Fractalkine Modulates Microglia Metabolism in Brain Ischemia

In the CNS, the chemokine CX3CL1 (fractalkine) is expressed on neurons while its specific receptor CX3CR1 is expressed on microglia and macrophages. Microglia play an important role in health and disease through CX3CL1/CX3CR1 signaling, and in many neurodegenerative disorders, microglia dysregulation has been associated with neuroinflammation. We have previously shown that CX3CL1 has neuroprotective effects against cerebral ischemia injury. Here, we investigated the involvement of CX3CL1 in the modulation of microglia phenotype and the underlying neuroprotective effect on ischemia injury. The expression profiles of anti- and pro-inflammatory genes showed that CX3CL1 markedly inhibited microglial activation both in vitro and in vivo after permanent middle cerebral artery occlusion (pMCAO), accompanied by an increase in the expression of anti-inflammatory genes. Moreover, CX3CL1 induces a metabolic switch in microglial cells with an increase in the expression of genes related to the oxidative pathway and a reduction in those related to the glycolytic pathway, which is the metabolic state associated to the pro-inflammatory phenotype for energy production. The data reported in this paper suggest that CX3CL1 protects against cerebral ischemia modulating the activation state of microglia and its metabolism in order to restrain inflammation and organize a neuroprotective response against the ischemic insult

KCa3.1 channels are involved in the infiltrative behavior of glioblastoma in vivo.

Glioblastoma multiforme (GBM) is a diffuse brain tumor characterized by high infiltration in the brain parenchyma rendering the tumor difficult to eradicate by neurosurgery. Efforts to identify molecular targets involved in the invasive behavior of GBM suggested ion channel inhibition as a promising therapeutic approach. To determine if the Ca(2+)-dependent K(+) channel KCa3.1 could represent a key element for GBM brain infiltration, human GL-15 cells were xenografted into the brain of SCID mice that were then treated with the specific KCa3.1 blocker TRAM-34 (1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole). After 5 weeks of treatment, immunofluorescence analyses of cerebral slices revealed reduced tumor infiltration and astrogliosis surrounding the tumor, compared with untreated mice. Significant reduction of tumor infiltration was also observed in the brain of mice transplanted with KCa3.1-silenced GL-15 cells, indicating a direct effect of TRAM-34 on GBM-expressed KCa3.1 channels. As KCa3.1 channels are also expressed on microglia, we investigated the effects of TRAM-34 on microglia activation in GL-15 transplanted mice and found a reduction of CD68 staining in treated mice. Similar results were observed in vitro where TRAM-34 reduced both phagocytosis and chemotactic activity of primary microglia exposed to GBM-conditioned medium. Taken together, these results indicate that KCa3.1 activity has an important role in GBM invasiveness in vivo and that its inhibition directly affects glioma cell migration and reduces astrocytosis and microglia activation in response to tumor-released factors. KCa3.1 channel inhibition therefore constitutes a potential novel therapeutic approach to reduce GBM spreading into the surrounding tissue

CX3CL1 Is Neuroprotective in Permanent Focal Cerebral Ischemia in Rodents

The chemokine CX3CL1 and its receptor CX3CR1 are constitutively expressed in the nervous system. In this study, we used in vivo murine models of permanent middle cerebral artery occlusion (pMCAO) to investigate the protective potential of CX3CL1. We report that exogenous CX3CL1 reduced ischemia-induced cerebral infarct size, neurological deficits, and caspase-3 activation. CX3CL1-induced neuroprotective effects were long lasting, being observed up to 50 d after pMCAO in rats. The neuroprotective action of CX3CL1 in different models of brain injuries is mediated by its inhibitory activity on microglia and, in vitro, requires the activation of adenosine receptor 1 (A₁R). We show that, in the presence of the A₁R antagonist 1,3-dipropyl-8-cyclopentylxanthine and in A₁R⁻/⁻ mice, the neuroprotective effect of CX3CL1 on pMCAO was abolished, indicating the critical importance of the adenosine system in CX3CL1 protection also in vivo. In apparent contrast with the above reported data but in agreement with previous findings, cx3cl1⁻/⁻ and cx3cr1(GFP/GFP) mice, respectively, deficient in CX3CL1 or CX3CR1, had less severe brain injury on pMCAO, and the administration of exogenous CX3CL1 increased brain damage in cx3cl1⁻/⁻ ischemic mice. We also report that CX3CL1 induced a different phagocytic activity in wild type and cx3cl1⁻/⁻ microglia in vitro during cotreatment with the medium conditioned by neurons damaged by oxygen-glucose deprivation. Together, these data suggest that acute administration of CX3CL1 reduces ischemic damage via an adenosine-dependent mechanism and that the absence of constitutive CX3CL1-CX3CR1 signaling changes the outcome of microglia-mediated effects during CX3CL1 administration to ischemic brain

Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors

Polymorphic variants of the gene encoding for metabotropic glutamate receptor 3 (mGlu3) are linked to schizophrenia. Because abnormalities of cortical GABAergic interneurons lie at the core of the pathophysiology of schizophrenia, we examined whether mGlu3 receptors influence the developmental trajectory of cortical GABAergic transmission in the postnatal life. mGlu3-/- mice showed robust changes in the expression of interneuron-related genes in the prefrontal cortex (PFC), including large reductions in the expression of parvalbumin (PV) and the GluN1 subunit of NMDA receptors. The number of cortical cells enwrapped by perineuronal nets was increased in mGlu3-/- mice, suggesting that mGlu3 receptors shape the temporal window of plasticity of PV+ interneurons. Electrophysiological measurements of GABAA receptor-mediated responses revealed a more depolarized reversal potential of GABA currents in the somata of PFC pyramidal neurons in mGlu3-/- mice at postnatal d 9 associated with a reduced expression of the K+/Cl- symporter. Finally, adult mGlu3-/- mice showed lower power in electroencephalographic rhythms at 1-45 Hz in quiet wakefulness as compared with their wild-type counterparts. These findings suggest that mGlu3 receptors have a strong impact on the development of cortical GABAergic transmission and cortical neural synchronization mechanisms corroborating the concept that genetic variants of mGlu3 receptors may predispose to psychiatric disorders.-Imbriglio, T., Verhaeghe, R., Martinello, K., Pascarelli, M. T., Chece, G., Bucci, D., Notartomaso, S., Quattromani, M., Mascio, G., Scalabrì, F., Simeone, A., Maccari, S., Del Percio, C., Wieloch, T., Fucile, S., Babiloni, C., Battaglia, G., Limatola, C., Nicoletti, F., Cannella, M. Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors

Microglia modulates hippocampal synaptic transmission and sleep duration along the light/dark cycle

Microglia, the brain's resident macrophages, actively contributes to the homeostasis of cerebral parenchyma by sensing neuronal activity and supporting synaptic remodeling and plasticity. While several studies demonstrated different roles for astrocytes in sleep, the contribution of microglia in the regulation of sleep/wake cycle and in the modulation of synaptic activity in the different day phases has not been deeply investigated. Using light as a zeitgeber cue, we studied the effects of microglial depletion with the colony stimulating factor-1 receptor antagonist PLX5622 on the sleep/wake cycle and on hippocampal synaptic transmission in male mice. Our data demonstrate that almost complete microglial depletion increases the duration of NREM sleep and reduces the hippocampal excitatory neurotransmission. The fractalkine receptor CX3CR1 plays a relevant role in these effects, because cx3cr1GFP/GFP mice recapitulate what found in PLX5622-treated mice. Furthermore, during the light phase, microglia express lower levels of cx3cr1 and a reduction of cx3cr1 expression is also observed when cultured microglial cells are stimulated by ATP, a purinergic molecule released during sleep. Our findings suggest that microglia participate in the regulation of sleep, adapting their cx3cr1 expression in response to the light/dark phase, and modulating synaptic activity in a phase-dependent manner

Adenosine A1 Receptors and Microglial Cells Mediate CX3CL1-Induced Protection of Hippocampal Neurons Against Glu-Induced Death

Fractalkine/CX3CL1 is a neuron-associated chemokine, which modulates microglia-induced neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 interaction modulates the release of cytokines from microglia, reducing the level of tumor necrosis factor-α, interleukin-1-β, and nitric oxide and induces the production of neurotrophic substances, both in vivo and in vitro. We have recently shown that blocking adenosine A1 receptors (A1R) with the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) abolishes CX3CL1-mediated rescue of neuronal excitotoxic death and that CX3CL1 induces the release of adenosine from microglia. In this study, we show that the presence of extracellular adenosine is mandatory for the neurotrophic effect of CX3CL1 as reducing adenosine levels in hippocampal cultures, by adenosine deaminase treatment, strongly impairs CX3CL1-mediated neuroprotection. Furthermore, we confirm the predominant role of microglia in mediating the neuronal effects of CX3CL1, because the selective depletion of microglia from hippocampal cultures treated with clodronate-filled liposomes causes the complete loss of effect of CX3CL1. We also show that hippocampal neurons obtained from A1R−/− mice are not protected by CX3CL1 whereas A2AR−/− neurons are. The requirement of functional A1R for neuroprotection is not unique for CX3CL1 as A1R−/− hippocampal neurons are not rescued from Glu-induced cell death by other neurotrophins such as brain-derived neurotrophic factor and erythropoietin, which are fully active on wt neurons