Purinergic mechanisms in neuroinflammation: An update from molecules to behavior.

The principle functions of neuroinflammation are to limit tissue damage and promote tissue repair in response to pathogens or injury. While neuroinflammation has utility, pathophysiological inflammatory responses, to some extent, underlie almost all neuropathology. Understanding the mechanisms that control the three stages of inflammation (initiation, propagation and resolution) is therefore of critical importance for developing treatments for diseases of the central nervous system. The purinergic signaling system, involving adenosine, ATP and other purines, plus a host of P1 and P2 receptor subtypes, controls inflammatory responses in complex ways. Activation of the inflammasome, leading to release of pro-inflammatory cytokines, activation and migration of microglia and altered astroglial function are key regulators of the neuroinflammatory response. Here, we review the role of P1 and P2 receptors in mediating these processes and examine their contribution to disorders of the nervous system. Firstly, we give an overview of the concept of neuroinflammation. We then discuss the contribution of P2X, P2Y and P1 receptors to the underlying processes, including a discussion of cross-talk between these different pathways. Finally, we give an overview of the current understanding of purinergic contributions to neuroinflammation in the context of specific disorders of the central nervous system, with special emphasis on neuropsychiatric disorders, characterized by chronic low grade inflammation or maternal inflammation. An understanding of the important purinergic contribution to neuroinflammation underlying neuropathology is likely to be a necessary step towards the development of effective interventions

Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes.

AIM: To carry out a systematic study on the effect of different storage conditions on the number as well as the physical and functional properties of antibacterial extracellular vesicles (EVs) derived from human neutrophilic granulocytes. METHODS: Production of EVs with antibacterial properties was initiated by opsonized Zymosan A particles. The number of released fluorescent EVs was determined by flow cytometry following careful calibration. Physical properties and size of EVs were investigated by flow cytometry, dynamic light scattering and electron microscopy. Functional properties of EVs were tested by bacterial survival assay. RESULTS: Storage at +20 degrees C or +4 degrees C resulted in a significant decrease of EV number and antibacterial effect after 1 day. Storage at -20 degrees C did not influence the EV number up to 28 days, but induced a shift in EV size and almost complete loss of antibacterial function by 28 days. Storage at -80 degrees C had no significant effect either on EV number or size and allowed partial preservation of the antibacterial function up to 28 days. Snap-freezing did not improve the results, whereas the widely used cryoprotectants induced EV lysis. CONCLUSION: Storage significantly alters both the physical and functional properties of EVs even if the number of EVs stays constant. If storage is needed, EVs should be kept at -80 degrees C, preferably not longer than 7 days. For functional tests, freshly prepared EVs are recommended

Involvement of P2Y 12 receptors in an NTG‐induced model of migraine in male mice

Background and purpose: P2Y12 receptors (P2Y12 Rs) are known to regulate different forms of pain and inflammation. In this study we investigated the participation of P2Y12 Rs in an animal model of migraine. Experimental approach: We tested the effect of the centrally administered selective P2Y12 R antagonist PSB-0739, and P2Y12 R gene deficiency in acute nitroglycerin (NTG)-treated mice. Additionally, platelet depletion was used to investigate the role of platelet P2Y12 Rs during migraine-like pain. Key results: NTG induced sensory hypersensitivity of C57BL/6 wild-type (P2ry12+/+ ) mice, accompanied by an increase in c-fos and CGRP expression in the upper cervical spinal cord (C1-C2) and trigeminal nucleus caudalis (TNC). Similar changes were also observed in P2Y12 R gene-deficient (P2ry12-/- ) mice. Prophylactic intrathecal application of PSB-0739 reversed thermal hyperalgesia and head grooming time in wild-type mice but had no effect in P2ry12-/- mice; furthermore, it was also effective when applied as a post-treatment. PSB-0739 administration suppressed the expression of c-fos in C1-C2 and TNC, and decrease C1-C2 levels of dopamine and serotonin in wild-type mice. NTG treatment itself did not change adenosine diphosphate (ADP)-induced platelet activation measured by CD62P upregulation in wild-type mice. Platelet depletion by anti-mouse CD41 antibody and clopidogrel attenuated NTG-induced thermal hypersensitivity and head grooming time in mice. Conclusion and implications: Taken together, our findings show that acute inhibition of P2Y12 Rs alleviates migraine-like pain in mice, by modulating the expression of c-fos, and platelet P2Y12 Rs might contribute to this effect. Hence, it is suggested that the blockade of P2Y12 Rs may have therapeutic potential against migraine

Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome

During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12, and the tyrosine kinases of the TAM family Mer tyrosine kinase (MerTK)/Axl. In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we per-formed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro. Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (http://www. mineco.gob.es) with FEDER funds to A.S. (BFU2012-32089 and RYC-2013-12817) to A.S. and J.V. (BFU2015-66689); a Leonardo Award from the BBVA Foundation to A.S. (IN16,_BBM_BAS_0260); a Basque Government Department of Education project to A.S. (PI_2016_1_0011; http://www.euskadi.eus/basque-government/department-educa- tion/); Ikerbasque start-up funds to J.V.; a Hungarian Research and Development Fund Grant (K116654) to B.S.; a Hungarian Brain Research Program Grant (2017-1.2.1-NKP-2017-00002) to B.S.; a National Institutes of Health Grant (AG060748) to G.L

Role of P2 receptors in normal brain development and in neurodevelopmental psychiatric disorders

The purinergic signaling system, including P2 receptors, plays an important role in various central nervous system (CNS) disorders. Over the last few decades, a substantial amount of accumulated data suggest that most P2 receptor subtypes (P2 × 1, 2, 3, 4, 6, and 7, and P2Y1, 2, 6, 12, and 13) regulate neuronal/neuroglial developmental processes, such as proliferation, differentiation, migration of neuronal precursors, and neurite outgrowth. However, only a few of these subtypes (P2 × 2, P2 × 3, P2 × 4, P2 × 7, P2Y1, and P2Y2) have been investigated in the context of neurodevelopmental psychiatric disorders. The activation of these potential target receptors and their underlying mechanisms mainly influence the process of neuroinflammation. In particular, P2 receptor-mediated inflammatory cytokine release has been indicated to contribute to the complex mechanisms of a variety of CNS disorders. The released inflammatory cytokines could be utilized as biomarkers for neurodevelopmental and psychiatric disorders to improve the early diagnosis intervention, and prognosis. The population changes in gut microbiota after birth are closely linked to neurodevelopmental/neuropsychiatric disorders in later life; thus, the dynamic expression and function of P2 receptors on gut epithelial cells during disease processes indicate a novel avenue for the evaluation of disease progression and for the discovery of related therapeutic compounds

P2X7 receptors drive poly(I:C) induced autism-like behavior in mice

Maternal immune activation (MIA) is a principal environmental risk factor contributing to autism spectrum disorder (ASD), which compromises fetal brain development at critical periods of pregnancy, and might be causally linked to ASD symptoms. We report that endogenous activation of the purinergic ion channel P2X7 (P2rx7) is necessary and sufficient to transduce MIA to autistic phenotype in male offspring. MIA induced by poly(I:C) injections to P2rx7 wild-type mouse dams elicited an autism-like phenotype in their offspring and these alterations were not observed in P2rx7 deficient mice, or following maternal treatment with a specific P2rx7 antagonist, JNJ7965567. Genetic deletion and pharmacological inhibition of maternal P2rx7s also counteracted the induction of IL-6 in the maternal plasma and fetal brain, and disrupted brain development, whilst postnatal P2rx7 inhibition alleviated behavioral and morphological alterations in the offspring. Administration of ATP to P2rx7 wild-type dams also evoked autistic phenotype, but not in knockout dams, implying that P2rx7 activation by ATP is sufficient to induce austim-like features in offspring. Our results point to maternal and offspring P2rx7s as potential therapeutic targets for the early prevention and treatment of ASD.SIGNIFICANCE STATEMENTAutism spectrum disorder (ASD) is a neurodevelopmental psychiatric disorder caused by genetic and environmental factors. Recent studies highlighted the importance of perinatal risks, in particular, maternal immune activation (MIA), showing strong association with the later emergence of ASD in the affected children. MIA could be mimicked in animal models via injection of a non-pathogenic agent poly(I:C) during pregnancy. This is the first report showing the key role of a ligand gated ion channel, the purinergic P2X7 receptor in MIA induced autism-like behavioral and biochemical features. We show that genetic or pharmacological inhibition of both maternal and offspring P2X7 receptors could reverse the compromised brain development and autistic phenotype pointing to new possibilities for prevention and treatment of ASD

Maternal P2X7 receptor inhibition prevents autism-like phenotype in male mouse offspring through the NLRP3-IL-1β pathway

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition caused by interactions of environmental and genetic factors. Recently we showed that activation of the purinergic P2X7 receptors is necessary and sufficient to convert maternal immune activation (MIA) to ASD-like features in male offspring mice. Our aim was to further substantiate these findings and identify downstream signaling pathways coupled to P2X7 upon MIA. Maternal treatment with the NLRP3 antagonist MCC950 and a neutralising IL-1β antibody during pregnancy counteracted the development of autistic characteristics in offspring mice. We also explored time-dependent changes of a widespread cytokine and chemokine profile in maternal blood and fetal brain samples of poly(I:C)/saline-treated dams. MIA-induced increases in plasma IL-1β, RANTES, MCP-1, and fetal brain IL-1β, IL-2, IL-6, MCP-1 concentrations are regulated by the P2X7/NLRP3 pathway. Offspring treatment with the selective P2X7 receptor antagonist JNJ47965567 was effective in the prevention of autism-like behavior in mice using a repeated dosing protocol. Our results highlight that in addition to P2X7, NLRP3, as well as inflammatory cytokines, may also be potential biomarkers and therapeutic targets of social deficits and repetitive behaviors observed in autism spectrum disorder

Microglia actively remodel adult hippocampal neurogenesis through the phagocytosis secretome

During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12; and the tyrosine kinases of the TAM family MerTK/Axl). In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro. Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.SIGNIFICANCE STATEMENTMicroglia are the brain professional phagocytes and, in the adult hippocampal neurogenic niche, they remove newborn cells naturally undergoing apoptosis. Here we show that phagocytosis of apoptotic cells triggers a coordinated transcriptional program that alters their secretome, limiting neurogenesis both in vivo and in vitro. In addition, chronic phagocytosis disruption in mice deficient for receptors P2Y12 and MerTK/Axl reduces adult hippocampal neurogenesis. In contrast, inducible MerTK downregulation transiently increases neurogenesis, suggesting that microglial phagocytosis provides a negative feedback loop that is necessary for the long-term maintenance of adult hippocampal neurogenesis. Therefore, we speculate that the effects of promoting engulfment/degradation of cell debris may go beyond merely removing corpses to actively promoting regeneration in development, ageing, and neurodegenerative diseases

The dualistic role of the purinergic P2Y12-receptor in an in vivo model of Parkinson's disease: Signalling pathway and novel therapeutic targets

Parkinson's disease (PD) is a chronic, progressive neurodegenerative condition; characterized with the degeneration of the nigrostriatal dopaminergic pathway and neuroinflammation. During PD progression, microglia, the resident immune cells in the central nervous system (CNS) display altered activity, but their role in maintaining PD development has remained unclear to date. The purinergic P2Y12-receptor (P2Y12R), which is expressed on the microglia in the CNS has been shown to regulate microglial activity and responses; however, the function of the P2Y12R in PD is unknown. Here we show that MPTP-induced PD symptoms in mice are associated with marked neuroinflammatory changes and P2Y12R contribute to the activation of microglia and progression of the disease. Surprisingly, while pharmacological or genetic targeting of the P2Y12R augments acute mortality in MPTP-treated mice, these interventions protect against the neurodegenerative cell loss and the development of neuroinflammation in vivo. Pharmacological inhibition of receptors during disease development reverses the symptoms of PD and halts disease progression. We found that P2Y12R regulates ROCK and p38 MAPK activity and control cytokine production. Our principal finding is that the receptor has a dualistic role in PD: functional P2Y12Rs are essential to initiate a protective inflammatory response, since the lack of the receptor leads to reduced survival; however, at later stages of neurodegeneration, P2Y12Rs are apparently responsible for maintaining the activated state of microglia and stimulating pro-inflammatory cytokine response. Understanding protective and detrimental P2Y12R-mediated actions in the CNS may reveal novel approaches to control neuroinflammation and modify disease progression in PD. Keywords: G-protein coupled receptor; Microglia; NeurodegenerationNeuroinflammation; P2Y(12)-receptor; Parkinson's disease