Glucocorticoids rescue cell surface trafficking of R451C Neuroligin3 and enhance synapse formation.

Neuroligins are synaptic cell adhesion proteins with a role in synaptic function, implicated in neurodevelopmental disorders. The autism spectrum disorder-associated substitution Arg451Cys (R451C) in NLGN3 promotes a partial misfolding of the extracellular domain of the protein leading to retention in the endoplasmic reticulum (ER) and the induction of the unfolded protein response (UPR). The reduced trafficking of R451C NLGN3 to the cell surface leads to altered synaptic function and social behavior. A screening in HEK-293 cells overexpressing NLGN3 of 2662 compounds (FDA-approved small molecule drug library), led to the identification of several glucocorticoids such as alclometasone dipropionate, desonide, prednisolone sodium phosphate, and dexamethasone (DEX), with the ability to favor the exit of full-length R451C NLGN3 from the ER. DEX improved the stability of R451C NLGN3 and trafficking to the cell surface, reduced the activation of the UPR, and increased the formation of artificial synapses between HEK-293 and hippocampal primary neurons. The effect of DEX was validated on a novel model system represented by neural stem progenitor cells and differentiated neurons derived from the R451C NLGN3 knock-in mouse, expressing the endogenous protein. This work shows a potential rescue strategy for an autism-linked mutation affecting cell surface trafficking of a synaptic protein.

Substrate stiffness effect on molecular crosstalk of epithelial-mesenchymal transition mediators of human glioblastoma cells.

The complexity of the microenvironment effects on cell response, show accumulating evidence that glioblastoma (GBM) migration and invasiveness are influenced by the mechanical rigidity of their surroundings. The epithelial-mesenchymal transition (EMT) is a well-recognized driving force of the invasive behavior of cancer. However, the primary mechanisms of EMT initiation and progression remain unclear. We have previously showed that certain substrate stiffness can selectively stimulate human GBM U251-MG and GL15 glioblastoma cell lines motility. The present study unifies several known EMT mediators to uncover the reason of the regulation and response to these stiffnesses. Our results revealed that changing the rigidity of the mechanical environment tuned the response of both cell lines through change in morphological features, epithelial-mesenchymal markers (E-, N-Cadherin), EGFR and ROS expressions in an interrelated manner. Specifically, a stiffer microenvironment induced a mesenchymal cell shape, a more fragmented morphology, higher intracellular cytosolic ROS expression and lower mitochondrial ROS. Finally, we observed that cells more motile showed a more depolarized mitochondrial membrane potential. Unravelling the process that regulates GBM cells' infiltrative behavior could provide new opportunities for identification of new targets and less invasive approaches for treatment.

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

Microglia, the brain's resident macrophages, actively contribute 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.

Neuro-Signals from Gut Microbiota: Perspectives for Brain Glioma.

Glioblastoma (GBM) is the most aggressive form of glioma tumor in adult brain. Among the numerous factors responsible for GBM cell proliferation and invasion, neurotransmitters such as dopamine, serotonin and glutamate can play key roles. Studies performed in mice housed in germ-free (GF) conditions demonstrated the relevance of the gut-brain axis in a number of physiological and pathological conditions. The gut-brain communication is made possible by vagal/nervous and blood/lymphatic routes and pave the way for reciprocal modulation of functions. The gut microbiota produces and consumes a wide range of molecules, including neurotransmitters (dopamine, norepinephrine, serotonin, gamma-aminobutyric acid [GABA], and glutamate) that reach their cellular targets through the bloodstream. Growing evidence in animals suggests that modulation of these neurotransmitters by the microbiota impacts host neurophysiology and behavior, and affects neural cell progenitors and glial cells, along with having effects on tumor cell growth. In this review we propose a new perspective connecting neurotransmitter modulation by gut microbiota to glioma progression.

Cytosolic serine hydroxymethyltransferase controls lung adenocarcinoma cells migratory ability by modulating AMP kinase activity.

Nutrient utilization and reshaping of metabolism in cancer cells is a well-known driver of malignant transformation. Less clear is the influence of the local microenvironment on metastasis formation and choice of the final organ to invade. Here we show that the level of the amino acid serine in the cytosol affects the migratory properties of lung adenocarcinoma (LUAD) cells. Inhibition of serine or glycine uptake from the extracellular milieu, as well as knockdown of the cytosolic one-carbon metabolism enzyme serine hydroxymethyltransferase (SHMT1), abolishes migration. Using rescue experiments with a brain extracellular extract, and direct measurements, we demonstrate that cytosolic serine starvation controls cell movement by increasing reactive oxygen species formation and decreasing ATP levels, thereby promoting activation of the AMP sensor kinase (AMPK) by phosphorylation. Activation of AMPK induces remodeling of the cytoskeleton and finally controls cell motility. These results highlight that cytosolic serine metabolism plays a key role in controlling motility, suggesting that cells are able to dynamically exploit the compartmentalization of this metabolism to adapt their metabolic needs to different cell functions (movement vs. proliferation). We propose a model to explain the relevance of serine/glycine metabolism in the preferential colonization of the brain by LUAD cells and suggest that the inhibition of serine/glycine uptake and/or cytosolic SHMT1 might represent a successful strategy to limit the formation of brain metastasis from primary tumors, a major cause of death in these patients.

Metabolic Reprograming of Microglia in the Regulation of the Innate Inflammatory Response.

Microglia sustain normal brain functions continuously monitoring cerebral parenchyma to detect neuronal activities and alteration of homeostatic processes. The metabolic pathways involved in microglia activity adapt at and contribute to cell phenotypes. While the mitochondrial oxidative phosphorylation is highly efficient in ATP production, glycolysis enables microglia with a faster rate of ATP production, with the generation of intermediates for cell growth and cytokine production. In macrophages, pro-inflammatory stimuli induce a metabolic switch from oxidative phosphorylation to glycolysis, a phenomenon similar to the Warburg effect well characterized in tumor cells. Modification of metabolic functions allows macrophages to properly respond to a changing environment and many evidence suggest that, similarly to macrophages, microglial cells are capable of a plastic use of energy substrates. Neuroinflammation is a common condition in many neurodegenerative diseases and the metabolic reprograming of microglia has been reported in neurodegeneration. Here we review the existing data on microglia metabolism and the connections with neuroinflammatory diseases, highlighting how metabolic changes contribute to module the homeostatic functions of microglia.

Gut microbiota alterations affect glioma growth and innate immune cells involved in tumor immunosurveillance in mice.

Glioma is a CNS tumor with few therapeutic options. Recently, host microbiota has been involved in the immune modulation of different tumors, but no data are available on the possible effects of the gut-immune axis on brain tumors. Here, we investigated the effect of gut microbiota alteration in a syngeneic (GL261) mouse model of glioma, treating mice with two antibiotics (ABX) and evaluating the effects on tumor growth, microbe composition, natural killer (NK) cells and microglia phenotype. We report that ABX treatment (i) altered the intestinal microbiota at family level, (ii) reduced cytotoxic NK cell subsets, and (iii) altered the expression of inflammatory and homeostatic proteins in microglia. All these findings could contribute to the increased growth of intracranial glioma that was observed after ABX treatment. These results demonstrate that chronic ABX administration alters microbiota composition and contributes to modulate brain immune state paving the way to glioma growth.

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 neuro-inflammation. 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.

1H-NMR metabolomics reveals the Glabrescione B exacerbation of glycolytic metabolism beside the cell growth inhibitory effect in glioma.

BACKGROUND: Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway. METHODS: We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used 1H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG5kDa-Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice. RESULTS: We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted 1H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition. CONCLUSIONS: Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor.

Kv1.3 activity perturbs the homeostatic properties of astrocytes in glioma.

Glial cells actively maintain the homeostasis of brain parenchyma, regulating neuronal excitability and preserving the physiological composition of the extracellular milieu. Under pathological conditions, some functions of glial cells could be compromised, exacerbating the neurotoxic processes. We investigated if the homeostatic activities of astrocytes and microglia could be modulated by the voltage-gated K+ channel Kv1.3. To this end we used in vitro and in vivo systems to model cell-to-cell interactions in tumoral conditions, using a specific inhibitor of Kv1.3 channels, 5-(4-phenoxybutoxy) psoralen (PAP-1). We demonstrated that PAP-1 increases astrocytic glutamate uptake, reduces glioma-induced neurotoxicity, and decreases microglial migration and phagocytosis. We also found in a tumor blood brain barrier model that Kv1.3 activity is required for its integrity. The crucial role of Kv1.3 channels as modulators of glial cell activity was confirmed in a mouse model of glioma, where PAP-1 treatment reduces tumor volume only in the presence of active glutamate transporters GLT-1. In the same mouse model, PAP-1 reduces astrogliosis and microglial infiltration. PAP-1 also reduces tumor cell invasion. All these findings point to Kv1.3 channels as potential targets to re-instruct glial cells toward their homeostatic functions, in the context of brain tumors.

Fractalkine receptor deficiency impairs microglial and neuronal responsiveness to chronic stress.

Chronic stress is one of the most relevant triggering factors for major depression. Microglial cells are highly sensitive to stress and, more generally, to environmental challenges. However, the role of these brain immune cells in mediating the effects of stress is still unclear. Fractalkine signaling - which comprises the chemokine CX3CL1, mainly expressed by neurons, and its receptor CX3CR1, almost exclusively present on microglia in the healthy brain - has been reported to critically regulate microglial activity. Here, we investigated whether interfering with microglial function by deleting the Cx3cr1 gene affects the brain's response to chronic stress. To this purpose, we housed Cx3cr1 knockout and wild-type adult mice in either control or stressful environments for 2weeks, and investigated the consequences on microglial phenotype and interactions with synapses, synaptic transmission, behavioral response and corticosterone levels. Our results show that hampering neuron-microglia communication via the CX3CR1-CX3CL1 pathway prevents the effects of chronic unpredictable stress on microglial function, short- and long-term neuronal plasticity and depressive-like behavior. Overall, the present findings suggest that microglia-regulated mechanisms may underlie the differential susceptibility to stress and consequently the vulnerability to diseases triggered by the experience of stressful events, such as major depression.

Fractalkine in the nervous system: neuroprotective or neurotoxic molecule?

Fractalkine (CX3CL1) is an intriguing chemokine that plays a central role in the nervous system. The expression of CX3CL1 on neurons and of its receptor CX3CR1 on microglia facilitates a privileged interaction, playing important roles in regulating the function and maturation of these cells. CX3CL1 is reported to have neuroprotective and anti-inflammatory activities in several experimental systems and animal models of disease, and its expression correlates with positive outcomes in human neuropathologies. However, a comparable amount of evidence shows that CX3CL1 sustains neuroinflammatory conditions and contributes to neurotoxicity. This review discusses the evidence in favor of the CX3CL1/CX3CR1 pair being neuroprotective and other evidence that it is neurotoxic. Our aim is to stimulate future research examining the molecular and cellular determinants responsible for this unique functional switch, which could be important for several neuropathologies.

Enriched environment reduces glioma growth through immune and non-immune mechanisms in mice.

Mice exposed to standard (SE) or enriched environment (EE) were transplanted with murine or human glioma cells and differences in tumour development were evaluated. We report that EE exposure affects: (i) tumour size, increasing mice survival; (ii) glioma establishment, proliferation and invasion; (iii) microglia/macrophage (M/Mφ) activation; (iv) natural killer (NK) cell infiltration and activation; and (v) cerebral levels of IL-15 and BDNF. Direct infusion of IL-15 or BDNF in the brain of mice transplanted with glioma significantly reduces tumour growth. We demonstrate that brain infusion of IL-15 increases the frequency of NK cell infiltrating the tumour and that NK cell depletion reduces the efficacy of EE and IL-15 on tumour size and of EE on mice survival. BDNF infusion reduces M/Mφ infiltration and CD68 immunoreactivity in tumour mass and reduces glioma migration inhibiting the small G protein RhoA through the truncated TrkB.T1 receptor. These results suggest alternative approaches for glioma treatment.

Partial block by riluzole of muscle sodium channels in myotubes from amyotrophic lateral sclerosis patients.

Denervated muscles undergo fibrillations due to spontaneous activation of voltage-gated sodium (Na(+)) channels generating action potentials. Fibrillations also occur in patients with amyotrophic lateral sclerosis (ALS). Riluzole, the only approved drug for ALS treatment, blocks voltage-gated Na(+) channels, but its effects on muscle Na(+) channels and fibrillations are yet poorly characterized. Using patch-clamp technique, we studied riluzole effect on Na(+) channels in cultured myotubes from ALS patients. Needle electromyography was used to study fibrillation potentials (Fibs) in ALS patients during riluzole treatment and after one week of suspension. Patients were clinically characterized in all recording sessions. In myotubes, riluzole (1 µM, a therapeutic concentration) reduced Na(+) current by 20%. The rate of rise and amplitude of spikes evoked by depolarizing stimuli were also reduced. Fibs were detected in all patients tested during riluzole treatment and riluzole washout had no univocal effect. Our study indicates that, in human myotubes, riluzole partially blocks Na(+) currents and affects action potentials but does not prevent firing. In line with this in vitro finding, muscle Fibs in ALS patients appear to be largely unaffected by riluzole.

Trasmembrane chemokines CX3CL1 and CXCL16 drive interplay between neurons, microglia and astrocytes to counteract pMCAO and excitotoxic neuronal death.

Upon noxious insults, cells of the brain parenchyma activate endogenous self-protective mechanisms to counteract brain damage. Interplay between microglia and astrocytes can be determinant to build a physiological response to noxious stimuli arisen from injury or stress, thus understanding the cross talk between microglia and astrocytes would be helpful to elucidate the role of glial cells in endogenous protective mechanisms and might contribute to the development of new strategy to mobilize such program and reduce brain cell death. Here we demonstrate that chemokines CX3CL1 and CXCL16 are molecular players that synergistically drive cross-talk between neurons, microglia and astrocytes to promote physiological neuroprotective mechanisms that counteract neuronal cell death due to ischemic and excitotoxic insults. In an in vivo model of permanent middle cerebral artery occlusion (pMCAO) we found that exogenous administration of soluble CXCL16 reduces ischemic volume and that, upon pMCAO, endogenous CXCL16 signaling restrains brain damage, being ischemic volume reduced in mice that lack CXCL16 receptor. We demonstrated that CX3CL1, acting on microglia, elicits CXCL16 release from glia and this is important to induce neroprotection since lack of CXCL16 signaling impairs CX3CL1 neuroprotection against both in vitro Glu-excitotoxic insult and pMCAO. Moreover the activity of adenosine receptor A3R and the astrocytic release of CCL2 play also a role in trasmembrane chemokine neuroprotective effect, since their inactivation reduces CX3CL1- and CXCL16 induced neuroprotection.

Fractalkine/CX3CL1 engages different neuroprotective responses upon selective glutamate receptor overactivation.

Neuronal death induced by overactivation of N-methyl-d-aspartate receptors (NMDARs) is implicated in the pathophysiology of many neurodegenerative diseases such as stroke, epilepsy and traumatic brain injury. This toxic effect is mainly mediated by NR2B-containing extrasynaptic NMDARs, while NR2A-containing synaptic NMDARs contribute to cell survival, suggesting the possibility of therapeutic approaches targeting specific receptor subunits. We report that fractalkine/CX3CL1 protects hippocampal neurons from NMDA-induced cell death with a mechanism requiring the adenosine receptors type 2A (A2AR). This is different from CX3CL1-induced protection from glutamate (Glu)-induced cell death, that fully depends on A1R and requires in part A3R. We show that CX3CL1 neuroprotection against NMDA excitotoxicity involves D-serine, a co-agonist of NR2A/NMDAR, resulting in cyclic AMP-dependent transcription factor cyclic-AMP response element-binding protein (CREB) phosphorylation.

Fractalkine/CX3CL1 modulates GABAA currents in human temporal lobe epilepsy.

PURPOSE: The chemokine fractalkine/CX3CL1 and its receptor CX3CR1 are widely expressed in the central nervous system (CNS). Recent evidence showed that CX3CL1 participates in inflammatory responses that are common features of CNS disorders, such as epilepsy. Mesial temporal lobe epilepsy (MTLE) is the prevalent form of focal epilepsy in adults, and hippocampal sclerosis (HS) represents the most common underlying pathologic abnormality, as demonstrated at autopsy and postresection studies. Relevant features of MTLE are a characteristic pattern of neuronal loss, as are astrogliosis and microglia activation. Several factors affect epileptogenesis in patients with MTLE, including a lack of γ-aminobutyric acid (GABA)ergic inhibitory efficacy. Therefore, experiments were designed to investigate whether, in MTLE brain tissues, CX3CL1 may influence GABAA receptor (GABAA R) mediated transmission, with a particular focus on the action of CX3CL1 on the use-dependent decrease (rundown) of the GABA-evoked currents (IGABA ), a feature underlying the reduction of GABAergic function in epileptic tissue. METHODS: Patch-clamp recordings were obtained from cortical pyramidal neurons in slices from six MTLE patients after surgery. Alternatively, the cell membranes from epileptic brain tissues of 17 MTLE patients or from surgical samples and autopsies of nonepileptic patients were microtransplanted into Xenopus oocytes, and IGABA were recorded using the standard two-microelectrode voltage-clamp technique. Immunohistochemical staining and double-labeling studies were carried out on the same brain tissues to analyze CX3CR1 expression. KEY FINDINGS: In native pyramidal neurons from cortical slices of patients with MTLE, CX3CL1 reduced IGABA rundown and affected the recovery of IGABA amplitude from rundown. These same effects were confirmed in oocytes injected with cortical and hippocampal MTLE membranes, whereas CX3CL1 did not influence IGABA in oocytes injected with nonepileptic tissues. Consistent with a specific effect of CX3CL1 on tissues from patients with MTLE, CX3CR1 immunoreactivity was higher in MTLE sclerotic hippocampi than in control tissues, with a prominent expression in activated microglial cells. SIGNIFICANCE: These findings indicate a role for CX3CL1 in MTLE, supporting recent evidence on the relevance of brain inflammation in human epilepsies. Our data demonstrate that in MTLE tissues the reduced GABAergic function can be modulated by CX3CL1. The increased CX3CR1 expression in microglia and the modulation by CX3CL1 of GABAergic currents in human epileptic brain suggests new therapeutic approaches for drug-resistant epilepsies based on the evidence that the propagation of seizures can be influenced by inflammatory processes.

CX3CL1 protects neurons against excitotoxicity enhancing GLT-1 activity on astrocytes.

In this paper we show for the first time that: i) astrocytes are required for the neuroprotective activity of CX3CL1 against excitotoxicity; ii) inhibition of the glutamate transporter 1 (GLT-1) prejudices CX3CL1-mediated neuroprotection; iii) CX3CL1 increases GLT-1 activity on astrocytes. The modulation of GLT-1 activity induced by CX3CL1 on astrocytes requires the presence and the activity of A1 adenosine receptor (A1R), being blocked by the specific antagonist DPCPX and absent in A1R(-/-) astrocytes. These data introduce the astrocytes as active players in CX3CL1-mediated signaling between microglia and neurons, identifying GLT-1 as a key mediator of the neuroprotective activity of CX3CL1.

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.