmacological interventions are also discussed.

Parkinson's disease (PD) is a neurodegenerative disorder, characterized by the loss of dopaminergic neurons in the substantia nigra and their projections to the striatum. Several processes have been described as potential inducers of the dopaminergic neuron death, such as inflammation, oxidative stress, and mitochondrial dysfunction. However, the death of dopaminergic neurons seems to be multifactorial, and its cause remains unclear. ATP-activating purinergic receptors influence various physiological functions in the CNS, including neurotransmission. Purinergic signaling is also involved in pathological scenarios, where ATP is extensively released and promotes sustained purinergic P2X7 receptor (P2X7R) activation and consequent induction of cell death. This effect occurs, among other factors, by oxidative stress and during the inflammatory response. On the other hand, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) is involved in energy metabolism and mitochondrial biogenesis. Expression and activity upregulation of this protein has been related with reduction of oxidative stress and neuroprotection. Therefore, P2X7R and PGC-1α are potential targets in the treatment of PD. Here hemiparkinsonism was induced by unilateral stereotactic injection of 6-OHDA in a rat model. After 7 days, the establishment of PD was confirmed and followed by treatment with the P2X7R antagonist Brilliant Blue G (BBG) or PGC-1α agonist fenofibrate. BBG, but not fenofibrate, reverted hemiparkinsonian behavior accompanied by an increase in tyrosine hydroxylase immunoreactivity in the substantia nigra. Our results suggest that the P2X7R may be a therapeutic target in Parkinson's disease.

Stress exposure is considered to be the main environmental cause associated with the development of depression. Due to the limitations of currently available antidepressants, a search for new pharmacological targets for treatment of depression is required. Recent studies suggest that adenosine triphosphate (ATP)-mediated signaling through the P2X7 receptor (P2X7R) might play a prominent role in regulating depression-related pathology, such as synaptic plasticity, neuronal degeneration, as well as changes in cognitive and behavioral functions. P2X7R is an ATP-gated cation channel localized in different cell types in the central nervous system (CNS), playing a crucial role in neuron-glia signaling. P2X7R may modulate the release of several neurotransmitters, including monoamines, nitric oxide (NO) and glutamate. Moreover, P2X7R stimulation in microglia modulates the innate immune response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome, consistent with the neuroimmune hypothesis of MDD. Importantly, blockade of P2X7R leads to antidepressant-like effects in different animal models, which corroborates the findings that the gene encoding for the P2X7R is located in a susceptibility locus of relevance to depression in humans. This review will discuss recent findings linked to the P2X7R involvement in stress and MDD neuropathophysiology, with special emphasis on neurochemical, neuroimmune, and neuroplastic mechanisms.

Promiscuous inhibition due to aggregate formation has been recognized as a major concern in drug discovery campaigns. Here, we report some aggregators identified in a virtual screening (VS) protocol to search for inhibitors of human ecto-5′-nucleotidase (ecto-5′-NT/CD73), a promising target for several diseases and pathophysiological events, including cancer, inflammation and autoimmune diseases. Four compounds (A, B, C and D), selected from the ZINC-11 database, showed IC50 values in the micromolar range, being at the same time computationally predicted as potential aggregators. To confirm if they inhibit human ecto-5′-NT via promiscuous mechanism, forming aggregates, enzymatic assays were done in the presence of 0.01% (v/v) Triton X-100 and an increase in the enzyme concentration by 10-fold. Under both experimental conditions, these four compounds showed a significant decrease in their inhibitory activities. To corroborate these findings, turbidimetric assays were performed, confirming that they form aggregate species. Additionally, aggregation kinetic studies were done by dynamic light scattering (DLS) for compound C. None of the identified aggregators has been previously reported in the literature. For the first time, aggregation and promiscuous inhibition issues were systematically studied and evaluated for compounds selected by VS as potential inhibitors for human ecto-5′-NT. Together, our results reinforce the importance of accounting for potential false-positive hits acting by aggregation in drug discovery campaigns to avoid misleading assay results.

The kallikrein-kinin system (KKS) is an endogenous pathway involved in many biological processes. Although primarily related to blood pressure control and inflammation, its activation goes beyond these effects. Neurogenesis and neuroprotection might be stimulated by bradykinin being of great interest for clinical applications following brain injury. This peptide is also an important player in spinal cord injury pathophysiology and recovery, in which bradykinin receptor blockers represent substantial therapeutic potential. Here, we highlight the participation of kinin receptors and especially bradykinin in mediating ischemia pathophysiology in the central and peripheral nervous systems. Moreover, we explore the recent advances on mechanistic and therapeutic targets for biological, pathological, and neural repair processes involving kinins.

Ecto-5'-nucleotidase/CD73 (ecto-5'-NT) participates in extracellular ATP catabolism by converting adenosine monophosphate (AMP) into adenosine. This enzyme affects the progression and invasiveness of different tumors. Furthermore, the expression of ecto-5'-NT has also been suggested as a favorable prognostic marker, attributing to this enzyme contradictory functions in cancer. Medulloblastoma (MB) is the most common brain tumor of the cerebellum and affects mainly children.The effects of ecto-5'-NT overexpression on human MB tumor growth were studied in an in vivo model. Balb/c immunodeficient (nude) 6 to 14-week-old mice were used for dorsal subcutaneous xenograph tumor implant. Tumor development was evaluated by pathophysiological analysis. In addition, the expression patterns of adenosine receptors were verified.The human MB cell line D283, transfected with ecto-5'-NT (D283hCD73), revealed reduced tumor growth compared to the original cell line transfected with an empty vector. D283hCD73 generated tumors with a reduced proliferative index, lower vascularization, the presence of differentiated cells and increased active caspase-3 expression. Prominent A1 adenosine receptor expression rates were detected in MB cells overexpressing ecto-5'-NT.This work suggests that ecto-5'-NT promotes reduced tumor growth to reduce cell proliferation and vascularization, promote higher differentiation rates and initiate apoptosis, supposedly by accumulating adenosine, which then acts through A1 adenosine receptors. Therefore, ecto-5'-NT might be considered an important prognostic marker, being associated with good prognosis and used as a potential target for therapy.

BackgroundNovel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo.Principal findingsP2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis.ConclusionsIn embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.