CX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathway

The exact roles of activated microglia and fractalkine (CX3CL1)/fractalkine receptor (CX3CR1) signaling are not fully understood in brain ischemic injury and the findings reported are controversial. Here, we investigated the effects of CX3CR1 siRNA on the expression of CX3CR1, p38 mitogen-activated protein kinase (p38MAPK), Protein Kinase C (PKC) and inflammatory cytokines, microglia activation, white matter lesions, and cognitive function in mice treated with bilateral common carotid artery stenosis (BCAS) in vivo as well as effects of exogenous CX3CL1, CX3CR1 siRNA, and SB2035080 on expression of inflammatory cytokines in BV2 microglia treated with oxygen–glucose deprivation (OGD) in vitro. We showed that CX3CR1 siRNA significantly inhibited the increased expression of CX3CR1, p38MAPK, PKC as well as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, and also attenuated microglia activation, white matter lesions, and cognitive deficits induced by BCAS in mice brain. We also showed that exogenous CX3CL1 could induce a further enhancement in TNF-α and IL-1β expression, which could be suppressed by CX3CR1 siRNA or by the p38MAPK inhibitor in OGD-treated BV2 microglial cells in vitro. Our findings indicated that CX3CL1/CX3CR1-mediated microglial activation plays a detrimental role in ischemic brain via p38MAPK/PKC signaling and also suggested that CX3CL1/CX3CR1 axis might be a putative therapeutic target to disrupt the cascade of deleterious events that lead to brain ischemic injury.     

CX3CL1/CX3CR1 regulates nerve injury‐induced pain hypersensitivity through the ERK5 signaling pathway

Peripheral nerve injury induces the cleavage of CX3CL1 from the membrane of neurons, where the soluble CX3CL1 subsequently plays an important role in the transmission of nociceptive signals between neurons and microglia. Here we investigated whether CX3CL1 regulates microglia activation through the phosphorylation of extracellular signal‐regulated protein kinase 5 (ERK5) in the spinal cord of rats with spinal nerve ligation (SNL). ERK5 and microglia were activated in the spinal cord after SNL. The knockdown of ERK5 by intrathecal injection of antisense oligonucleotides suppressed the hyperalgesia and nuclear impact of nuclear factor‐κB induced by SNL. The blockage of CX3CR1, the receptor of CX3CL1, significantly reduced the level of ERK5 activation following SNL. In addition, the antisense knockdown of ERK5 reversed the CX3CL1‐induced hyperalgesia and spinal microglia activation. Our study suggests that CX3CL1/CX3CR1 regulates nerve injury‐induced pain hypersensitivity through the ERK5 signaling pathway. © 2013 Wiley Periodicals, Inc.     

An initial investigation of spinal mechanisms underlying pain enhancement induced by fractalkine, a neuronally released chemokine

Fractalkine is a chemokine that is tethered to the extracellular surface of neurons. Fractalkine can be released, forming a diffusible signal. Spinal fractalkine (CX3CL1) is expressed by sensory afferents and intrinsic neurons, whereas its receptor (CX3CR1) is predominantly expressed by microglia. Pain enhancement occurs in response both to intrathecally administered fractalkine and to spinal fractalkine endogenously released by peripheral neuropathy. The present experiments examine whether fractalkine-induced pain enhancement is altered by a microglial inhibitor (minocycline) and/or by antagonists/inhibitors of three putative glial products implicated in pain enhancement: interleukin-1 (IL1), interleukin-6 (IL6) and nitric oxide (NO). In addition, it extends a prior study that demonstrated that intrathecal fractalkine-induced mechanical allodynia is blocked by a neutralizing antibody to the rat fractalkine receptor, CX3CR1. Here, intrathecal anti-CX3CR1 also blocked fractalkine-induced thermal hyperalgesia. Furthermore, blockade of microglial activation with minocycline prevented both fractalkine-induced mechanical allodynia (von Frey test) and thermal hyperalgesia (Hargreaves test). Microglial activation appears to lead to the release of IL1, given that pretreatment with IL1 receptor antagonist blocked both fractalkine-induced mechanical allodynia and thermal hyperalgesia. IL1 is not the only proinflammatory cytokine implicated, as a neutralizing antibody to rat IL6 also blocked fractalkine-induced pain facilitation. Lastly, NO appears to be importantly involved, as l-NAME, a broad-spectrum NO synthase inhibitor, also blocked fractalkine-induced effects. Taken together, these data support that neuronally released fractalkine enhances pain via activation of spinal cord glia. Thus, fractalkine may be a neuron-to-glia signal triggering pain facilitation.     

Analysis of CX3CR1 haplodeficiency in male and female APPswe/PSEN1dE9 mice along Alzheimer disease progression

Microglia, the resident immune cells of the brain, have recently emerged as key players in Alzheimer Disease (AD) pathogenesis, but their roles in AD remain largely elusive and require further investigation. Microglia functions are readily altered when isolated from their brain environment, and microglia reporter mice thus represent valuable tools to study the contribution of these cells to neurodegenerative diseases such as AD. The CX3CR1^+/eGFP mice is one of the most popular microglia reporter mice, and has been used in numerous studies to investigate in vivo microglial functions, including in the context of AD research. However, until now, the impact of CX3CR1 haplodeficiency on the typical features of Alzheimer Disease has not been studied in depth.To fill this gap, we generated APP^swe/PSEN1^dE9:CX3CR1^+/eGFP mice and analyzed these mice for Alzheimer’s like pathology and neuroinflammation hallmarks. More specifically, using robust multifactorial statistical and multivariate analyses, we investigated the impact of CX3CR1 deficiency in both males and females, at three typical stages of the pathology progression: at early stage when Amyloid-β (Aβ) deposition just starts, at intermediate stage during Aβ accumulation phase and at more advanced stages when Aβ plaque number stabilizes. We found that CX3CR1 haplodeficiency had little impact on the progression of the pathology in the APP^swe/PSEN1^dE9 model and demonstrated that the APP^swe/PSEN1^dE9:CX3CR1^+/eGFP line is a relevant and useful model to study the role of microglia in Alzheimer Disease. In addition, although Aβ plaques density is higher in females compared to age-matched males, we show that their glial reaction, inflammation status and memory deficits are not different.     

Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions

Fractalkine is a unique chemokine reported to be constitutively expressed by neurons. Its only receptor, CX3CR1, is expressed by microglia. Little is known about the expression of fractalkine and CX3CR1 in spinal cord. Given that peripheral nerve inflammation and/or injury gives rise to neuropathic pain, and neuropathic pain may be partially mediated by spinal cord glial activation and consequent glial proinflammatory cytokine release, there must be a signal released by affected neurons that triggers the activation of glia. We sought to determine whether there is anatomical evidence implicating spinal fractalkine as such a neuron-to-glia signal. We mapped the regional and cellular localization of fractalkine and CX3CR1 in the rat spinal cord and dorsal root ganglion, under basal conditions and following induction of neuropathic pain, employing both an inflammatory (sciatic inflammatory neuropathy; SIN) as well as a traumatic (chronic constriction injury; CCI) model. Fractalkine immunoreactivity and mRNA were observed in neurons, but not glia, in the rat spinal cord and dorsal root ganglia, and levels did not change following either CCI or SIN. By contrast, CX3CR1 was expressed by microglia in the basal state, and the microglial cellular concentration was up-regulated in a regionally specific manner in response to neuropathy. CX3CR1-expressing cells were identified as microglia by their cellular morphology and positive OX-42 and CD4 immunostaining. The cellular distribution of fractalkine and CX3CR1 in the spinal circuit associated with nociceptive transmission supports a potential role in the mechanisms that contribute to the exaggerated pain state in these models of neuropathy.     

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 (A?R). We show that, in the presence of the A?R antagonist 1,3-dipropyl-8-cyclopentylxanthine and in A?R?/? 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.     

Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, during acute and chronic inflammation in the rodent CNS

In this study, we investigate the expression of fractalkine (CX3CL1) and the fractalkine receptor (CX3CR1) in the naive rat and mouse central nervous system (CNS). We determine if the expression of this chemokine and its receptor are altered during chronic or acute inflammation in the CNS. In addition, we determine if CX3CL1, which has been reported to be chemoattractant to leukocytes in vitro, is capable of acting as a chemoattractant in the CNS in vivo. Immunohistochemistry was performed using primary antibodies recognizing soluble and membrane-bound CX3CL1 and the N-terminus of the CX3CR1. We found that neurons in the naive rodent brain are immunoreactive for CX3CL1 and CX3CR1, both showing a perinuclear staining pattern. Resident microglia associated with the parenchyma and macrophages in the meninges and choroid plexus constituitively express CX3CR1. In a prion model of chronic neurodegeneration and inflammation, CX3CL1 immunoreactivity is upregulated in astrocytes and CX3CR1 expression is elevated on microglia. In surviving neurons, expression of CX3CL1 appears unaltered relative to normal neurons. There is a decrease in neuronal CX3CR1 expression. Acute inflammatory responses in the CNS, induced by stereotaxic injections of lipopolysaccharide or kainic acid, results in activation of microglia and astrocytes but no detectable changes in the glial expression of CX3CL1 or CX3CR1. The expression of CX3CL1 and CX3CR1 by glial cells during inflammation in the CNS may be influenced by the surrounding cytokine milieu, which has been shown to differ in acute and chronic neuroinflammation.     

Evidence that exogenous and endogenous fractalkine can induce spinal nociceptive facilitation in rats

Recent evidence suggests that spinal cord glia can contribute to enhanced nociceptive responses. However, the signals that cause glial activation are unknown. Fractalkine (CX3C ligand-1; CX3CL1) is a unique chemokine expressed on the extracellular surface of spinal neurons and spinal sensory afferents. In the dorsal spinal cord, fractalkine receptors are primarily expressed by microglia. As fractalkine can be released from neurons upon strong activation, it has previously been suggested to be a neuron-to-glial signal that induces glial activation. The present series of experiments provide an initial investigation of the spinal pain modulatory effects of fractalkine. Intrathecal fractalkine produced dose-dependent mechanical allodynia and thermal hyperalgesia. In addition, a single injection of fractalkine receptor antagonist (neutralizing antibody against rat CX3C receptor-1; CX3CR1) delayed the development of mechanical allodynia and/or thermal hyperalgesia in two neuropathic pain models: chronic constriction injury (CCI) and sciatic inflammatory neuropathy. Intriguingly, anti-CX3CR1 reduced nociceptive responses when administered 5-7 days after CCI, suggesting that prolonged release of fractalkine may contribute to the maintenance of neuropathic pain. Taken together, these initial investigations of spinal fractalkine effects suggest that exogenous and endogenous fractalkine are involved in spinal sensitization, including that induced by peripheral neuropathy.     

Microglial phagocytosis and activation underlying photoreceptor degeneration is regulated by CX3CL1‐CX3CR1 signaling in a mouse model of retinitis pigmentosa

Retinitis pigmentosa (RP), a disease characterized by the progressive degeneration of mutation‐bearing photoreceptors, is a significant cause of incurable blindness in the young worldwide. Recent studies have found that activated retinal microglia contribute to photoreceptor demise via phagocytosis and proinflammatory factor production, however mechanisms regulating these contributions are not well‐defined. In this study, we investigate the role of CX3CR1, a microglia‐specific receptor, in regulating microglia‐mediated degeneration using the well‐established rd10 mouse model of RP. We found that in CX3CR1‐deficient (CX3CR1^GFP/GFP) rd10 mice microglial infiltration into the photoreceptor layer was significantly augmented and associated with accelerated photoreceptor apoptosis and atrophy compared with CX3CR1‐sufficient (CX3CR1^GFP/+) rd10 littermates. CX3CR1‐deficient microglia demonstrated increased phagocytosis as evidenced by (1) having increased numbers of phagosomes in vivo, (2) an increased rate of phagocytosis of fluorescent beads and photoreceptor cellular debris in vitro, and (3) increased photoreceptor phagocytosis dynamics on live cell imaging in retinal explants, indicating that CX3CR1 signaling in microglia regulates the phagocytic clearance of at‐risk photoreceptors. We also found that CX3CR1 deficiency in retinal microglia was associated with increased expression of inflammatory cytokines and microglial activation markers. Significantly, increasing CX3CL1‐CX3CR1 signaling in the rd10 retina via exogenous intravitreal delivery of recombinant CX3CL1 was effective in (1) decreasing microglial infiltration, phagocytosis and activation, and (2) improving structural and functional features of photoreceptor degeneration. These results indicate that CX3CL1‐CX3CR1 signaling is a molecular mechanism capable of modulating microglial‐mediated degeneration and represents a potential molecular target in therapeutic approaches to RP. GLIA 2016;64:1479–1491     

Altered microglia morphology and higher resilience to stress-induced depression-like behavior in CX3CR1-deficient mice

Microglia are suggested to be involved in several neuropsychiatric diseases. Indeed changes in microglia morphology have been reported in different mouse models of depression. A crucial regulatory system for microglia function is the well-defined CX3C axis. Thus, we aimed to clarify the role of microglia and CX3CR1 in depressive behavior by subjecting CX3CR1-deficient mice to a particular chronic despair model (CDM) paradigm known to exhibit face validity to major depressive disorder. In wild-type mice we observed the development of chronic depressive-like behavior after 5 days of repetitive swim stress. 3D-reconstructions of Iba-1-labeled microglia in the dentate molecular layer revealed that behavioral effects were associated with changes in microglia morphology towards a state of hyper-ramification. Chronic treatment with the anti-depressant venlafaxine ameliorated depression-like behavior and restored microglia morphology. In contrast, CX3CR1 deficient mice showed a clear resistance to either (i) stress-induced depressive-like behavior, (ii) changes in microglia morphology and (iii) antidepressant treatment. Our data point towards a role of hyper-ramified microglia in the etiology of chronic depression. The lack of effects in CX3CR1 deficient mice suggests that microglia hyper-ramification is controlled by neuron-microglia signaling via the CX3C axis. However, it remains to be elucidated how hyper-ramified microglia contribute to depressive-like behavior.     

Differential effects of stress on microglial cell activation in male and female medial prefrontal cortex

Susceptibility to stress-linked psychological disorders, including post-traumatic stress disorder and depression, differs between men and women. Dysfunction of medial prefrontal cortex (mPFC) has been implicated in many of these disorders. Chronic stress affects mPFC in a sex-dependent manner, differentially remodeling dendritic morphology and disrupting prefrontally mediated behaviors in males and females. Chronic restraint stress induces microglial activation, reflected in altered microglial morphology and immune factor expression, in mPFC in male rats. Unstressed females exhibit increased microglial ramification in several brain regions compared to males, suggesting both heightened basal activation and a potential for sex-dependent effects of stress on microglial activation. Therefore, we assessed microglial density and ramification in the prelimbic region of mPFC, and immune-associated genes in dorsal mPFC in male and female rats following acute or chronic restraint stress. Control rats were left unstressed. On the final day of restraint, brains were collected for either qPCR or visualization of microglia using Iba-1 immunohistochemistry. Microglia in mPFC were classified as ramified, primed, reactive, or amoeboid, and counted stereologically. Expression of microglia-associated genes (MHCII, CD40, IL6, CX3CL1, and CX3CR1) was also assessed using qPCR. Unstressed females showed a greater proportion of primed to ramified microglia relative to males, alongside heightened CX3CL1–CX3CR1 expression. Acute and chronic restraint stress reduced the proportion of primed to ramified microglia and microglial CD40 expression in females, but did not significantly alter microglial activation in males. This sex difference in microglial activation could contribute to the differential effects of stress on mPFC structure and function in males versus females.     

Microglia modulate hippocampal neural precursor activity in response to exercise and aging

Exercise has been shown to positively augment adult hippocampal neurogenesis; however, the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here, we used transgenic Csf1r-GFP mice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely, microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX(3)CL1, a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX(3)CL1 receptor, CX(3)CR1, but not control IgG, dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX(3)CL1 was observed following running, reduced levels of this chemokine were found in the aged brain. Lower levels of CX(3)CL1 with advancing age correlated with the natural decline in neural precursor cell activity, a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus, and that signaling through the CX(3)CL1-CX(3)CR1 axis critically contributes toward this process.     

Activation of cannabinoid receptor 2 attenuates mechanical allodynia and neuroinflammatory responses in a chronic post‐ischemic pain model of complex regional pain syndrome type I in rats

Complex regional pain syndrome type 1 (CRPS‐I) remains one of the most clinically challenging neuropathic pain syndromes and its mechanism has not been fully characterized. Cannabinoid receptor 2 (CB2) has emerged as a promising target for treating different neuropathic pain syndromes. In neuropathic pain models, activated microglia expressing CB2 receptors are seen in the spinal cord. Chemokine fractalkine receptor (CX3CR1) plays a substantial role in microglial activation and neuroinflammation. We hypothesized that a CB2 agonist could modulate neuroinflammation and neuropathic pain in an ischemia model of CRPS by regulating CB2 and CX3CR1 signaling. We used chronic post‐ischemia pain (CPIP) as a model of CRPS‐I. Rats in the CPIP group exhibited significant hyperemia and edema of the ischemic hindpaw and spontaneous pain behaviors (hindpaw shaking and licking). Intraperitoneal administration of MDA7 (a selective CB2 agonist) attenuated mechanical allodynia induced by CPIP. MDA7 treatment was found to interfere with early events in the CRPS‐I neuroinflammatory response by suppressing peripheral edema, spinal microglial activation and expression of CX3CR1 and CB2 receptors on the microglia in the spinal cord. MDA7 also mitigated the loss of intraepidermal nerve fibers induced by CPIP. Neuroprotective effects of MDA7 were blocked by a CB2 antagonist, AM630. Our findings suggest that MDA7, a novel CB2 agonist, may offer an innovative therapeutic approach for treating neuropathic symptoms and neuroinflammatory responses induced by CRPS‐I in the setting of ischemia and reperfusion injury.     

Interactions between chemokine and mu-opioid receptors: anatomical findings and electrophysiological studies in the rat periaqueductal grey

Opioids have immunomodulatory functions and may alter susceptibility to immune disorders. Behavioral studies also indicate that chemokines, molecules expressed by immune cells, block opioid-induced analgesia in the periaqueductal grey (PAG). Bi-directional heterologous desensitization of opioid and chemokine receptors has been described in cell systems. We report the anatomical and functional interactions of chemokine receptors with the mu-opioid receptor (MOR) in the rat brain. The chemokine receptors, CXCR4 and CX3CR1, as well as their chemokine substrates, CXCL12 and CX3CL1, are widely expressed in the central nervous system (CNS). Immunohistochemical techniques were utilized to investigate MOR-CXCR4 and MOR-CX3CR1 receptor colocalization in multiple brain areas. Our results demonstrate co-expression of these receptors on individual neurons in several regions including cingulate cortex, hippocampus, and PAG, suggesting functional receptor interactions. Whole-cell patch-clamp recordings of PAG neurons in a rat brain slice preparation were used to examine morphine or chemokine (CXCL12, CX3CL1) effects alone, or in combination on neuronal membrane properties. Morphine (10 μM) hyperpolarized and reduced input resistance of PAG neurons. CXCL12 and CX3CL1 (10 nM) had no impact on either parameter. In the presence of CXCL12, morphine’s electrophysiological effects were blocked in all neurons examined, whereas with CX3CL1, morphine’s effects were blocked in 57% of neurons studied. The data provide electrophysiological evidence for MOR-CXCR4 and MOR-CX3CR1 heterologous desensitization in the PAG at the single-cell level. These interactions may contribute to the limited utility of opioid analgesics for inflammatory pain treatment and supports chemokines as neuromodulators.     

The roles of fractalkine/CX3CR1 system in neuronal death following pilocarpine-induced status epilepticus

Although fractalkine is one of chemokines involved in mediation of neuronal/microglial interaction, it is not known whether fractalkine/CX3CR1-mediated pathogenesis occurs in the rat brain following epileptogenic insults. In order to elucidate the roles of the fractalkine/CX3CR1 system in microglial activation and neurodegeneration induced by status epilepticus (SE), we investigated changes in fractalkine/CX3CR1 system within the rat hippocampus following SE. In non-SE induced animals, fractalkine and CX3CR1 immunoreactivity was detected in neurons and microglia, respectively. Following SE, fractalkine immunoreactivity was transiently increased in neurons and astrocytes. CX3CR1 immunoreactivity was also transiently detected in neurons (particularly in CA1 pyramidal cells). Intracerebroventricular infusions of recombinant rat fractalkine aggravated SE-induced neuronal damage, while fractalkine IgG or CX3CR1 IgG infusion alleviated it, compared to saline-infused animals. These findings suggest that fractalkine/CX3CR1 system may play an important role in SE-induced neuronal damages via neuron-microglial interactions.     

CX3CR1 Disruption Differentially Influences Dopaminergic Neuron Degeneration in Parkinsonian Mice Depending on the Neurotoxin and Route of Administration

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons accompanied by an inflammatory reaction. The neuron-derived chemokine fractalkine (CX3CL1) is an exclusive ligand for the receptor CX3CR1 expressed on microglia. The CX3CL1/CX3CR1 signaling is important for sustaining microglial activity. Using a recently developed PD model, in which the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin is delivered intranasally, we hypothesized that CX3CR1 could play a role in neurotoxicity and glial activation. For this, we used CX3CR1 knock-in mice and compared results with those obtained using the classical PD models through intraperitonal MPTP or intrastriatal 6-hydroxydopamine (6-OHDA). The striatum from all genotypes (CX3CR1^+/+, CX3CR1^+/GFP and CX3CR1-deficient mice) showed a significant dopaminergic depletion after intranasal MPTP inoculation. In contrast to that, we could not see differences in the number of dopaminergic neurons in the substantia nigra of CX3CR1-deficient animals. Similarly, after 6-OHDA infusion, the CX3CR1 deletion decreased the amphetamine-induced turning behavior observed in CX3CR1^+/GFP mice. After the 6-OHDA inoculation, a minor dopaminergic neuronal loss was observed in the substantia nigra from CX3CR1-deficient mice. Distinctly, a more extensive neuronal cell loss was observed in the substantia nigra after the intraperitoneal MPTP injection in CX3CR1 disrupted animals, corroborating previous results. Intranasal and intraperitoneal MPTP inoculation induced a similar microgliosis in CX3CR1-deficient mice but a dissimilar change in the astrocyte proliferation in the substantia nigra. Nigral astrocyte proliferation was observed only after intraperitoneal MPTP inoculation. In conclusion, intranasal MPTP and 6-OHDA lesion in CX3CR1-deficient mice yield no nigral dopaminergic neuron loss, linked to the absence of astroglial proliferation.     

Cx3cr1‐deficiency exacerbates alpha‐synuclein‐A53T induced neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the degeneration of dopaminergic neurons of the substantia nigra and the accumulation of protein aggregates, called Lewy bodies, where the most abundant is alpha‐synuclein (α‐SYN). Mutations of the gene that codes for α‐SYN (SNCA), such as the A53T mutation, and duplications of the gene generate cases of PD with autosomal dominant inheritance. As a result of the association of inflammation with the neurodegeneration of PD, we analyzed whether overexpression of wild‐type α‐SYN (α‐SYN^WT) or mutated α‐SYN (α‐SYN^A53T) are involved in the neuronal dopaminergic loss and inflammation process, along with the role of the chemokine fractalkine (CX3CL1) and its receptor (CX3CR1). We generated in vivo murine models overexpressing human α‐SYN^WT or α‐SYN^A53T in wild type (Cx3cr1^+/+) or deficient (Cx3cr1^–/–) mice for CX3CR1 using unilateral intracerebral injection of adeno‐associated viral vectors. No changes in CX3CL1 levels were observed by immunofluorescence or analysis by qRT‐PCR in this model. Interestingly, the expression α‐SYN^WT induced dopaminergic neuronal death to a similar degree in both genotypes. However, the expression of α‐SYN^A53T produced an exacerbated neurodegeneration, enhanced in the Cx3cr1^–/– mice. This neurodegeneration was accompanied by an increase in neuroinflammation and microgliosis as well as the production of pro‐inflammatory markers, which were exacerbated in Cx3cr1^–/– mice overexpressing α‐SYN^A53T. Furthermore, we observed that in primary microglia CX3CR1 was a critical factor in the modulation of microglial dynamics in response to α‐SYN^WT or α‐SYN^A53T. Altogether, our study reveals that CX3CR1 plays an essential role in neuroinflammation induced by α‐SYN^A53T.