Modulating neurotoxicity through CX3CL1/CX3CR1 signaling

1The CX3CL1/CX3CR1 axis, together with CD200/CD200R, have mainly been studied in the context of their involvement in halting potentially toxic activated microglial phenotypes. The phenotypes embodied by the term “microglia activation” have been hotly debated. Recent nomenclatures proposed M1- and M2-like microglial phenotypes, characterized by the production of pro- or anti-inflammatory cytokines and markers, named after the alternative activation states of macrophages. The transition between these two forms, however, is not all or none and several intermediate microglia phenotypes have been described, together with the identification of overlapping features between alternatively activated phenotypes. Furthermore, it’s now evident that no M1/M2 polarization can be supported, even for peripheral macrophages.

2Given contemporary approaches to genome wide expression profiling, we now face the welcome prospect of devising a robust, meaningful account of microglial reactive phenotypes.

3In spite of this complex picture, there is a general consensus on the relatively unique role of the CX3C system in mediating key microglial activities, mainly because of its privileged position at the interface with neurons. Microglia-neuron interaction is dynamic, as revealed by in vivo video microscopy using cx3cr1GFP/+ mice and disclosing that microglia branches continuously survey neuronal surfaces and the cerebral microenvironment in the healthy brain, presumably to sense dysfunctional synapses, damaged neurons, or the presence of potentially dangerous agents. Thus, in the adult brain, microglia may exert a sentinel function for neurons and, when neuronal damage occurs, microglia rapidly react to protect or to eliminate neurons, if irreversibly damaged. CX3C signaling is deeply involved in this rescue process, directly modulating different aspects of microglia biology important for neuron protection, like the production of soluble factors directly involved in neuron survival, the modulation of phagocytic activity, but also indirectly affecting other cell types (resident or infiltrating) present in brain parenchyma that, in turn, might influence neuron survival.

4CX3CR1 was localized to microglia and CX3CL1 to neurons using in situ hybridization, by. Availability of mice with a GFP fluorescent reporter for CX3CR1 transcription confirmed the former finding in adult mice and subsequent studies showed that CX3CR1 is characteristic of microglia throughout embryogenesis and during the murine lifespan. Subsequently, developed a CX3CL1 reporter and showed a similar neuronal distribution of CX3CL1 in all regions of the adult brain. These expression patterns are somewhat modulated but do not fundamentally alter in disease models. Therefore, CX3CL1/CX3CR1 signaling provides insight into microglial–neuronal interactions throughout the lifespan (Table 1) and in an immense variety of pathological conditions. This review summarizes a portion of this research.

5The CX3CL1/CX3CR1 signaling participates in the control of production and release of several cytokines from microglia. Earlier in vitro studies demonstrated that LPS- and IFNγ-induced release of cytokines such as interleukin-1β (IL-1β), TNFα, 8-isoprostane, NO, and IL-6 in cultured microglia was efficiently blocked by CX3CL1 stimulation. Since then several papers confirmed and provided further in vivo evidence supporting the hypothesis that, together with other molecules like CD200, CD22 and CD172, CX3CL1 signaling in the brain reduces microglia reactivity to toxic stimuli, maintaining microglia in a modulated state. This hypothesis can be evaluated even though the status of “microglial activation” is profoundly uncertain [see for a recent review].

6Interleukin-1β is an inflammatory cytokine playing pivotal roles in local and systemic processes and is a key inducer of peripheral and central immune responses to infection or injury. Inhibition of IL-1β signaling has beneficial effects in a variety of experimental paradigms of acute brain damage. Despite controversial data on its neurotoxic effects in vivo and in vitro, IL-1β is considered a promising clinical target in several neuropathologies. In paradigms of brain injury where IL-1β increases, CX3CL1 reduces IL-1β levels, correlating with protection from damage.

7Increased production of IL-1β by microglial cells isolated from the brain of Cx3cr1-/- mice upon systemic LPS challenge was described by. Cx3cr1-/- microglial cells from LPS-challenged mice provided a toxic insult when transplanted in the forebrain of wild type mice, resulting in neuronal TUNEL labeling. Interestingly these toxic effects were prevented eliminating IL1R signaling either with IL-1 receptor antagonist a (IL-1Ra) or in IL1-/- mice.

8also showed enhanced dopaminergic cell loss in Cx3cr1-/- or Cx3cl1-/- mice after peripheral MPTP administration. asked two follow-on questions: what was the role of cytokine production? and which CX3CL1 isoform (soluble or membrane-associated) was responsible for the neuroprotective effect? They reported that IL-1β signaling was blunted by soluble AAV-CX3CL1, injected in the substantia nigra pars compacts of MPTP-injected cx3cl1-/- mice, so that less IL-1β and TNFα were produced in the ventral mesencephalon and microglial CD68 expression was reduced. These alterations in cytokine profile induced by soluble AAV-CX3CL1 correlated with reduced death of dopaminergic TH positive neurons and improved motor coordination on accelerating rotarod. A protective effect of soluble CX3CL1 on dopaminergic neurons was demonstrated by the same authors in a rat model of PD, induced by intrastriatal 6-OHDA injection.

9CX3CL1 decreased in the brain of aged mice possibly due to neuronal loss, and this change is accompanied by an altered effector state of microglia. In aged mice, the response to systemic infection is amplified by the microglia effector response: LPS administration evokes an increased IL-1β and a reduced TGFβ expression, in comparison with young adult animals.

10Aging is reported to decrease neurogenesis in the hippocampal region of the brain. Similarly to aging, deficits in CX3CL1 signaling due to gene deletion or caused by CX3CR1 blocking antibodies reduced the number of progenitor cells in the hippocampal dentate gyrus. CX3CL1 administration reverted the age-related decrease in neurogenesis and chronic blockade of CX3CR1 function reduced neurogenesis and increased IL-1β in young adult mice. IL-1β was implicated in the reduced survival and proliferation rate of neuronal progenitors in the hippocampal region of CX3CR1-/- mice, since IL-1Ra reverted the observed phenotypes.

11The involvement of IL-1 in CX3CL1-induced effects in experimental models of AD is suggested by several lines of evidence. Mouse deleted of cx3cr1 showed a strong increase of microtubule-associated protein tau (MAPT) phosphorylation upon LPS challenge, and humanized MAPT transgenic mice crossed with cx3cr1-/- mice exhibited accelerated behavioral impairment along with increased MAPT hyperphosphorylation and aggregation. These data, together with the observation that microglia-induced MAPT phosphorylation in neurons can be blocked by IL-1Ra and p38 inhibitors further supports the hypothesis that inhibiting production of IL-1β constitutes a central mechanism for CX3CL1 neuroprotective signaling. Additional possible targets of CX3CL1 signaling in AD have been suggested: demonstrated that hAPP/cx3cr1-/- mice have exacerbated neuronal and cognitive function defects and increased inflammatory responses in comparison with hAPP/cx3cr1+/+ mice. The lack of cx3cr1 in this AD mouse model (hAPP-J20) did not modify plaque deposition but increased tau phosphorylation, cognitive deficits, and microglia activation, along with enhanced IL-6 and TNF-α levels. In line with these observations, AD autopsy brain sections showed reduced CX3CL1 levels in the cortex and hippocampus regions. Further supporting a potential role for CX3CR1 signaling in AD pathogenesis, reported that in two different mouse models of AD (APPS1 and R1.40), characterized by different velocity of β-amyloid deposition, crossing with cx3cr1-/- mice reduced the extent of amyloid deposition, the number of dystrophic neurons and of plaque-associated microglia cells. This protected phenotype was accompanied by increased CCL2 and TNF-α and elevated IL-1β expression. By contrast, a potentially toxic effect for CX3CL1 was suggested by demonstrating that the deficits induced by β-amyloid injection in hippocampal CA1, as well as the increased IL-1β expression and microglia activation, were all reverted by central CX3CR1 suppression by siRNA delivery. A toxic effect for CX3CR1 signaling in AD was also proposed by that found a reduced neuronal loss when the 3xTg AD mice were crossed with cx3cr1-/- mice. In summary, CX3CR1/CX3CL1 signaling modifies AD-related pathology quite consistently but a unified concept of the net outcome of the effects has not been conclusively proven.

12Alteration of IL-1β levels was reported in cx3cr1-deficient mice upon focal cerebral ischemia. demonstrated a reduction in IL-1β and TNF-α production in brains of cx3cr1-/- mice upon induction of cerebral ischemia, together with reduced ischemic volume and less neuronal cell death. Similar experiments in a focal brain ischemia model were performed by in cx3cl1 deficient mouse and confirmed lower ischemic volume in the absence of CX3CL1/CX3CR1 signaling. This result was later confirmed in a pMCAO model. In apparent contrast, when CX3CL1 was exogenously administrated to wt mice and followed by pMCAO, a significant reduction of ischemic volume was observed while in cx3cl1-/- mice CX3CL1 administration worsened the effects of ischemia.The mechanisms underlying divergent effects of CX3CL1 administration to wt and Cx3cl1-/- mice remain uncertain. In favor of a protective effect of CX3CL1 in cerebral ischemia, reported that stroke patients exhibited a positive correlation between high plasma CX3CL1 levels and a better clinical outcome. Interestingly, demonstrated a protective role for CX3CR1 in neonatal hypoxic ischemia in female mice, with a possible gender specific protective effect. The authors demonstrated that hippocampal CX3CL1 was reduced by ischemia and that Cx3cr1-/- mice showed worse learning deficits and hippocampal damage after ischemia.

13In an in vitro genetic model of amyotrophic lateral sclerosis (ALS), where the mutated SOD1G93A is specifically expressed by astrocytes, reported that conditioned medium from mesenchymal stem cell (MSC) cultures increased CX3CL1 and reduced TNFα, IL6, and iNOS expression on astrocytes, and mediated a protective effect towards primary motor neurons. In vivo, MSC administration improved the functional outcome of mutated mice. In cultured microglia, the presence of mutated SOD1G93A also increased CX3CR1 levels.

14Taken together, these data demonstrate that CX3CL1 attenuates inflammatory cytokine production, strongly modulating the neuroprotective activity of microglia in several brain diseases.

15Adenosine is a well-established modulator of synaptic transmission and has protective effects in the nervous system, primarily mediated through adenosine type 1 receptor (A1R). Adenosine is released by neurons and glial cells and its level in the brain is regulated essentially by the activity of adenosine kinase (ADK), and 5′ nucleotidase acting on extracellular ATP.

16Upon CX3CL1 treatment of cultured microglia, increased extracellular adenosine release is observed. In in vitro models of excitotoxicity and in vivo models of cerebral ischemia, the neuroprotective activity of CX3CL1 requires A1R activation. This neuroprotective mechanism does not derive from a direct microglia-neuron cross talk, requiring (at least in vitro) the involvement of astrocytes whose A1Rs are stimulated by adenosine released from microglia, increasing astrocyte glutamate transporter (GLT-1) expression and activity.

17The neuroprotective activity of adenosine has been correlated to its ability to hamper the release of excitatory glutamate at pre-synaptic terminals. In this regard, it is interesting to note that CX3CL1 has a wide spectrum of modulatory activities on glutamatergic neurotransmission, interfering with: (i) pre-synaptic glutamate release, (ii) the amplitude of AMPA currents, thereby altering GluR1 phosphorylation state (post-synaptic modulation), (iii) LTP expression and LTD induction in hippocampal CA1 region, (iv) synaptic NMDAR currents through D-serine release from glia; (v) glutamatergic synapse maturation during development.

18All together these data demonstrate that excitatory neurotransmission, which is deeply implicated in the most common neurotoxic pathways, is affected by CX3CL1/CX3CR1 signaling, with permissive cooperativity of the adenosine system.

19Microglia produce a number of growth factors providing trophic support to developing or damaged brain circuits. During brain development, in the first postnatal week, CX3CR1 expression on microglia is important for the survival of cortical neurons of layer V, by regulating the production of insulin-like growth factor-1 (IGF-1; ). In particular, increased death of layer V neurons was observed in the cerebral cortex of cx3cr1GFP/GFP mice, in which microglia produced a significantly reduced amount of the growth factor IGF-1. So during brain development, CX3CR1-mediated IGF-1 secretion from microglia modulates neuronal survival in postnatal cortical layer V.

20A specific BDNF polymorphism (Val66Met) is associated with memory deficits and increased vulnerability to anxiety and depressive disorders both in humans and mice. Interestingly, this polymorphism also associates with impaired CX3CL1/CX3CR1 hippocampal signaling, reducing expression of CX3CL1 protein and mRNA in dorsal hippocampus. Chronic CX3CL1 infusion in the hippocampal region of Val66Met mice recovered memory deficits, restored neurogenesis in the DG and increased Akt phosphorylation levels.

21One of the most intriguing functions of microglia in the central nervous system is certainly its activity as the resident phagocyte. In development and in pathological neuroinflammatory conditions, microglial phagocytosis is important for the elimination of dead and damaged neurons, myelin residues and β-amyloid peptides [reviewed in]. During normal brain development, microglial phagocytosis is involved in eliminating supernumerary neurons, but also in synaptic pruning and circuit refinement. Among the key factors that emerged as potential modulators of neuron phagocytosis by microglia during development is the complement system, with the proteins C1q and C3 expressed on synapses and microglial CR3/CD11b mediating the elimination of immature and weakly active synapses. TGF-β produced by astrocytes signals to retinal ganglion cells for C1q expression, thus favoring retinogeniculate refining, and inserting astrocytes in microglia-neuron communication.

22Cell damage causes high-grade ATP release (both from damaged cells and through astrocyte exocytosis) regulating microglial process extension and also contributing to inflammasome activation within microglia. Under stress, neurons manifest the phosphatidylserine (PS) “eat me” signal which can be recognized by several microglia receptor/adaptor complexes including Gas6/MerTK and MFGE8/vitronectin receptor. In ischemic conditions potentially viable neurons reversibly express PS on their surface and are vulnerable to be taken up and destroyed by microglia. Selective elimination of MFGE8/vitronectin receptor “eat me” signaling strongly improved experimental stroke outcome.

23CX3CR1 signaling also modulates microglia phagocytosis of neurons and their processes in both physiological and pathological conditions. During development, CX3CL1/CX3CR1 signaling contributes to refinement of synaptic elements in varied CNS regions during the postnatal “critical period”. This fine-tuning of anatomical connections is important to establish and build an optimal functional circuitry.

24In Alzheimer’s disease (AD), CX3CL1/CX3CR1 blockade modulates microglia phagocytic activity in a way that markedly attenuates amyloid deposition. In particular, CX3CR1 deficient microglia show increased phagocytic activity when crossed with different AD mouse models CRND8 or APPPS1 and R1.40, resulting in attenuated Aβ deposition. Based on results that look beyond amyloid deposition in various models, the overall outcome of eliminating CX3CR1 signaling remains in doubt.

25Neurons damaged by toxic insults, like glutamate, release more CX3CL1 that increases MFG-E8 expression on microglia. As noted above, MFG-E8 is a PS receptor that recognize PS on injured neurons and acts as an adaptor recognized by microglial vitronectin receptor, inducing damaged-cell uptake. CX3CL1 up-regulation of MFG-E8 expression was associated to increased heme oxygenase 1 (HO-1) expression through the MAPKs ERK and JNK and the nuclear factor erythroid 2 related factor (NFE2LE). reported that CX3CL1 reduced Tau-induced microgliosis via up-regulating transcription factor NRF2/NFE2LE, along with increased HO-1 expression. Since the increased expression of HO-1 is reported to mediate anti inflammatory activities, this pathway could be involved in the reported neuroprotective effects of CX3CL1 in different Tau pathology models.

26These data indicate that the clearance and phagocytic activity of microglia can be modulated by CX3CL1 signaling through varied mechanisms, and that the final outcome in term of neuroprotection or neurotoxicity might result from the presence of co-stimulatory signals and, consequently, from the effector programs of microglia.

27In vitro, CX3CL1 promoted survival of neural precursor cells upon growth factor withdrawal from the culture medium. Further, cx3cr1-/- mice have reduced neurogenesis in the hippocampal DG region but this reduction was accompanied by contrasting effects on memory deficits and synaptic plasticity: Maggi et al., reported that cx3cr1-/- mice had increased hippocampal LTP and performed better in the Morris water maze test, while, in the same mouse strain, described reduced hippocampal LTP and deficits in the Morris water maze. At least in part, these conflicting data reflect the lack of simple relations between DG neurogenesis, plasticity processes in the hippocampal CA1 region and learning behavior.

28More recently, demonstrated that CX3CR1 deficiency impairs neurogenesis both in young and aged mice and that voluntary physical exercise increased brain CX3CL1 levels and DG neurogenesis, both effects being abolished by intrahippocampal CX3CR1 antibody injection.

29In the olfactory bulb, CX3CR1 deletion increased olfactory sensory neuron death upon bulbectomy and cx3cr1-/- mice showed reduced proliferation of intraepithelial stem progenitor cells, increased macrophage recruitment in the lesioned epithelium and increased TNF-α and IL-6 levels.

30All together these data suggest that neuroprotective effects of CX3CL1 might arise partly from an increased proliferation or survival of progenitor elements in selected brain regions where neurogenesis continues throughout adult life.

31In the CNS, the unique expression of CX3CR1 on microglia clearly suggests that the neuroprotective effects of CX3CL1 depend primarily on the direct communication between microglia and neurons. Recent evidence suggested that CX3CL1/CX3CR1 signaling in neuroprotection again excitotoxic insult might not be restricted to a direct microglia-neuron communication but also involves indirectly modulation of astrocyte activity., in fact, demonstrated that CX3CL1, acting on microglia, induced the production and release of soluble factors that exerted their effects on astrocytes, inducing the functional up regulation and the increased expression of the excitatory amino acid transporter GLT-1. As already reported in in vitro and in vivo systems, this cross-talk requires the “permissive” presence of adenosine, specifically acting on astrocyte A1R. These data demonstrated for the first time a role for astrocytes in mediating the neuroprotection induced by CX3CL1/CX3CR1 signaling between microglia and neurons.

32Neuroprotective effects of CX3CL1 could also arise from hematogenous leukocytes that enter the brain upon specific, injury-induced challenge.

33In mouse experimental allergic encephalomyelitis (EAE) demonstrated that CX3CR1 deficient mice had an exacerbated phenotype, with severe spastic paralysis and hemorrhagic inflammation in the CNS, resulting in higher mortality. The increased toxicity resulted from an altered control of NK cells entry into the brain of EAE-affected animals. These findings were extended by, who showed that CX3CR1 deficiency limited to bone marrow cells of mice with EAE resulted in more severe pathology, with increased demyelination, axonal damage and reduced calbindin positive neurons in the cerebellum. These effects correlated with increased CD115+, Ly6ClowCD11c+ dendritic cell infiltration in the CNS and increased IL-17 and IFNγ expression in the cerebellum, forebrain and spinal cord.

34reported that in CD73-/- mice, where the ectonucleotidase responsible for extracellular adenosine accumulation was absent, EAE induction produced a milder phenotype. More recently, the same group reported that extracellular adenosine increased CX3CL1 expression and that CX3CL1 promoted lymphocyte entry into the brain of EAE mice. Therefore, in CD73 deficient mice, extracellular adenosine cannot be produced, CX3CL1 is not upregulated and a reduced amount of lymphocytes enter cerebral parenchyma, resulting in a protected phenotype.

35In focal cerebral ischemia, reported a reduced leukocyte (CD45+ cells) infiltration in the brain of cx3cr1-/- mice and a consistently reduced damaging of the blood brain barrier. This correlated with a protected phenotype, with reduced ischemic volume and lesser numbers of apoptotic cells and a better performance in the behavioral test of adhesive tape removal.

36All together these data highlight that CX3CL1/CX3CR1 signaling modulates the entry of hematogenous leukocytes, with beneficial or deleterious consequences contingent on context.

37This review highlights the manifold effects of altering CX3CR1/CX3CL1 signaling, which can be observed throughout the lifespan from early embryogenesis through diseases of the aging brain. For the most part, CX3CR1/CX3CL1 can be viewed as a fulcrum with which to modify microglial physiology to probe into the panoply of functions of these versatile and essential cells. Adding to complexity but also lending heuristic value, the effects of modifying CX3CR1/CX3CL1 pathways are extremely context dependent. Nevertheless in each case insights about upstream and downstream signaling from CX3CR1/CX3CL1 have been informative and may point to new therapeutic directions for brain disease. Finally, it is now clear that macroglia (such as astrocytes) and hematogenous leukocytes also contribute to the varied physiology of the CX3CR1/CX3CL1 signaling system.

38The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.