Neurotransmitter signaling in the pathophysiology of microglia

Erratun publicado en Frontiers in Cellular Neuroscience 7 : (2013) // Article ID 107Microglial cells are the resident immune cells of the central nervous system. In the resting state, microglia are highly dynamic and control the environment by rapidly extending and retracting motile processes. Microglia are closely associated with astrocytes and neurons, particularly at the synapses, and more recent data indicate that neurotransmission plays a role in regulating the morphology and function of surveying/resting microglia, as they are endowed with receptors for most known neurotransmitters. In particular, microglia express receptors for ATP and glutamate, which regulate microglial motility. After local damage, the release of ATP induces microgliosis and activated microglial cells migrate to the site of injury, proliferate, and phagocytose cells, and cellular compartments. However, excessive activation of microglia could contribute to the progression of chronic neurodegenerative diseases, though the underlying mechanisms are still unclear. Microglia have the capacity to release a large number of substances that can be detrimental to the surrounding neurons, including glutamate, ATP, and reactive oxygen species. However, how altered neurotransmission following acute insults or chronic neurodegenerative conditions modulates microglial functions is still poorly understood. This review summarizes the relevant data regarding the role of neurotransmitter receptors in microglial physiology and pathology.Our work is funded by Fundacion Mutua Madrilena, MINECO, ERANET-Neuron (Nanostroke), CIBERNED, Gobierno Vasco and Universidad del Pais Vasco. Nuria Vazquez-Villoldo holds a fellowship from Gobierno Vasco

Inflammation in stroke: the role of cholinergic, purinergic and glutamatergic signaling

The inflammatory response is a major factor in stroke pathophysiology and contributes to secondary neuronal damage in both acute and chronic stages of the ischemic injury. Recent work in experimental cerebral ischemia has demonstrated the involvement of neurotransmitter signaling in the modulation of neuroinflammation. The present review discusses recent findings on the therapeutic potential and diagnostic perspectives of cholinergic, purinergic and glutamatergic receptors and transporters in experimental stroke. It provides evidence of the role of neurotransmission signaling as a promising inflammatory biomarker in stroke. Finally, recent molecular imaging studies using positron emission tomography of cholinergic receptors and glutamatergic transporters are outlined along with their potential as novel anti-inflammatory therapy to reduce the outcome of cerebral ischemia.We acknowledge financial support by MINECO SAF2014-54070-JIN (A.M.) and SAF2016-75292-R (C.M.)

Contribution of P2X4 Receptors to CNS Function and Pathophysiology

The release and extracellular action of ATP are a widespread mechanism for cell-to-cell communication in living organisms through activation of P2X and P2Y receptors expressed at the cell surface of most tissues, including the nervous system. Among ionototropic receptors, P2X4 receptors have emerged in the last decade as a potential target for CNS disorders such as epilepsy, ischemia, chronic pain, anxiety, multiple sclerosis and neurodegenerative diseases. However, the role of P2X4 receptor in each pathology ranges from beneficial to detrimental, although the mechanisms are still mostly unknown. P2X4 is expressed at low levels in CNS cells including neurons and glial cells. In normal conditions, P2X4 activation contributes to synaptic transmission and synaptic plasticity. Importantly, one of the genes present in the transcriptional program of myeloid cell activation is P2X4. Microglial P2X4 upregulation, the P2X4+ state of microglia, seems to be common in most acute and chronic neurodegenerative diseases associated with inflammation. In this review, we summarize knowledge about the role of P2X4 receptors in the CNS physiology and discuss potential pitfalls and open questions about the therapeutic potential of blocking or potentiation of P2X4 for different pathologies.The authors work are supported by grants from Merck Serono (a business of Merck KGaA, Darmstadt, Germany), Spanish Ministry of Education and Science (SAF2016-75292-R and PID2019-109724RB-I00), Basque Government (IT1203/19 and PI-2016-1-0016) and Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED)

Functional and Metabolic Characterization of Microglia Culture in a Defined Medium

Microglia are the endogenous immune cells of the brain and act as sensor of infection and pathologic injury to the brain, leading to a rapid plastic process of activation that culminates in the endocytosis and phagocytosis of damaged tissue. Microglia cells are the most plastic cells in the brain. Microglia isolation from their environment as well as culturing them in the presence of serum alter their function and lead to a rapid loss of their signature gene expression. Previous studies have identified pivotal factors allowing microglia culture in the absence of serum. Here, we have further characterized the function, expression of markers, metabolic status and response to pro and anti-inflammatory stimulus of microglia isolated by magnetic-activated cell sorting and cultured in a chemically defined medium. We have compared this new method with previous traditional protocols of culturing microglia that use high concentrations of serum.This work was supported by the Spanish Ministry of Education and Science (SAF2013-45084-R and SAF2016-75292-R, including a fellowship to AM), the University of the Basque Country (UPV/EHU; fellowship to AZ), and Centro de Investigacion Biomedica en Red, Enfermedades Neurodegenerativas (CIBERNED) (grant no. CB06/05/0076)

Increased expression of cystine/glutamate antiporter in multiple sclerosis

<p>Abstract</p> <p>Background</p> <p>Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter x_c^-, an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system x_c^-in glutamate homeostasis alterations in MS pathology.</p> <p>Methods</p> <p>Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients.</p> <p>Results and discussion</p> <p>We show here that human activated monocytes release glutamate through cystine/glutamate antiporter x_c^-and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes.</p> <p>Conclusions</p> <p>Together, these results reveal that increased expression of the cystine/glutamate antiporter system x_c^-in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.</p

Role of Mitochondrial Dynamics in Microglial Activation and Metabolic Switch

Microglia act as sensors of injury in the brain, favoring its homeostasis. Their activation and polarization toward a proinflammatory phenotype are associated with injury and disease. These processes are linked to a metabolic reprogramming of the cells, characterized by high rates of glycolysis and suppressed oxidative phosphorylation. This metabolic switch can be reproduced in vitro by microglial stimulation with LPS plus IFN-gamma. To understand the mechanisms regulating mitochondrial respiration abolishment, we examined potential alterations in mitochondrial features during this switch using rat primary microglia. Cells did not show any change in mitochondrial membrane potential, suggesting a limited impact in the mitochondrial viability. We provide evidence that reverse operation of F0F1-ATP synthase contributes to mitochondrial membrane potential. In addition, we studied the possible implication of mitochondrial dynamics in the metabolic switch using the mitochondrial division inhibitor-1 (Mdivi-1), which blocks dynamin-related protein 1 (Drp1)-dependent mitochondrial fission. Mdivi-1 significantly reduced the expression of proinflammatory markers in LPS plus IFN-gamma-treated microglia. However, this inhibition did not lead to a recovery of the oxidative phosphorylation ablation by LPS plus IFN-gamma or to a microglia repolarization. Altogether, these results suggest that Drp1-dependent mitochondrial fission, although potentially involved in microglial activation, does not play an essential role in metabolic reprogramming and repolarization of microglia

P2X7 Receptors as a Therapeutic Target in Cerebrovascular Diseases

Shortage of oxygen and nutrients in the brain induces the release of glutamate and ATP that can cause excitotoxicity and contribute to neuronal and glial damage. Our understanding of the mechanisms of ATP release and toxicity in cerebrovascular diseases is incomplete. This review aims at summarizing current knowledge about the participation of key elements in the ATP-mediated deleterious effects in these pathologies. This includes pannexin-1 hemichannels, calcium homeostasis modulator-1 (CALHM1), purinergic P2X7 receptors, and other intermediaries of CNS injury downstream of ATP release. Available data together with recent pharmacological developments in purinergic signaling may constitute a new opportunity to translate preclinical findings into more effective therapies in cerebrovascular diseases.This study was supported by grants from CONACYT-Mexico No. 252121 and PAPIITUNAM-Mexico No. IN203519 to ROA laboratory; by Spanish Ministry of Education and Science/FEDER (SAF2016-75292-R), Basque Government (IT1203/19), CIBERNED, Eranet-Neuron and Universidad del Pais Vasco to CM's laboratory. AC-M is a researcher from Catedras-CONACYT commissioned at Instituto de Neurobiologia at Universidad Nacional Autonoma de Mexico (UNAM)

Clemastine Induces an Impairment in Developmental Myelination

[EN] Abnormalities in myelination are associated to behavioral and cognitive dysfunction in neurodevelopmental psychiatric disorders. Thus, therapies to promote or accelerate myelination could potentially ameliorate symptoms in autism. Clemastine, a histamine H1 antagonist with anticholinergic properties against muscarinic M1 receptor, is the most promising drug with promyelinating properties. Clemastine penetrates the blood brain barrier efficiently and promotes remyelination in different animal models of neurodegeneration including multiple sclerosis, ischemia and Alzheimer's disease. However, its role in myelination during development is unknown. We showed that clemastine treatment during development increased oligodendrocyte differentiation in both white and gray matter. However, despite the increase in the number of oligodendrocytes, conduction velocity of myelinated fibers of corpus callosum decreased in clemastine treated mice. Confocal and electron microscopy showed a reduction in the number of myelinated axons and nodes of Ranvier and a reduction of myelin thickness in corpus callosum. To understand the mechanisms leading to myelin formation impairment in the presence of an excess of myelinating oligodendrocytes, we focused on microglial cells that also express muscarinic M1 receptors. Importantly, the population of CD11c(+) microglia cells, necessary for myelination, as well as the levels of insulin growth factor-1 decrease in clemastine-treated mice. Altogether, these data suggest that clemastine impact on myelin development is more complex than previously thought and could be dependent on microglia-oligodendrocyte crosstalk. Further studies are needed to clarify the role of microglia cells on developmental myelination.This work was supported by Spanish Ministry of Education and Science (SAF 2016-75292- R); Spanish Ministry of Science andInnovation (PID 2019-109724RB-I00); Basque Government (PI-2016-1-0016); the University of the Basque Country (UPV/EHU);and Centro de Investigacion Biomedica en Red, EnfermedadesNeurodegenerativas (CIBERNED; grant CB06/05/0076). AP hasa predoctoral fellowship from the University of the Basque Country (UPV/EHU), AM has a predoctoral fellowship fromthe Spanish Ministry of Education and Science and AO-de-A hasa postdoctoral fellowship from the Basque Governmen

In vivo multimodal imaging of adenosine A1 receptors in neuroinflammation after experimental stroke

Adenosine A(l) receptors (A(l)ARs) are promising imaging biomarkers and targets for the treatment of stroke. Nevertheless, the role of A(l)ARs on ischemic damage and its subsequent neuroinflammatory response has been scarcely explored so far. Methods: In this study, the expression of A(1)ARs after transient middle cerebral artery occlusion (MCAO) was evaluated by positron emission tomography (PET) with [F-18]CPFPX and immunohistochemistry (IHC). In addition, the role of AIARs on stroke inflammation using pharmacological modulation was assessed with magnetic resonance imaging (MRI), PET imaging with [F-18]DPA-714 (TSPO) and [F-18]FLT (cellular proliferation), as well as IHC and neurofunctional studies. Results: In the ischemic territory, [F-18]CPFPX signal and IHC showed the overexpression of A(l)ARs in microglia and infiltrated leukocytes after cerebral ischemia. Ischemic rats treated with the AAR agonist ENBA showed a significant decrease in both [F-18]DPA-714 and [F-18]FLT signal intensities at day 7 after cerebral ischemia, a feature that was confirmed by IHC results. Besides, the activation of A(l)AR promoted the reduction of the brain lesion, as measured with T2W-MRI, and the improvement of neurological outcome including motor, sensory and reflex responses. These results show for the first time the in vivo PET imaging of A(l)AR expression after cerebral ischemia in rats and the application of [F-18]FLT to evaluate glial proliferation in response to treatment. Conclusion: Notably, these data provide evidence for A(l)AR playing a key role in the control of both the activation of resident glia and the de novo proliferation of microglia and macrophages after experimental stroke in rats.The authors would like to thank A. Leukona and V. Salinas for technical support in the radiosynthesis. This study was funded by grants from the Spanish Ministry of Education and Science/FEDER RYC-201722412, SAF2016-75292-R, SAF2017-87670-R and PID2019-107989RB-I00, the Basque Government (IT1203/19, BIO18/IC/006) and CIBERNED. Maria Ardaya holds a fellowship from the University of Pais Vasco. Ana Joya acknowledges funding from Fundacio La Marato de TV3 (17/C/2017). Juan Jose Gutierrez acknowledges funding from Euskampus Fundazioa. Jordi Llop also acknowledges The Spanish Ministry of Economy and Competitiveness (Grant CTQ2017-87637-R). Part of the work has been performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant No. MDM-2017-0720)