Hyperactivation of P2X7 receptors as a culprit of COVID-19 neuropathology.

Scientists and health professionals are exhaustively trying to contain the coronavirus disease 2019 (COVID-19) pandemic by elucidating viral invasion mechanisms, possible drugs to prevent viral infection/replication, and health cares to minimize individual exposure. Although neurological symptoms are being reported worldwide, neural acute and long-term consequences of SARS-CoV-2 are still unknown. COVID-19 complications are associated with exacerbated immunoinflammatory responses to SARS-CoV-2 invasion. In this scenario, pro-inflammatory factors are intensely released into the bloodstream, causing the so-called "cytokine storm". Both pro-inflammatory factors and viruses may cross the blood-brain barrier and enter the central nervous system, activating neuroinflammatory responses accompanied by hemorrhagic lesions and neuronal impairment, which are largely described processes in psychiatric disorders and neurodegenerative diseases. Therefore, SARS-CoV-2 infection could trigger and/or worse brain diseases. Moreover, patients with central nervous system disorders associated to neuroimmune activation (e.g. depression, Parkinson's and Alzheimer's disease) may present increased susceptibility to SARS-CoV-2 infection and/or achieve severe conditions. Elevated levels of extracellular ATP induced by SARS-CoV-2 infection may trigger hyperactivation of P2X7 receptors leading to NLRP3 inflammasome stimulation as a key mediator of neuroinvasion and consequent neuroinflammatory processes, as observed in psychiatric disorders and neurodegenerative diseases. In this context, P2X7 receptor antagonism could be a promising strategy to prevent or treat neurological complications in COVID-19 patients.

High P2X6 receptor expression in human bladder cancer predicts good survival prognosis.

As alterations in purinergic signaling have been observed in bladder diseases, we aimed to assess the potential prognostic role of purinergic receptors in bladder cancer in a translational approach based on clinical databases and in vitro data. The prognostic role of purinergic receptors in the survival of patients with bladder cancer and the expression profile of the altered P2 receptors in normal and in tumor samples were determined using The Cancer Genome Atlas databank. In T24 and RT4 human bladder cancer cell lines, the P2 purinergic receptors were characterized by RT-PCR and RT-qPCR analysis including radiotherapy exposure as treatment. The cell number and the cumulative population doubling were also assessed. The expression profile of P2X6 receptor in the cancer pathological stage and in the nodal metastasis status was in agreement with Kaplan-Meier analysis, indicating that high expression of this receptor was related to an increased survival rate in patients with bladder cancer. Of all the P2 receptors expressed on T24 cell line, P2X6 presented high expression after radiotherapy, while it was not altered in RT4 cells. In addition, irradiation promoted a decrease of T24 cell number, but did not change the cell number of RT4 after the same time and radiation dose. Along 7 days after irradiation exposure, both cells regrew. However, while P2X6 receptor was downregulated in T24 cells, it was upregulated in RT4 cells. Our findings indicated that high P2X6 receptor expression induced by radiation in T24 cell line may predict a good survival prognostic factor.

Mesenchymal stem cell-glioblastoma interactions mediated via kinin receptors unveiled by cytometry.

Glioblastoma (GBM) is one of the most malignant and devastating brain tumors. The presence of highly therapy-resistant GBM cell subpopulations within the tumor mass, rapid invasion into brain tissues and reciprocal interactions with stromal cells in the tumor microenvironment contributes to an inevitable fatal prognosis for the patients. We highlight the most recent evidence of GBM cell crosstalk with mesenchymal stem cells (MSCs), which occurs either by direct cell-cell interactions via gap junctions and microtubules or cell fusion. MSCs and GBM paracrine interactions are commonly observed and involve cytokine signaling, regulating MSC tropism toward GBM, their intra-tumoral distribution, and immune system responses. MSC-promoted effects depending on their cytokine and receptor expression patterns are considered critical for GBM progression. MSC origin, tumor heterogeneity and plasticity may also determine the outcome of such interactions. Kinins and kinin-B1 and -B2 receptors play important roles in information flow between MSCs and GBM cells. Kinin-B1 receptor activity favors tumor migration and fusion of MSCs and GBM cells. Flow and image (tissue) cytometry are powerful tools to investigate GBM cell and MSC crosstalk and are applied to analyze and characterize several other cancer types.

Targeting Purinergic Signaling and Cell Therapy in Cardiovascular and Neurodegenerative Diseases.

Extracellular purines exert several functions in physiological and pathophysiological mechanisms. ATP acts through P2 receptors as a neurotransmitter and neuromodulator and modulates heart contractility, while adenosine participates in neurotransmission, blood pressure, and many other mechanisms. Because of their capability to differentiate into mature cell types, they provide a unique therapeutic strategy for regenerating damaged tissue, such as in cardiovascular and neurodegenerative diseases. Purinergic signaling is pivotal for controlling stem cell differentiation and phenotype determination. Proliferation, differentiation, and apoptosis of stem cells of various origins are regulated by purinergic receptors. In this chapter, we selected neurodegenerative and cardiovascular diseases with clinical trials using cell therapy and purinergic receptor targeting. We discuss these approaches as therapeutic alternatives to neurodegenerative and cardiovascular diseases. For instance, promising results were demonstrated in the utilization of mesenchymal stem cells and bone marrow mononuclear cells in vascular regeneration. Regarding neurodegenerative diseases, in general, P2X7 and A2A receptors mostly worsen the degenerative state. Stem cell-based therapy, mainly through mesenchymal and hematopoietic stem cells, showed promising results in improving symptoms caused by neurodegeneration. We propose that purinergic receptor activity regulation combined with stem cells could enhance proliferative and differentiation rates as well as cell engraftment.

Characterization of Comments About bioRxiv and medRxiv Preprints.

Importance: Preprints have been increasingly used in biomedical science, and a key feature of many platforms is public commenting. The content of these comments, however, has not been well studied, and it is unclear whether they resemble those found in journal peer review. Objective: To describe the content of comments on the bioRxiv and medRxiv preprint platforms. Design, Setting, and Participants: In this cross-sectional study, preprints posted on the bioRxiv and medRxiv platforms in 2020 were accessed through each platform's application programming interface on March 29, 2021, and a random sample of preprints containing between 1 and 20 comments was evaluated independently by 3 evaluators using an instrument to assess their features and general content. Main Outcome and Measures: The numbers and percentages of comments from authors or nonauthors were assessed, and the comments from nonauthors were assessed for content. These nonauthor comments were assessed to determine whether they included compliments, criticisms, corrections, suggestions, or questions, as well as their topics (eg, relevance, interpretation, and methods). Nonauthor comments were also analyzed to determine whether they included references, provided a summary of the findings, or questioned the preprint's conclusions. Results: Of 52 736 preprints, 3850 (7.3%) received at least 1 comment (mean [SD] follow-up, 7.5 [3.6] months), and the 1921 assessed comments (from 1037 preprints) had a median length of 43 words (range, 1-3172 words). The criticisms, corrections, or suggestions present in 694 of 1125 comments (61.7%) were the most prevalent content, followed by compliments (n = 428 [38.0%]) and questions (n = 393 [35.0%]). Criticisms usually regarded interpretation (n = 286), methodological design (n = 267), and data collection (n = 238), while compliments were mainly about relevance (n = 111) and implications (n = 72). Conclusions and Relevance: In this cross-sectional study of preprint comments, topics commonly associated with journal peer review were frequent. However, only a small percentage of preprints posted on the bioRxiv and medRxiv platforms in 2020 received comments on these platforms. A clearer taxonomy of peer review roles would help to describe whether postpublication peer review fulfills them.

Exploring CD39 and CD73 Expression as Potential Biomarkers in Prostate Cancer.

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.

Purinergic signaling in cognitive impairment and neuropsychiatric symptoms of Alzheimer's disease.

About 10 million new cases of dementia develop worldwide each year, of which up to 70% are attributable to Alzheimer's disease (AD). In addition to the widely known symptoms of memory loss and cognitive impairment, AD patients frequently develop non-cognitive symptoms, referred to as behavioral and psychological symptoms of dementia (BPSDs). Sleep disorders are often associated with AD, but mood alterations, notably depression and apathy, comprise the most frequent class of BPSDs. BPSDs negatively affect the lives of AD patients and their caregivers, and have a significant impact on public health systems and the economy. Because treatments currently available for AD are not disease-modifying and mainly aim to ameliorate some of the cognitive symptoms, elucidating the mechanisms underlying mood alterations and other BPSDs in AD may reveal novel avenues for progress in AD therapy. Purinergic signaling is implicated in the pathophysiology of several central nervous system (CNS) disorders, such as AD, depression and sleep disorders. Here, we review recent findings indicating that purinergic receptors, mainly the A1, A2A, and P2X7 subtypes, are associated with the development/progression of AD. Current evidence suggests that targeting purinergic signaling may represent a promising therapeutic approach in AD and related conditions. This article is part of the Special Issue on "Purinergic Signaling: 50 years".

The Purinergic System as a Target for the Development of Treatments for Bipolar Disorder.

The neurobiological and neurochemical mechanisms underlying the pathophysiology of bipolar disorder are complex and not yet fully understood. From circadian disruption to neuroinflammation, many pathways and signaling molecules are important contributors to bipolar disorder development, some specific to a disease subtype or a cycling episode. Pharmacological agents for bipolar disorder have shown only partial efficacy, including mood stabilizers and antipsychotics. The purinergic hypothesis for bipolar disorder emerges in this scenario as a promising target for further research and drug development, given its role in neurotransmission and neuroinflammation that results in behavioral and mood regulation. Here, we review the basic concepts of purinergic signaling in the central nervous system and its contribution to bipolar disorder pathophysiology. Allopurinol and novel P2X7 receptor antagonists are promising candidates for treating bipolar disorder. We further explore currently available pharmacotherapies and the emerging new purinergic targets for drug development in bipolar disorder.

Dysfunctional purinergic signaling correlates with disease severity in COVID-19 patients.

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.

Role of P2X7 Receptors in Immune Responses During Neurodegeneration.

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.

Lithium-induced neuroprotective activity in neuronal and microglial cells: A purinergic perspective.

Lithium is the mainstay of pharmacotherapy for treating bipolar disorder (BD). However, despite its wide use for over 60 years in the clinic, its mechanisms of action are not yet well defined. Elucidating lithium's mechanism of action will not only shed light on the pathophysiology of BD, but also potentially uncover new treatment targets. Previous studies suggest that the purinergic system may be involved in lithium's neuroprotective action; thus, the specific aim of this study is to better understand the neuroprotective action of lithium against ATP-induced cellular effect in both neuronal and microglial cellular lineages. We used PC12 neuronal and N9 microglial cells, evaluating cell death by cell counting and Annexin/PI cytometry assay, P2 × 7R immunocontent and ectonucleotidases activity, together with cytokine and nitrite assessment for microglial activity determination. Our results indicate that cells of different neural origins are responsive to ATP, in the sense of neuronal excitotoxicity and microglial switch into an activated M1-like phenotype respectively. Lithium, in turn, modulates the response in neuronal PC12 cells, preventing ATP-induced cell death. On the other hand, in N9 microglial cells, lithium was unable to prevent ATP-induced activation via P2 × 7R, indicating that lithium protective action against the effects of ATP more likely occurs in neurons rather than in microglia. Further studies are needed to better characterize the involvement of the purinergic system in the mechanism of action of lithium against neuronal death and microglial activation, in order to uncover new therapeutic adjunctive targets, such as antagonism of P2 × 7R, as potential approach for bipolar disorder treatment.

Post-weaning social isolation impairs purinergic signaling in rat brain.

Early life stressors, such as social isolation (SI), can disrupt brain development contributing to behavioral and neurochemical alterations in adulthood. Purinergic receptors and ectonucleotidases are key regulators of brain development in embryonic and postnatal periods, and they are involved in several psychiatric disorders, including schizophrenia. The extracellular ATP drives purinergic signaling by activating P2X and P2Y receptors and it is hydrolyzed by ectonucleotidases in adenosine, which activates P1 receptors. The purpose of this study was to investigate if SI, a rodent model used to replicate abnormal behavior relevant to schizophrenia, impacts purinergic signaling. Male Wistar rats were reared from weaning in group-housed or SI conditions for 8 weeks. SI rats exhibited impairment in prepulse inhibition and social interaction. SI presented increased ADP levels in cerebrospinal fluid and ADP hydrolysis in the hippocampus and striatum synaptosomes. Purinergic receptor expressions were upregulated in the prefrontal cortex and downregulated in the hippocampus and striatum. A2A receptors were differentially expressed in SI prefrontal cortex and the striatum, suggesting distinct roles in these brain structures. SI also presented decreased ADP, adenosine, and guanosine levels in the cerebrospinal fluid in response to D-amphetamine. Like patients with schizophrenia, uric acid levels were prominently increased in SI rats after D-amphetamine challenge. We suggest that the SI-induced deficits in prepulse inhibition might be related to the SI-induced changes in purinergic signaling. We provide new evidence that purinergic signaling is markedly affected in a rat model relevant to schizophrenia, pointing out the importance of purinergic system in psychiatry conditions.

The P2X7 Receptor: Central Hub of Brain Diseases.

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.

P2X7 Purinergic Receptor Is Involved in the Pathophysiology of Mania: a Preclinical Study.

The pathophysiology of bipolar disorder remains incompletely elucidated. The purinergic receptor, P2X7 (P2X7R), plays a central role in neuroinflammation, the establishment, and maintenance of microglial activation and neuronal damage/death, all characteristics of bipolar disorder pathology. The present study aims to explore the participation of the P2X7R in a preclinical pharmacological model of mania. We analyzed the modulatory effects of the P2X7R antagonist, brilliant blue, on behavior, monoamines, gene expression, serum purine levels, and cell typing in a pharmacological model of mania induced by D-amphetamine (AMPH) in mice. Our results corroborate an association between the P2X7 receptor and the preclinical animal model of mania, as demonstrated by the decreased responsiveness to AMPH in animals with pharmacologically blocked P2X7R. This study further suggests a possible dopaminergic mechanism for the action of P2X7 receptor antagonism. Additionally, we observed increased peripheral levels of adenosine, a neuroprotective molecule, and increased central expression of Entpd3 and Entpd1 leading to the hydrolysis of ATP, a danger signal, possibly as an attempt to compensate for the damage induced by AMPH. Lastly, P2X7R antagonism in the AMPH model was found to potentially modulate astrogliosis. Our results support the hypothesis that P2X7R plays a vital role in the pathophysiology of mania, possibly by modulating the dopaminergic pathway and astrogliosis, as reflected in the behavioral changes observed. Taken together, this study suggests that a purinergic system imbalance is associated with the AMPH-induced preclinical animal model of mania. P2X7R may represent a promising molecular therapeutic target for bipolar disorder.

Purinergic Receptors in Basal Ganglia Diseases: Shared Molecular Mechanisms between Huntington's and Parkinson's Disease.

Huntington's (HD) and Parkinson's diseases (PD) are neurodegenerative disorders caused by the death of GABAergic and dopaminergic neurons in the basal ganglia leading to hyperkinetic and hypokinetic symptoms, respectively. We review here the participation of purinergic receptors through intracellular Ca2+ signaling in these neurodegenerative diseases. The adenosine A2A receptor stimulates striatopallidal GABAergic neurons, resulting in inhibitory actions on GABAergic neurons of the globus pallidus. A2A and dopamine D2 receptors form functional heteromeric complexes inducing allosteric inhibition, and A2A receptor activation results in motor inhibition. Furthermore, the A2A receptor physically and functionally interacts with glutamate receptors, mainly with the mGlu5 receptor subtype. This interaction facilitates glutamate release, resulting in NMDA glutamate receptor activation and an increase of Ca2+ influx. P2X7 receptor activation also promotes glutamate release and neuronal damage. Thus, modulation of purinergic receptor activity, such as A2A and P2X7 receptors, and subsequent aberrant Ca2+ signaling, might present interesting therapeutic potential for HD and PD.

Bipolar disorder and 1513A>C P2RX7 polymorphism frequency.

Although the etiology of Bipolar Disorder (BD) remains unknown, a strong genetic component to the pathogenesis and risk for this disorder has been widely hypothesized. Several risk genes for BD have been identified; of these, the purinergic P2 × 7 receptor (P2 × 7R) constitutes a pro-inflammatory receptor and a potential risk gene candidate. The purpose of the present study was to assess the frequency of the 1513 A > C P2RX7 polymorphism (rs3751143; Glu496Ala), which leads to receptor loss-of-function, in 154 BD patients versus 184 control subjects. The existence of a differential modulation of P2 × 7R was also analyzed in 22 euthymic BD patients, in comparison to 18 healthy controls. Our data show a decrease in 1513C allele frequency (p = 0.045) and a potential increase in 1513 A A/AC (p = 0.055) genotype frequency in BD patients, compared to controls, indicating an enhanced function of the pro-inflammatory P2 × 7 receptor in BD subjects. Interestingly, no differences in P2RX7 gene and protein expression were found between euthymic BD patients and matched healthy controls. In conclusion, our results suggest that P2 × 7R might play a role in the pathophysiology of BD and add new information regarding this receptor as a potential biomarker for the prediction and diagnosis of the disorder.

Effects of N-acetylcysteine on amphetamine-induced sensitization in mice.

OBJECTIVE: N-acetylcysteine (NAC) is beneficial in psychiatric conditions, including schizophrenia. Patients with schizophrenia exhibit mesolimbic dopamine hyperfunction consequent to an endogenous sensitization process. This sensitization can be modeled in rodents by repeated exposure to psychostimulants, provoking an enduring amplified response at subsequent exposure. The aim of this study was to investigate the effects of NAC on amphetamine sensitization in mice. METHODS: D-amphetamine was administered to C57BL/6 mice three times a week for 3 weeks; the dose was increased weekly from 1 to 3 mg/kg. NAC (60 mg/kg) or saline was administered intraperitoneally before saline or amphetamine during the second and third weeks. After a 4-week washout period, latent inhibition (LI) and the locomotor response to amphetamine 2 mg/kg were assessed. RESULTS: Sensitization disrupted LI and amplified the locomotor response; NAC disrupted LI in control mice. In sensitized animals, NAC attenuated the enhanced locomotion but failed to prevent LI disruption. CONCLUSION: NAC warrants consideration as a candidate for early intervention in ultra-high risk subjects due to its safety profile and the relevance of its mechanism of action. Supplementing this proposition, we report that NAC attenuates sensitization-induced locomotor enhancement in mice. The finding that NAC disrupted LI incites a cautionary note and requires clarification.

Effects of N-acetylcysteine on amphetamine-induced sensitization in mice

Objective: N-acetylcysteine (NAC) is beneficial in psychiatric conditions, including schizophrenia. Patients with schizophrenia exhibit mesolimbic dopamine hyperfunction consequent to an endogenous sensitization process. This sensitization can be modeled in rodents by repeated exposure to psychostimulants, provoking an enduring amplified response at subsequent exposure. The aim of this study was to investigate the effects of NAC on amphetamine sensitization in mice. Methods: D-amphetamine was administered to C57BL/6 mice three times a week for 3 weeks; the dose was increased weekly from 1 to 3 mg/kg. NAC (60 mg/kg) or saline was administered intraperitoneally before saline or amphetamine during the second and third weeks. After a 4-week washout period, latent inhibition (LI) and the locomotor response to amphetamine 2 mg/kg were assessed. Results: Sensitization disrupted LI and amplified the locomotor response; NAC disrupted LI in control mice. In sensitized animals, NAC attenuated the enhanced locomotion but failed to prevent LI disruption. Conclusion: NAC warrants consideration as a candidate for early intervention in ultra-high risk subjects due to its safety profile and the relevance of its mechanism of action. Supplementing this proposition, we report that NAC attenuates sensitization-induced locomotor enhancement in mice. The finding that NAC disrupted LI incites a cautionary note and requires clarification.