Fractalkine Signaling Research Articles

CNS-Wide over Expression of Fractalkine Improves Cognitive Functioning in a Tauopathy Model

Accumulating evidence increasingly implicates regulation of neuroinflammation as a potential therapeutic target in Alzheimer’s disease and other neurodegenerative disorders. Fractalkine (FKN) is a unique chemokine that is expressed and secreted by neurons and reduces expression of pro-inflammatory genes. To further demonstrate the utility of agents that increase FKN signaling throughout the central nervous system as possible therapies for AD, we assessed the impact of soluble FKN (sFKN) over expression on cognition in tau depositing rTg450 mice after the onset of cognitive deficits. Using adeno-associated virus serotype 4, we infected cells lining the ventricular system with soluble FKN to increase FKN signaling over a larger fraction of the brain than achieved with intraparenchymal injections. We found that soluble FKN over expression by cells lining the ventricles significantly improved cognitive performance on the novel mouse recognition and radial arm water maze tasks. These benefits were achieved without detectable reductions in tau hyperphosphorylation, hippocampal atrophy, or microglial CD45 expression. Utilizing qPCR, we report a significant increase in Vegfa expression, indicating an increase in trophic support and possible neovascularization in AAV-sFKN-injected mice. To our knowledge, this is the first demonstration that FKN over expression can rescue cognitive function in a tau depositing mouse line.Graphical Regulating neuroinflammation is an attractive therapeutic target for Alzheimer’s disease. Microglial activation can not only drive pathology but also accelerate cognitive decline. The chemokine fractalkine regulates the microglial phenotype, increasing trophic support of neurons, and significantly improving cognitive functioning in the rTg4510 mouse model of tauopathy.

Bidirectional Microglia-Neuron Communication in Health and Disease.

Microglia are ramified cells that exhibit highly motile processes, which continuously survey the brain parenchyma and react to any insult to the CNS homeostasis. Although microglia have long been recognized as a crucial player in generating and maintaining inflammatory responses in the CNS, now it has become clear, that their function are much more diverse, particularly in the healthy brain. The innate immune response and phagocytosis represent only a little segment of microglia functional repertoire that also includes maintenance of biochemical homeostasis, neuronal circuit maturation during development and experience-dependent remodeling of neuronal circuits in the adult brain. Being equipped by numerous receptors and cell surface molecules microglia can perform bidirectional interactions with other cell types in the CNS. There is accumulating evidence showing that neurons inform microglia about their status and thus are capable of controlling microglial activation and motility while microglia also modulate neuronal activities. This review addresses the topic: how microglia communicate with other cell types in the brain, including fractalkine signaling, secreted soluble factors and extracellular vesicles. We summarize the current state of knowledge of physiological role and function of microglia during brain development and in the mature brain and further highlight microglial contribution to brain pathologies such as Alzheimer’s and Parkinson’s disease, brain ischemia, traumatic brain injury, brain tumor as well as neuropsychiatric diseases (depression, bipolar disorder, and schizophrenia).

Genetic disruption of fractalkine signaling leads to enhanced loss of cochlear afferents following ototoxic or acoustic injury.

Cochlear hair cells are vulnerable to a variety of insults like acoustic trauma and ototoxic drugs. Such injury can also lead to degeneration of spiral ganglion neurons (SGNs), but this occurs over a period of months to years. Neuronal survival is necessary for the proper function of cochlear prosthetics, therefore, it is of great interest to understand the mechanisms that regulate neuronal survival in deaf ears. We have recently demonstrated that selective hair cell ablation is sufficient to attract leukocytes into the spiral ganglion, and that fractalkine signaling plays a role in macrophage recruitment and in the survival of auditory neurons. Fractalkine (CX3 CL1), a chemokine that regulates adhesion and migration of leukocytes is expressed by SGNs and signals to leukocytes via its receptor CX3 CR1. The present study has extended the previous findings to more clinically relevant conditions of sensorineural hearing loss by examining the role of fractalkine signaling after aminoglycoside ototoxicity or acoustic trauma. Both aminoglycoside treatment and acoustic overstimulation led to the loss of hair cells as well as prolonged increase in the numbers of cochlear leukocytes. Lack of CX3 CR1 did not affect macrophage recruitment after injury, but resulted in increased loss of SGNs and enhanced expression of the inflammatory cytokine interleukin-1β, when compared to mice with intact CX3 CR1. These data indicate that the dysregulation of macrophage response caused by the absence of CX3 CR1 may contribute to inflammation-mediated neuronal loss in the deafened ear, suggesting a key role for inflammation in the long-term survival of target-deprived afferent neurons.

Experience-Dependent Synaptic Plasticity in V1 Occurs without Microglial CX3CR1.

Brief monocular deprivation (MD) shifts ocular dominance and reduces the density of thalamic synapses in layer 4 of the mouse primary visual cortex (V1). We found that microglial lysosome content is also increased as a result of MD. Previous studies have shown that the microglial fractalkine receptor CX3CR1 is involved in synaptic development and hippocampal plasticity. We therefore tested the hypothesis that neuron-to-microglial communication via CX3CR1 is an essential component of visual cortical development and plasticity in male mice. Our data show that CX3CR1 is not required for normal development of V1 responses to visual stimulation, multiple forms of experience-dependent plasticity, or the synapse loss that accompanies MD in layer 4. By ruling out an essential role for fractalkine signaling, our study narrows the search for understanding how microglia respond to active synapse modification in the visual cortex.SIGNIFICANCE STATEMENT Microglia in the visual cortex respond to monocular deprivation with increased lysosome content, but signaling through the fractalkine receptor CX3CR1 is not an essential component in the mechanisms of visual cortical development or experience-dependent synaptic plasticity.

The microglial fractalkine receptor is not required for activity-dependent plasticity in the mouse visual system.

Microglia have recently been implicated as key regulators of activity-dependent plasticity, where they contribute to the removal of inappropriate or excess synapses. However, the molecular mechanisms that mediate this microglial function are still not well understood. Although multiple studies have implicated fractalkine signaling as a mediator of microglia-neuron communications during synaptic plasticity, it is unclear whether this is a universal signaling mechanism or whether its role is limited to specific brain regions and stages of the lifespan. Here, we examined whether fractalkine signaling mediates microglial contributions to activity-dependent plasticity in the developing and adolescent visual system. Using genetic ablation of fractalkine's cognate receptor, CX3 CR1, and both ex vivo characterization and in vivo imaging in mice, we examined whether fractalkine signaling is required for microglial dynamics and modulation of synapses, as well as activity-dependent plasticity in the visual system. We did not find a role for fractalkine signaling in mediating microglial properties during visual plasticity. Ablation of CX3 CR1 had no effect on microglial density, distribution, morphology, or motility, in either adolescent or young adult mice across brain regions that include the visual cortex. Ablation of CX3 CR1 also had no effect on baseline synaptic turnover or contact dynamics between microglia and neurons. Finally, we found that fractalkine signaling is not required for either early or late forms of activity-dependent visual system plasticity. These findings suggest that fractalkine is not a universal regulator of synaptic plasticity, but rather has heterogeneous roles in specific brain regions and life stages.

The chemokine fractalkine (CX3CL1) attenuates H2O2-induced demyelination in cerebellar slices

BackgroundFractalkine/CX3CR1 signalling has been implicated in many neurodegenerative and neurological diseases of the central nervous system (CNS). This signalling pathway plays an important role in regulating reactive oxygen species (ROS), as well as itself being altered in conditions of oxidative stress. Here, we investigated the effects of recombinant fractalkine (rCX3CL1) in models of hydrogen peroxide (H2O2)-induced demyelination and astrocyte toxicity, within organotypic cerebellar slice cultures.MethodsOrganotypic cerebellar slice cultures were generated from postnatal day 10 C57BL/6J mice to assess myelination. Immunohistochemistry was used to measure the degree of myelination. Fluorescent images were obtained using a leica SP8 confocal microscope and data analysed using ImageJ software.ResultsWe show here, for the first time, that rCX3CL1 significantly attenuated bolus H2O2-induced demyelination as measured by expression of myelin basic protein (MBP) and attenuated reduced vimentin expression. Using the GOX-CAT system to continuously generate low levels of H2O2 and induce demyelination, we observed similar protective effects of rCX3CL1 on MBP and MOG fluorescence, although in this model, the decrease in vimentin expression was not altered.ConclusionsThis data indicates possible protective effects of fractalkine signalling in oxidative stress-induced demyelination in the central nervous system. This opens up the possibility of fractalkine receptor (CX3CR1) modulation as a potential new target for protecting against oxidative stress-induced demyelination in both inflammatory and non-inflammatory nervous system disorders.

Impaired microglia fractalkine signaling affects stress reaction and coping style in mice

Microglia, resident immune cells of the CNS are sensitive to various perturbations of the environment, such as stress exposure, and may be involved in translating these changes to behavior. Among the pathways mediating stress-related neuronal cues to microglia, the fractalkine-fractalkine receptor (CX3CR1) signaling plays a crucial role. Using mice, in which the CX3CR1 gene was deleted, we explored hormonal and behavioral responses to acute and chronic stress along with changes in hypothalamic microglia. CX3CR1−/− animals display active escape in forced swim- and tail suspension tests, exaggerated neuronal activation in the hypothalamic paraventricular nucleus and increased corticosterone release in response to restraint. Analysis of Iba1 immunostaining of hypothalamic sections revealed stress-related reduction of microglia in CX3CR1−/− mice. Because microglia also contribute to energy balance regulation, we characterized metabolic phenotype of CX3CR1−/− mice. Comparison of respiratory exchange ratio did not show genotype effect on fuel preference, however, the energy expenditure was increased in CX3CR1−/− mice, which may be related to their active coping behavior.Microglia and fractalkine signaling has been repeatedly shown to be involved chronic stress-induced depressive state. CX3CR1−/− mice did not become anhedonic in the “two hit” chronic stress paradigm, confirming resistance of these animals to chronic stress-induced mood alterations. However, there was no difference in stress hormone levels, open field performance and hypothalamic microglia distribution between the genotypes. These results highlight differential involvement of microglia fractalkine signaling in controlling/integrating hormonal-, metabolic and behavioral responses to acute and chronic stress challenges.

Fractalkine Signaling Attenuates Perivascular Clustering of Microglia and Fibrinogen Leakage during Systemic Inflammation in Mouse Models of Diabetic Retinopathy.

Fractalkine (FKN) is a chemokine expressed constitutively by healthy neurons and signals to microglia upon interaction with the FKN receptor, CX3CR1. Signaling between FKN and CX3CR1 transduces inhibitory signals that ameliorate microglial activation and proinflammatory cytokine release in neuroinflammatory conditions. The aim of this study is to determine the mechanisms associated with microglial activation and vascular leakage during diabetic retinopathy (DR) and under conditions of low-level endotoxemia, common in diabetic patients. Utilizing the Ins2Akita strain (Akita), a mouse model of type 1 diabetes, our results show that leakage of the blood-protein fibrin(ogen) into the retina occurs as a result of chronic (4 months) but not acute (1.5 months) hyperglycemia. Conversely, inducing endotoxin-mediated systemic inflammation during acute diabetes resulted in fibrinogen deposition in the retina, a phenotype that was exacerbated in mice lacking CX3CR1 signaling. Systemic inflammation in Cx3cr1−/− mice led to robust perivascular clustering of proliferating microglia in areas of fibrinogen extravasation, and induced IL-1β expression in microglia and astrocytes. Lastly, we determined a protective effect of modulating FKN/CX3CR1 signaling in the diabetic retina. We show that intravitreal (iv) administration of recombinant FKN into diabetic FKN-KO mice, reduced fibrinogen deposition and perivascular clustering of microglia in the retina during systemic inflammation. These data suggest that dysregulated microglial activation via loss of FKN/CX3CR1 signaling disrupts the vascular integrity in retina during systemic inflammation.

Spermatozoal Fractalkine Signaling Pathway Is Upregulated in Subclinical Varicocele Patients with Normal Seminogram and Low-Level Leucospermia.

Background Fractalkine is produced in seminal plasma in small amounts and correlates with sperm motility. Purpose To investigate the possible effect of low-level leucospermia on spermatozoa oxidative stress and sDNA fragmentation in patients with subclinical varicocele and apparently normal seminogram, and also to study the role of spermatozoal fractalkine and its receptor (CX3CR1) gene expression as a marker of spermatozoa inflammatory response. Methods This study included 80 patients with subclinical varicocele (45 fertile and 35 infertile) and 45 age-matched fertile volunteers. In semen samples, fractalkine and CX3CR1 gene expression were investigated by qRT-PCR. Moreover, seminal plasma malondialdehyde (MDA) and total antioxidant capacity (TAC) were measured. Results There are significant decrease in semen quality and significant increase in seminal leucocytes count in subclinical varicocele. Our results show a significant increase in MDA and TAC levels, DNA fragmentation, and expression levels of fractalkine and its receptor (CX3CR1) in subclinical varicocele groups. Conclusion Subclinical varicocele induces seminal and spermatozoal subclinical inflammatory response in the form of low-level leucospermia and increased mRNA expression of the fractalkine signaling pathway, leading to increased spermatozoal ROS production, oxidative stress, and DNA fragmentation. These could cooperate in the pathogenesis of delayed fertility in males with subclinical varicocele.

Loss of Fractalkine Signaling Exacerbates Axon Transport Dysfunction in a Chronic Model of Glaucoma.

Neurodegeneration in glaucoma results in decline and loss of retinal ganglion cells (RGCs), and is associated with activation of myeloid cells such as microglia and macrophages. The chemokine fractalkine (FKN or Cx3cl1) mediates communication from neurons to myeloid cells. Signaling through its receptor Cx3cr1 has been implicated in multiple neurodegenerative diseases, but the effects on neuronal pathology are variable. Since it is unknown how FKN-mediated crosstalk influences RGC degeneration in glaucoma, we assessed this in a chronic mouse model, DBA/2J. We analyzed a DBA/2J substrain deficient in Cx3cr1, and compared compartmentalized RGC degeneration and myeloid cell responses to those in standard DBA/2J mice. We found that loss of FKN signaling exacerbates axon transport dysfunction, an early event in neurodegeneration, with a significant increase in RGCs with somal accumulation of the axonal protein phosphorylated neurofilament, and reduced retinal expression of genes involved in axon transport, Kif1b, and Atp8a2. There was no change in the loss of Brn3-positive RGCs, and no difference in the extent of damage to the proximal optic nerve, suggesting that the loss of fractalkine signaling primarily affects axon transport. Since Cx3cr1 is specifically expressed in myeloid cells, we assessed changes in retinal microglial number and activation, changes in gene expression, and the extent of macrophage infiltration. We found that loss of fractalkine signaling led to innate immune changes within the retina, including increased infiltration of peripheral macrophages and upregulated nitric oxide synthase-2 (Nos-2) expression in myeloid cells, which contributes to the production of NO and can promote axon transport deficits. In contrast, resident retinal microglia appeared unchanged either in number, morphology, or expression of the myeloid activation marker ionized calcium binding adaptor molecule 1 (Iba1). There was also no significant increase in the proinflammatory gene interleukin 1 beta (Il1β). We conclude that loss of fractalkine signaling causes a selective worsening of axon transport dysfunction in RGCs, which is linked to enhanced Nos-2 expression in myeloid cells. Our findings suggest that distinct mechanisms may contribute to different aspects of RGC decline in glaucoma, with axonal transport selectively altered after loss of Cx3cr1 in microglia and/or macrophages.

Regulation of Physical Microglia-Neuron Interactions by Fractalkine Signaling after Status Epilepticus.

Microglia, the resident immune cells of the brain, perform elaborate surveillance in which they physically interact with neuronal elements. A novel form of microglia–neuron interaction named microglial process convergence (MPC) toward neuronal axons and dendrites has recently been described. However, the molecular regulators and pathological relevance of MPC have not been explored. Here, using high-resolution two-photon imaging in vivo and ex vivo, we observed a dramatic increase in MPCs after kainic acid– or pilocarpine-induced experimental seizures that was reconstituted after glutamate treatment in slices from mice. Interestingly, a deficiency of the fractalkine receptor (CX3CR1) decreased MPCs, whereas fractalkine (CX3CL1) treatment increased MPCs, suggesting that fractalkine signaling is a critical regulator of these microglia–neuron interactions. Furthermore, we found that interleukin-1β was necessary and sufficient to trigger CX3CR1-dependent MPCs. Finally, we show that a deficiency in fractalkine signaling corresponds with increased seizure phenotypes. Together, our results identify the neuroglial CX3CL1–CX3CR1 communication axis as a modulator of potentially neuroprotective microglia–neuron physical interactions during conditions of neuronal hyperactivity.

Brown adipose tissue in obesity: Fractalkine-receptor dependent immune cell recruitment affects metabolic-related gene expression

Brown adipose tissue (BAT) plays essential role in metabolic- and thermoregulation and displays morphological and functional plasticity in response to environmental and metabolic challenges. BAT is a heterogeneous tissue containing adipocytes and various immune-related cells, however, their interaction in regulation of BAT function is not fully elucidated. Fractalkine is a chemokine synthesized by adipocytes, which recruits fractalkine receptor (CX3CR1)-expressing leukocytes into the adipose tissue. Using transgenic mice, in which the fractalkine receptor, Cx3cr1 gene was replaced by Gfp, we evaluated whether deficiency in fractalkine signaling affects BAT remodeling and function in high-fat-diet - induced obesity. Homo- and heterozygote male CX3CR1-GFP mice were fed with normal or fat enriched (FatED) diet for 10weeks. Interscapular BAT was collected for molecular biological analysis. Heterozygous animals in which fractalkine signaling remains intact, gain more weight during FatED than CX3CR1 deficient gfp/gfp homozygotes. FatED in controls resulted in macrophage recruitment to the BAT with increased expression of proinflammatory mediators (Il1a, b, Tnfa and Ccl2). Local BAT inflammation was accompanied by increased expression of lipogenic enzymes and resulted in BAT “whitening”. By contrast, fractalkine receptor deficiency prevented accumulation of tissue macrophages, selectively attenuated the expression of Tnfa, Il1a and Ccl2, increased BAT expression of lipolytic enzymes (Atgl, Hsl and Mgtl) and upregulated genes involved thermo-metabolism (Ucp1, Pparg Pgc1a) in response to FatED. These results highlight the importance of fractalkine-CX3CR1 interaction in recruitment of macrophages into the BAT of obese mice which might contribute to local tissue inflammation, adipose tissue remodeling and regulation of metabolic-related genes.

Spinal Microgliosis Due to Resident Microglial Proliferation Is Required for Pain Hypersensitivity after Peripheral Nerve Injury

Peripheral nerve injury causes neuropathic pain accompanied by remarkable microgliosis in the spinal cord dorsal horn. However, it is still debated whether infiltrated monocytes contribute to injury-induced expansion of the microglial population. Here, we found that spinal microgliosis predominantly results from local proliferation of resident microglia but not from infiltrating monocytes after spinal nerve transection (SNT) by using two geneticmouse models (CCR2(RFP/+):CX3CR1(GFP/+) and CX3CR1(creER/+):R26(tdTomato/+) mice) as well as specific staining of microglia and macrophages. Pharmacological inhibition of SNT-induced microglial proliferation correlated with attenuated neuropathic pain hypersensitivities. Microglial proliferation is partially controlled by purinergic and fractalkine signaling, as CX3CR1(-/-) and P2Y12(-/-) mice show reduced spinal microglial proliferation and neuropathic pain. These results suggest that local microglial proliferation is the sole source of spinal microgliosis, which represents a potential therapeutic target for neuropathic pain management.

Elevated Fractalkine (CX3CL1) Levels in the Trigeminal Ganglion Mechanically Sensitize Temporalis Muscle Nociceptors.

It has been proposed that after nerve injury or tissue inflammation, fractalkine (CX3CL1) released from dorsal root ganglion neurons acts on satellite glial cells (SGCs) through CX3C receptor 1 (CX3CR1) to induce neuroplastic changes. The existence and importance of fractalkine/CX3CR1 signaling in the trigeminal ganglia has not yet been clarified. This study investigated (1) whether trigeminal ganglion neurons that innervate temporalis muscle and their associated SGCs contain fractalkine and/or express CX3CR1, (2) if intraganglionic injection of fractalkine increases the mechanical sensitivity of temporalis muscle afferent fibers, (3) whether complete Freund's adjuvant (CFA)-induced inflammation of the temporalis muscle alters the expression of fractalkine or its receptor in the trigeminal ganglion, and (4) if intraganglionic administration of CX3CR1 antibodies alters afferent mechanical sensitivity. Immunohistochemistry and in vivo electrophysiological recordings in male and female rats were used to address these questions. It was found that ∼50% of temporalis ganglion neurons and ∼25% of their associated SGCs express CX3CR1, while only neurons expressed fractalkine. Temporalis muscle inflammation increased the expression of fractalkine, but only in male rats. Intraganglionic injection of fractalkine (25g/ml; 3μl) induced prolonged afferent mechanical sensitization. Intraganglionic injection of CX3CR1 antibody increased afferent mechanical threshold, but this effect was greater in controls than in rats with CFA-induced muscle inflammation. These findings raise the possibility that basal fractalkine signalling within the trigeminal ganglion plays an important role in mechanical sensitivity of masticatory muscle sensory afferent fibers and that inhibition of CX3CR1 signaling within the trigeminal ganglia may induce analgesia through a peripheral mechanism.

Beneficial impact of intracerebroventricular fractalkine administration on behavioral and biochemical changes induced by prenatal stress in adult rats: Possible role of NLRP3 inflammasome pathway

Several lines of evidence indicate that adverse experience in early life may be a triggering factor for pathological inflammatory processes and lead to the development of depression. Fractalkine (CX3CL1), a chemokine, plays an important role not only in the migration, differentiation and proliferation of neuronal and glial cells but also in the regulation of neuronal–microglial signaling and the production of pro-inflammatory factors.In the present study, we examined the impact of a prenatal stress procedure on the expression of fractalkine in the hippocampus and frontal cortex of young and adult male rats. Furthermore, we measured the age-dependent effect of stress during pregnancy on the expression of pro-inflammatory factors IL-1β, IL-18, TNF-α, IL-6, and CCL2 in both brain structures. Next, to illustrate the link between fractalkine signaling and the behavioral and biochemical changes induced by prenatal stress, adult prenatally stressed offspring were injected intracerebroventricularly (icv) with exogenous fractalkine.We reported that prenatal stress leads to long-lasting deficits in fractalkine signaling and enhanced inflammatory activation. The study demonstrates that icv administration of fractalkine attenuates the behavioural changes evoked by prenatal stress procedure in adult animals. Moreover, fractalkine administration, exhibits anti-inflammatory action, mainly in the frontal cortex of adult prenatally stressed rats. The effect of fractalkine is related to inhibition of NLRP3 inflammasome. However, its action on the other members of NOD-like receptor family (NLR) cannot be excluded.These findings provide new in vivo evidence that the behavioral and inflammatory disturbances observed in adult prenatally stressed rats may be related to long-lasting malfunctions in fractalkine signaling.

Fractalkine signaling during systemic endotoxemia inhibits perivascular microglial lesion formation in the diabetic retina

Abstract It’s known that diabetics are more susceptible to infections and there is an association between diabetes and chronic inflammation, but whether systemic inflammation contributes to retinopathy remains unknown. Microglial-mediated inflammation has gained recognition as a key contributor to diabetic retinopathy (DR) pathogenesis. We have shown that signaling between the chemokine fractalkine (FKN) and CX3CR1 on microglia is important to maintain neuroprotection and that CX3CR1-deficiency directs microglial-mediated inflammation and neurotoxicity in the diabetic retina. To extend these studies, we tested the hypothesis that acute endotoxemia perpetuates microglial activation and that CX3CR1-deficent mice will be more susceptible to retinal pathology due to dysregulated microglial responses. Systemic endotoxemia was induced in nondiabetic and diabetic CX3CR1-HET and CX3CR1-KO mice by administration of four-daily injections of LPS (1 mg/kg/mouse; i.p.; n=12). Confocal analysis of retinal tissue revealed that in CX3CR1-KO mice, endotoxemia induced a robust-cellular activation represented by morphological changes, IL-1β, iNOS expression, and intense perivascular clusters of microglia. This phenotype was exacerbated in the diabetic CX3CR1-KO retina. These microglial lesions observed in CX3CR1-KO mice coincided with perivascular accumulation of the blood-protein fibrinogen. Lastly, fractalkine treatment (intraocular; 30 ng) into FKN-KO mice dampened microglial activation and astrogliosis during acute endotoxemia. These data suggest that systemic inflammation influences breakdown of the blood-retinal barrier, and may provide therapeutic advances by fractalkine treatment to mitigate DR pathogenesis.

Brain Slice Culture Demonstrates Expedited Cell Traficking in the Presence of Fractalkine in a Medulloblastoma Tumor Microenvironment Model

Abstract 20% of childhood brain tumors are medulloblastomas (MB), and the five-year survival rate can be as low as 60%. The interaction between MB and native immune cells are not well understood, and identifying methods of crosstalk could produce better outcomes for patients. Fractalkine (FKN) is a ligand for the receptor CX3CR1 that plays an inhibitory role in the brain’s immune system by keeping microglia quiescent. Normally, FKN is surface bound and expressed by neurons and astrocytes, but under inflammatory conditions FKN is secreted to serve as a chemoattractant. Preliminary work in our lab shows that FKN is produced by MB and may act as a decoy to maintain microglial quiescence leading to immune escape. Therefore we investigated FKN signaling and microglial kinetics ex vivo utilizing organotypic brain slice culture coupled with real time 2-photon laser scanning microscopy. To differentiate microglia from MB, CX3CR1+/GFP or CX3CR1GFP/GFP knock-in mice were sacrificed, and the brain was removed and sectioned. Slices were collected and cultured on inserts in an incubation chamber during imaging. To determine baseline activity, CX3CR1+/GFP and CX3CR1GFP/GFP brain slices were imaged over 48 hours. MB tumor cells, labelled with SNARF-1, were added to the cultures and imaged over the same time course. Images were processed to differentiate the motility of microglia and tumors. We found that microglia from CX3CR1+/GFP moved slower than microglia from CX3CR1GFP/GFP at baseline and in the presence of tumor. Furthermore, we found that MB moved faster in CX3CR1GFP/GFP slices, suggesting that fractalkine plays a role in the migration of MB.

Dark microglia: A new phenotype predominantly associated with pathological states.

The past decade has witnessed a revolution in our understanding of microglia. These immune cells were shown to actively remodel neuronal circuits, leading to propose new pathogenic mechanisms. To study microglial implication in the loss of synapses, the best pathological correlate of cognitive decline across chronic stress, aging, and diseases, we recently conducted ultrastructural analyses. Our work uncovered the existence of a new microglial phenotype that is rarely present under steady state conditions, in hippocampus, cerebral cortex, amygdala, and hypothalamus, but becomes abundant during chronic stress, aging, fractalkine signaling deficiency (CX3CR1 knockout mice), and Alzheimer's disease pathology (APP‐PS1 mice). Even though these cells display ultrastructural features of microglia, they are strikingly distinct from the other phenotypes described so far at the ultrastructural level. They exhibit several signs of oxidative stress, including a condensed, electron‐dense cytoplasm and nucleoplasm making them as “dark” as mitochondria, accompanied by a pronounced remodeling of their nuclear chromatin. Dark microglia appear to be much more active than the normal microglia, reaching for synaptic clefts, while extensively encircling axon terminals and dendritic spines with their highly ramified and thin processes. They stain for the myeloid cell markers IBA1 and GFP (in CX3CR1‐GFP mice), and strongly express CD11b and microglia‐specific 4D4 in their processes encircling synaptic elements, and TREM2 when they associate with amyloid plaques. Overall, these findings suggest that dark microglia, a new phenotype that we identified based on their unique properties, could play a significant role in the pathological remodeling of neuronal circuits, especially at synapses. GLIA 2016;64:826–839

Editorial: Cytokines as Players of Neuronal Plasticity and Sensitivity to Environment in Healthy and Pathological Brain.

In this e-Book, we collected recent evidence on the role of cytokines (including chemokines) in the interplay between environmental stimulation and central responses in both physiological and pathological conditions. The e-Book includes original studies and review articles focused on cytokine function during brain development as well as in the mature brain. Cytokines, together with neurotransmitters and hormones, are signaling molecules playing a key role in the maintenance of neuro-immune-endocrine system homeostasis. The cytokine systems (constituted by cytokines, their receptors and regulators of their activity) are expressed throughout the brain and their expression is regulated during brain development until aging. Brain cells, including neurons as well as glia cells, can release and/or be responsive to cytokines, therefore these molecules can enable communication between different cell types. Under physiological conditions, cytokines typically participate in brain development and plasticity by translating environmental into molecular signals. However, once the allostatic equilibrium is compromised, cytokine systems, if over- or chronically-activated, may participate in mediating toxic effects in the brain. Indeed, a central role for cytokines in neuropsychiatric as well as neurodegenerative disorders is now well recognized. The major source of cytokines release in the brain is microglia cells that are actively involved in adult brain homeostasis and in neural loss and synaptic maturation during development. The contribution of Pagani et al. addresses the role of fractalkine (CX3CL1) signaling in the developmental profile of morphological features and physiological properties of microglia using mice lacking the fractalkine receptor (who is expresses only in microglia within the healthy brain). Sheridan et al. investigated the role for fractalkine in synaptic plasticity showing that the levels of hippocampal fractalkine increases after a memory task and that the chemokine regulates glutamate-mediated neurotransmission tone. A comprehensive review on the role of fractalkine in regulating microglia properties, brain plasticity and behavior is provided by Paolicelli et al.. Another cytokine known to modulate memory-related processes is interferon (IFN)-γ. One of the article included in this e-book describe the effects induced by the lack of IFN-γ on memory function under basal or stressful conditions (Litteljohn et al.). This study emphasizes the importance of considering the “brain state” (healthy or disease) when the modulation of neurobehavioral processes by the cytokine systems it is evaluated. Interestingly, in the commentary of von Bohlen and Halbach, the astrocytic secreted lipocalin 2 is described as a mediator of the dialogue between astrocytes and neuron that in turn affects synaptic plasticity. Neuroimmune factors are particularly relevant to a number of neurophatologies. They play important pro-survival and/or pro-death roles by regulating targets in specific brain regions. Sutinem et al. reported that in pathological conditions, such as Alzheimer's disease, the cytokine interleukin (IL)-18 seems potentially involved in driving protein changes relevant for the pathogenesis, whereas Chiavegato et al. described the role of IL-1 beta and the high mobility group B1 (HMGB1) in preventing seizure-like discharges in models of focal epilepsy. Cytokines affect brain functions through different molecular mechanisms. Groul et al. show that up-regualtion of astrocytic CCL2 and Il-6 differentially affect the levels of specific proteins in cerebellum and hippocampus. The review of Guyon described how the stimulation of the receptor CXCR4 by the chemokine CXCL12 regulates the synaptic release of glutamate and γ-aminobutyric acid (GABA). Calabrese et al. proposed the neurotrophin brain-derived neurotrophic factor (BDNF) as a bridge between increased levels of pro-inflammatory cytokines and impaired neuroplasticity while the contribution of Cattaneo et al. is centered on epigenetics mechanisms that, by altering inflammation-immune systems and neuronal plasticity, may increase vulnerability to develop psychiatric disorders following early life stressful events. Finally, in their review, Singhal et al. analyze the neuroimmune mechanisms associated with neurobiological and behavioral changes following different environmental stimulations. In particular, they described how environmental enrichment (mainly physical exercise) affects neuroimmune targets and behavior. Overall, the work presented herein provides an insight into important aspects of the role of neuroimmune factors on brain activity and behavior. It represent a small windows on a complex panorama that still remain to be fully elucidated, especially with regard to the interplay between cytokine action, environmental stimulation and neuronal outcome.

The Role of C Fibers in Spinal Microglia Induction and Possible Relation with TRPV3 Expression During Chronic Inflammatory Arthritis in Rats.

Introduction:Stimulation of peptidergic fibers activates microglia in the dorsal horn. Microglia activation causes fractalkine (FKN) release, a neuron-glia signal, which enhances pain. The transient vanilloid receptor 1 (TRPV1) mediates the release of neuropeptides, which can subsequently activate glia. TRPV1 and TRPV2 are generally expressed on C and Aδ fibers, respectively. Expression of both proteins is upregulated during inflammation, but expression of TRPV3 after induction of inflammation is unclear.Methods:Adult male Wistar rats were used in all experiments. Arthritis was induced in them by single subcutaneous injection of complete Freund’s adjuvant (CFA) in their right hindpaws. Resiniferatoxin (RTX) was used to eliminate peptidergic fibers. We examined the relation between FKN and TRPV3 expression by administration of anti-FKN antibody.Results:Our study findings indicated that 1) spinal TRPV3 was mostly expressed on nonpeptidergic fibers, 2) expression of spinal TRPV3 increased following inflammation, 3) elimination of peptidergic fibers decreased spinal TRPV3 expression, 4) alteration of hyperalgesia was compatible with TRPV3 changes in RTX-treated rat, and 5) anti-FKN antibody reduced spinal TRPV3 expression.Discussion:It seems that the hyperalgesia variation during different phases of CFA-induced arthritis correlates with spinal TRPV3 expression variation on peptidergic fibers. Moreover, spinal microglial activation during CFA inflammation is involved in TRPV3 expression changes via FKN signaling.