control of microglial neurotoxicity by the fractalkine receptor
TRANSCRIPT
Control of microglial neurotoxicity by thefractalkine receptor
Astrid E Cardona1, Erik P Pioro1, Margaret E Sasse1, Volodymyr Kostenko1, Sandra M Cardona1,Ineke M Dijkstra1, DeRen Huang1, Grahame Kidd1, Stephen Dombrowski2, RanJan Dutta1, Jar-Chi Lee3,Donald N Cook4, Steffen Jung5,6, Sergio A Lira7, Dan R Littman6 & Richard M Ransohoff1
Microglia, the resident inflammatory cells of the CNS, are the only CNS cells that express the fractalkine receptor (CX3CR1).
Using three different in vivo models, we show that CX3CR1 deficiency dysregulates microglial responses, resulting in
neurotoxicity. Following peripheral lipopolysaccharide injections, Cx3cr1–/– mice showed cell-autonomous microglial neurotoxicity.
In a toxic model of Parkinson disease and a transgenic model of amyotrophic lateral sclerosis, Cx3cr1–/– mice showed more
extensive neuronal cell loss than Cx3cr11 littermate controls. Augmenting CX3CR1 signaling may protect against microglial
neurotoxicity, whereas CNS penetration by pharmaceutical CX3CR1 antagonists could increase neuronal vulnerability.
CX3CR1 is expressed by monocytes, dendritic cells (DCs), and subsetsof T cells and natural killer cells in the circulation and by microglia inthe central nervous system (CNS)1. Fractalkine (CX3CL1), the exclu-sive ligand for CX3CR1, is synthesized as a transmembrane glycopro-tein, from which a soluble chemokine can be proteolytically released2.CX3CR1/CX3CL1 signaling exerts distinct functions in different tissuecompartments: CX3CR1 deficiency impairs the morphogenesis ofmyeloid DCs, which occupy the lamina propria of the small intestine3;further, CX3CR1 contributes to the migration of circulating monocytesto noninflamed tissues, where they differentiate into macrophages andDCs. However, peripheral immune and inflammatory reactions aremostly unaltered in Cx3cr1–/– or Cx3cl1–/– mice4,5. CX3CR1 is respon-sible for recruiting natural killer cells to cardiac allografts6 and to theinflamed CNS of mice with experimental autoimmune encephalomye-litis7. In vitro, CX3CL1 promotes neuronal survival and inhibitsmicroglial apoptosis8, but the roles of CX3CL1/CX3CR1 in the intactCNS are enigmatic.
Microglia, which have characteristics of immature myeloid cells,sample the extracellular space of the healthy CNS through continuousextension, retraction and remodeling of cellular processes9,10. Inresponse to injury, microglial cells undergo rapid morphological andfunctional activation and acquire properties of mature myeloid cells,including antigen presentation, reactive species generation, matrixmetalloproteinase (MMP) expression and phagocytosis, as well ascytokine and growth factor secretion11. Microglial production ofreactive species, MMPs and inflammatory cytokines have been impli-cated in neurotoxicity in vitro. However, control of microglial neuro-toxicity in vivo remains poorly understood. Altered microglial function
can cause CNS disease in humans: homozygous deficiency of eithertype 2 triggering receptor expressed on myeloid cells (TREM2) or itsintracellular adaptor TYROBP causes adult onset dementing leuko-encephalopathy, which is recapitulated in Tyrobp–/– mice12. Knock-down of TREM2 in mouse microglia impairs their phagocytic activityand enhances inflammatory gene expression, suggesting a mechanismfor the human disorder13.
To investigate the role of CX3CR1 in the intact CNS, we used mice inwhich the Cx3cr1 gene was replaced with a cDNA encoding greenfluorescent protein (GFP), such that heterozygous Cx3cr1+/GFP
(Cx3cr1+/–) mice expressed the GFP reporter in cells that retainedreceptor function, whereas Cx3cr1GFP/GFP (Cx3cr1–/–) cells were labeledand also lacked CX3CR1 (ref. 5). The CX3CR1-GFP reporter identifiesmicroglia in vivo5,9,10 and permits their isolation and purificationex vivo. Our results indicated that in the absence of CX3CR1/CX3CL1 signaling, microglia had altered responses, both to inflam-matory and neurotoxic stimuli. In particular, CX3CR1 deficiency wasassociated with neuronal cell death after systemic lipopolysaccharide(LPS) challenge. Cx3cr1–/– mice demonstrated more neuronal cell lossin a toxin-induced model of Parkinson disease and in a model ofgenetic motor neuron disease. The results identified CX3CL1 as thefirst soluble factor that regulates microglial neurotoxicity.
RESULTS
Neuronal damage in Cx3cr1–/– mice after systemic LPS
We first examined the GFP+ population in the CNS of Cx3cr1GFP miceand found that green fluorescent cells overlapped precisely with braincells expressing microglial markers such as ionized calcium-binding
Received 6 March; accepted 5 May; published online 28 May 2006; corrected online 11 June 2006; doi:10.1038/nn1715
1Neuroinflammation Research Center and Department of Neurosciences, Lerner Research Institute, 2Department of Neurosurgery, and 3Department of Quantitative HealthSciences, Cleveland Clinic, Cleveland, Ohio 44195, USA. 4National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.5Department of Immunology, Weizman Institute of Science, Rehovot 76100, Israel. 6Howard Hughes Medical Institute and Skirball Institute for Biomolecular Medicine, NewYork University School of Medicine, New York, New York 10016, USA. 7Immunobiology Center, Mount Sinai School of Medicine, New York, New York 10029, USA.Correspondence should be addressed to R.M.R. ([email protected]).
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adapter molecule-1 (IBA-1)14 (Fig. 1a–c) and Griffonia simplicifoliaisolectin B4 (data not shown). This indicated that all CNS microgliawere GFP+ inCx3cr1+/GFP and Cx3cr1GFP/GFP mice, designated below asCx3cr1+/– and Cx3cr1–/–, respectively. According to a previous report15,various CNS cells express CX3CR1 immunoreactivity. We found nocells that coexpressed GFP and neuron-specific nuclear protein (NeuN)(Fig. 1d–f), the proteoglycan NG2 (Fig. 1g–i) or glial fibrillary acidicprotein (GFAP) (Fig. 1j–l), indicating that neither CNS neurons, NG2+
glia nor astrocytes expressed the CX3CR1 transcription unit in vivo bythis analysis.
To examine microglial activation in the absence of CX3CR1, weinduced a systemic inflammatory reaction by means of intraperitoneal(i.p.) injections of LPS (ref. 16). Cx3cr1+/– mice showed moderatemorphological transformation of microglia (Fig. 2a), whereasCx3cr1–/– mice showed intense and widespread microglial activation(Fig. 2b). Cx3cr1–/– but not Cx3cr1+/– mice showed numerous annexinV–positive cells with neuronal morphology, throughout the cortexand hippocampus (Fig. 2c–f). We quantified the annexin V–positivecells in the dentate gyrus and found that they were significantlymore numerous in Cx3cr1–/– mice (Fig. 2g; P ¼ 0.0012) that receivedLPS than in controls (Fig. 2g). We obtained compatible results fromTdT-mediated dUTP nick end labeling (TUNEL) analyses (data notshown). Based on these findings, we formed the hypothesis thatsystemic inflammation, induced by LPS, activated the neurotoxicpotential of microglia in Cx3cr1–/– mice. We asked whether directlocal effects of LPS in Cx3cr1–/– mice might differ from those inCx3cr1+/– mice. RNase protection assay (RPA) of peritoneal macro-phages from LPS-challenged Cx3cr1+/– and Cx3cr1–/– mice demon-strated equivalent production of representative inflammatory cytokines(Fig. 2h). This finding suggested that direct local effects of LPS wereequivalent in heterozygous and knockout mice and that the neurotoxicconsequences of peripheral LPS injections in Cx3cr1–/– mice arosewithin the CNS.
Cell-autonomous neurotoxicity of Cx3cr1–/– microglia
To address whether microglial neurotoxicity induced by systemicinflammation was cell autonomous, we developed an adoptive transferprotocol using purified populations of GFP-labeled activated micro-glial cells (Fig. 3a). These were isolated from LPS-injected Cx3cr1–/– orCx3cr1+/– mice (n ¼ 6–8 donor mice per inoculum), washed andmicroinjected into the frontal cortex of wild-type recipient mice(Fig. 3b). At 36 h after transfer, recipient brains were analyzed for
the presence of microglia in serial free-floatinghorizontal sections through the needle track,and 1–2 mm ventral to the end of the needlemark. The injection sites were defined as thelast section containing a visible needle trackand the next serial section without the needleartifact. Examination of injection sites(Fig. 3c–f) showed divergent phenotypes forCx3cr1+/– (Fig. 3c) as compared withCx3cr1–/– microglial cells (Fig. 3d). Microgliafrom Cx3cr1+/– mice, as previously describedfor myeloid cells17, were not observed atinjection sites (Fig. 3c) and migrated widelythroughout the CNS parenchyma, preferen-tially in white matter tracts (Fig. 3g). Incontrast, microglial cells from Cx3cr1–/– miceremained localized at the injection site(Fig. 3d) and migrating cells (Fig. 3g–j)were not detected in wild-type recipients of
activated Cx3cr1–/– microglia (Fig. 3h). Furthermore, we analyzedneuronal cell death (Fig. 3k–n) and readily identified TUNEL/NeuNdouble-positive neurons near the injection site (60–200 mm deep) inwild-type recipients of Cx3cr1–/– microglia (Fig. 3l) but not inrecipients of Cx3cr1+/– microglia (Fig. 3k). The CNS tissues of wild-type recipients of Cx3cr1–/– microglia contained significantly fewermigrating cells (Fig. 3o; P¼ 0.02 compared with Cx3cr1+/– microglia).Additionally, significantly more TUNEL-positive neurons weredetected in the CNS of wild-type recipients of Cx3cr1–/– microglialcells (Fig. 3p; P ¼ 0.02). The microglial phenotype in these adoptivetransfer experiments was dependent both on genotype and activationstatus, as unactivated microglia from Cx3cr1–/– control mice (thatreceived i.p. saline injections) distributed throughout the white mattertracts of recipient wild-type mice (data not shown). These findingsdemonstrated that activated Cx3cr1–/– microglia are neurotoxic in theCNS of wild-type mice and provided an assay for evaluating putativemediators of this toxic effect.
Our initial results suggested that CX3CR1 modulated the response ofCNS microglia to systemic inflammation. This hypothesis was furthertested by ex vivo RPA, using highly enriched preparations of microglia(Fig. 3a) from Cx3cr1+/– or Cx3cr1–/– mice, purified from CNS tissuesafter induction of systemic inflammation by i.p. LPS injections. Theseexperiments demonstrated increased expression of interleukin (IL)-1b,but not tumor necrosis factor (TNF)-a, IL-6 or lymphotoxin, bymicroglia from LPS-injected Cx3cr1–/– mice (Fig. 3q, P ¼ 0.02).
Microglia from LPS-injected Cx3cr1–/– mice also demonstratedelevated production of IL-1b–associated signaling intermediates suchas Myd88 (data not shown). These findings were noteworthy, becauseIL-1 action through IL-1 receptor type 1 (IL-1R1) has been consistentlyassociated with neurodegeneration in vivo18. We tested whether IL-1was a mediator of neurotoxicity caused byCx3cr1–/– microglia, by usingIL-1 receptor antagonist (IL-1RA), which blocks the assembly of the IL-1R1/IL-1 receptor accessory protein (IL-1RAcP) signaling complex.When IL-1RA was included in the adoptive transfer inoculum alongwith activated microglia from LPS-injected Cx3cr1–/– mice, weobserved a reversal of the knockout phenotype (compare Fig. 3d,h,lwith Fig. 3e,i,m). In particular, blockade of IL-1 signaling restoredmigration of Cx3cr1–/– microglia throughout the CNS of wild-typerecipients (Fig. 3o; P ¼ 0.03 comparing Cx3cr1–/– microglia with andwithout IL-1RA), and the number of TUNEL-positive neurons wassignificantly reduced (Fig. 3p; P¼ 0.02 comparing wild-type recipientsof Cx3cr1–/– cells with and without IL-1RA). Coinjection of Cx3cr1–/–
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Figure 1 Microglial cells comprise the CX3CR1/GFP+ population. (a–l) Brain sections from Cx3cr1+/–
mice immunostained with antibodies to IBA-1 (a–c), NeuN (d–f), NG2 (g–i) and GFAP (j–l), showing
that the GFP+ population overlaps precisely with IBA-1+ microglial cells (c, merged image). Cellsexpressing the CX3CR1-GFP reporter (d,g,j) did not colocalize to markers of neurons (e: NeuN+ cells,
red), NG2+ glial cells (h: NG2+, red) or astrocytes (k: GFAP+, red) as shown in merged images (f,i,l).
Scale bar, 25 mm.
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microglia with inert carrier protein did not enhance microglial migra-tion or reduce microglial neurotoxicity (Fig. 3o,p).
It remained uncertain whether neurotoxic properties of IL-1 in theseexperiments proceeded through autocrine effects of IL-1 on Cx3cr1–/–
microglia or paracrine effects on recipient microglia, astrocytes andneurons, as IL-1RA blocked signaling to all cells within the injectionsite. Adoptive transfer of activated Cx3cr1–/– cells into the CNS ofIl1r1–/– recipients demonstrated that defective migration by Cx3cr1–/–
microglial cells could be partially rescued by abrogation of the hostresponse to IL-1. In Il1r1–/– recipients, transferred Cx3cr1–/– microglialcells remained partially localized at the injection site (Fig. 3f) butmigrating donor-derived GFP+ cells were readily detected (Fig. 3j) andwere significantly more numerous than in the wild-type recipients ofCx3cr1–/– microglia (Fig. 3o; P¼ 0.04 comparing Il1r1–/– and wild-typerecipients of Cx3cr1–/– cells). Notably, TUNEL-positive cells werevirtually absent from the CNS of Il1r1–/– recipients of Cx3cr1–/–
microglia (Fig. 3n,p; P ¼ 0.02 comparing Cx3cr1–/– microglia trans-ferred to wild-type and Il1r1–/– mice).
To exclude the possibility that these results emerged from variabilityin the placement of the microinjected cells, we performed stereotacticadoptive transfer experiments. These experiments required that8-week-old recipients be used, in order to accommodate the stereo-tactic apparatus (Fig. 4a). We observed that Cx3cr1–/– microglia(Fig. 4b) but not Cx3cr1+/– microglia (Fig. 4c) remained at the
injection sites. Cx3cr1+/– microglia but notCx3cr1–/– microglia were found migratingthroughout the white matter of recipients(Fig. 4d,e). Following transfer of Cx3cr1–/–
microglia but not Cx3cr1+/– microglia, wedetected annexin V–positive neurons (com-pare Figs. 4f and g).
Together, these results established thatCX3CR1 governs critical components of themicroglial response to systemic inflammationin vivo, as suggested by previous in vitrostudies8,19, and that IL-1 is a mediatorof microglial neurotoxicity induced bysystemic inflammation.
SNpc neurons in Cx3cr1–/– or Cx3cl1–/–
mice after MPTP
Microglial neurotoxicity has also been pro-posed to augment the severity of neurodegen-erative processes including Parkinson disease.We addressed a potential role for CX3CR1 inthe microglial reaction to neurodegenerationby evaluating the responses of gender-matchedlittermate Cx3cr1–/–, Cx3cr1+/– and Cx3cr1+/+
mice to the administration of the dopaminergicneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP), which recapitulatesselected features of Parkinson disease.CX3CR1 deficiency aggravated the pathologicaloutcomes of MPTP injection in mice. Weperformed a stereological analysis of the sub-stantia nigra pars compacta (SNpc) of saline-(Fig. 5a–c) and MPTP-injected (Fig. 5d–i)Cx3cr1+/+ and Cx3cr1–/– littermate mice 7 dafter challenge (Table 1). We found a signifi-cant MPTP-induced loss of tyrosine hydroxy-lase–immunoreactive (TH-IR) cells and a
similar loss of Nissl-stained neurons in the SNpc of Cx3cr1+/+ mice,with no change in the percent of Nissl-stained cells that were TH-IR.The loss of both TH-IR cells and Nissl-stained cells was significantly(P o 0.001) worse in Cx3cr1–/– mice (Table 1). We also evaluated theeffect of CX3CL1 ligand deficiency in this model: the results were nearlyidentical to those observed in receptor-deficient Cx3cr1–/– mice(Table 1). Saline-injected mice showed no genotype-related differences(P ¼ 0.86 for TH-IR cells; P ¼ 0.47 for Nissl-stained cells). Transportand metabolism of MPTP were not altered in Cx3cr1–/– mice, asconcentrations of MPTP in the SN were equivalent in heterozygousand knockout mice, and striatal concentrations of 1-methyl-4-phenyl-pyridinium (MPP+) were not significantly different, either 90 min or3 h after injection (data not shown). Inspection of coronal sections ofthe SNpc ofCx3cr1+/– andCx3cr1–/– mice (Fig. 5j,k) suggested a greaterdegree of morphological transformation of knockout microglia, ascompared to those in heterozygous mice, in response to MPTPinjections. Taken together, these results demonstrated that the responseto MPTP in mice lacking microglial CX3CR1 signaling caused greaterloss of TH-IR neurons.
Worsened disease in Cx3cr1–/–SOD1G93A transgenic mice
Microglial cells have also been implicated in the loss of motor neuronsduring the fatal neurodegenerative disorder amyotrophic lateral sclero-sis (ALS). Transgenic mice that overexpress a mutant form of the
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Figure 2 Cx3cr1–/– mice show increased microglial activation and enhanced neuronal damage after
systemic inflammation. (a,b) IBA-1 immunohistochemistry revealed highly ramified microglia in the
hippocampus of LPS-treated Cx3cr1+/– mice (a), compared to increased microglial activation in
Cx3cr1–/– mice (b) whose cells have shorter and thicker processes and bigger cell bodies. (c–f) Annexin V
immunostaining showed numerous annexin V–immunoreactive cells in Cx3cr1–/– mice (d,f: brown
staining, arrows) with the nuclear morphology of neurons, but not in Cx3cr1+/– mice. (g) Quantitation of
annexin V–positive cells showed a significant (P ¼ 0.0012) increase of annexin V–positive cells in the
dentate gyrus of Cx3cr1–/– mice after LPS administration. (h) RNase protection analysis of RNA from
peritoneal macrophages showed low cytokine levels in saline-injected Cx3cr1+/– mice (gray), and equal
responses in LPS-injected Cx3cr1+/– (white) and Cx3cr1–/– mice (black). Error bars represent s.d.
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human SOD1 gene (SOD1G93A) encoding copper/zinc superoxidedismutase show age-dependent degeneration of motor neurons accom-panied by limb weakness and provide a useful model of ALS (ref. 20).We established a colony of mice that were SOD1G93A/Cx3cr1+/+,SOD1G93A/Cx3cr1+/–, SOD1G93A/Cx3cr1–/– or nontrangenic/Cx3cr1–/–,and performed serial neurobehavioral and survival assessment ofSOD1G93A/Cx3cr1+/– and SOD1G93A/Cx3cr1–/– mice, with histologicalanalysis of a cohort of surviving mice from all four genotypes at 133 d.Microglial reaction, as judged by IBA-1 immunohistochemistry,showed morphological transformation in the lumbar spinal cords ofall transgenic mice (Fig. 6a–d), without significant genotype-relateddifferences in the tissue area showing IBA-1 immunoreactivity (46% in
SOD1G93A/Cx3cr1–/– compared to 39% in SOD1G93A/Cx3cr1+/+ mice;P ¼ 0.12). SOD1G93A/Cx3cr1–/– mice showed decreased neuronal celldensity at this time point (Figs. 6e–i) when compared with eitherlittermate SOD1G93A/Cx3cr1+/+ (P ¼ 0.02) or SOD1G93A/Cx3cr1+/–
(P ¼ 0.03) mice (Fig. 6i). Comparisons of neuronal cell densitybetween transgenic heterozygous and transgenic wild-type mice didnot show statistically significant differences (Fig. 6i; P ¼ 0.09 compar-ing SOD1G93A/Cx3cr1+/– and SOD1G93A/Cx3cr1+/+ mice).
Behavior and survival analyses also revealed differences betweenSOD1G93A/Cx3cr1+/– and SOD1G93A/Cx3cr1–/– mice. Hindlimb gripstrength showed evident decline after 7–9 weeks in all SOD1G93A
mice, as compared to nontransgenic/Cx3cr1–/– littermate control mice
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Figure 3 Adoptive transfer studies of microglia by intracranial microinjection. (a) Flow cytometry shows microglial preparations containing 490% GFP+ cells
(purple overlay, GFP– Cx3cr1+/+ microglia), used for RNA isolation or adoptive transfer. (b) Recipient brains (shown in axial section) were stained using NeuN
antibodies (blue) and TUNEL (red). (c–j) Merged GFP-NeuN images show injection sites within dotted ovals (c–f) and points of migration (g–j). (k–n) Apoptotic
neurons in merged GFP-NeuN-TUNEL images. Cx3cr1+/– microglia (c) were not detected at the injection site, but were distributed along white matter tracts
(g, arrows), whereas Cx3cr1–/– microglia (d) remained localized at injection site, did not distribute throughout the brain (h) and were associated with apoptotic
neurons 60–200 mm from the injection site (i, arrows). When LPS-activated Cx3cr1–/– microglia were transferred with IL-1RA into wild-type recipients (e,i,m),few GFP+ microglia were found at the injection site (e) and migrating cells were seen (i, arrowhead) without neuronal apoptosis near the injection site (m). In
Il1r1–/– recipients (f,j,n), many Cx3cr1–/– microglia persisted at the injection site (f), but were also detected at points of migration (j, arrowhead) without
associated neuronal cell death (n) near the injection site. (o) Number of migrating microglia, counted at sites shown in b (asterisks). (p) Apoptotic neurons
were counted in serial sections at a depth of 60–200 mm from the injection site, using transfer preparations and recipients as indicated. Scale bars, 25 mm.
(q) Cytokine profiling by RPA revealed an increased expression of IL-1b by activated Cx3cr1–/– microglia. Error bars represent s.d.
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which became stronger as they attained adulthood (data not shown).Between weeks 15 through 20, when weakness became progressive,random coefficient modeling disclosed a strong age � group interaction(P o 0.01) indicating that SOD1G93A/Cx3cr1–/– mice had a fasterdecline in hindlimb grip strength than SOD1G93A/Cx3cr1+/– mice.With further analysis of the data, we found that the male Cx3cr1–/–
group showed a much steeper decline (slope ¼ –19.92 ± 2.1 (s.e.m.),n¼ 5) than the other groups (male Cx3cr1+/–: –6.01 ± 2.5, n¼ 5; femaleCx3cr1+/–: –6.72 ± 3.5, n¼ 6; and female Cx3cr1–/–: –8.41 ± 2.4, n¼ 7),accounting for the group differences (Fig. 6j). Male SOD1G93A/Cx3cr1–/– mice also showed faster loss of body weight (P o 0.01) andforelimb grip strength (P ¼ 0.02) than male SOD1G93A/Cx3cr1+/– mice(data not shown). Using a defined indicator of terminal state andKaplan-Meier analysis (Fig. 6k), survival was significantly (log rank P¼0.003) reduced in male SOD1G93A/Cx3cr1–/– mice as compared withmale SOD1G93A/Cx3cr1+/– mice and with female SOD1G93A/Cx3cr1+/–
or SOD1G93A/Cx3cr1–/– mice. Notably, there is precedent for a gendereffect in this model, as absence of the p75NTR receptor was selectivelybeneficial for female, but not male, SOD1G93A mice21. In the aggregate,
the results of these experiments further supported the role of CX3CR1as a key regulator of microglial neurotoxicity in the contexts of eitherinflammation or neurodegeneration.
DISCUSSION
We demonstrated a role for the chemokine receptor CX3CR1 inmicroglial neurotoxicity in three clinically relevant models: CNSresponse to systemic inflammation, the MPTP model of Parkinsondisease, and the SOD1G93A model of ALS. Based on complementaryexpression of CX3CL1 on neurons and CX3CR1 on microglia, it hasbeen proposed that neuron signaling to microglia might be mediatedthrough this receptor1. Previous in vitro results support this concept:excitotoxic injury is a potent stimulus for release of CX3CL1 fromcultured neurons22; CX3CR1 supports neuronal survival23; microgliacultured with CX3CL1 are protected from Fas-mediated apoptosis8;and CX3CL1 suppresses neuronal cell death in LPS- and IFN-g–stimulated microglial and neuronal cocultures24. Our currentfindings confirm this hypothesis in vivo and establish models forinvestigating mechanisms of microglial neurotoxicity. For example,we used an adoptive transfer protocol to examine neurotoxicmechanisms in LPS-injected Cx3cr1–/– mice: the GFP reporter wasused to verify purification of relatively large numbers of microgliathat had been activated in vivo, and our results implicated IL-1 in
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a Figure 4 Adoptive transfer studies using stereotaxic placement of microglial
cells. (a–g) Thirty-six hours after stereotaxic placement of microglial cells
(a, stereotactic coordinate), recipient brains were analyzed for the presence of
GFP+ microglia (green microglial cells in b,c; merged GFP–4,6-diamidino-2-
phenylindole (DAPI) images in d,e). Sections were stained for NeuN (blue)
and annexin V (red), shown in merged GFP-NeuN–annexin V images (f,g).
Cx3cr1–/– microglia (b) remained clustered at the injection site (dotted oval),
without evidence of migration along white matter or subventricular zones (d),and apoptotic neurons near the injection site were easily detected
(f, arrowheads). In contrast, Cx3cr1+/– microglial clusters were not detected
at the injection site (c), no evident association with neuronal cell death was
observed (g), and these microglia distributed along white matter tracts and
subventricular zones (e). Scale bars in d–g, 25 mm.
Figure 5 Enhanced neurotoxic effects of MPTP in Cx3cr1–/– mice.
(a–i) Matched sections at the level of the SNpc were TH-immunostained
and counterstained with cresyl violet in saline- (a–c) and MPTP-treated
mice (d–i). Seven days after MPTP administration, we observed a reduction
in the number of TH-IR cells in the SNpc (outlined) of Cx3cr1+/+ mice
(d–f), with more pronounced effects in mice lacking CX3CR1 (g–i).
(j,k) Immunofluorescent labeling of TH neurons (red) shows decreasednumbers of neurons and robust microglial activation (green) in the SNpc
(arrows) of Cx3cr1–/– mice (inset in k, showing high-power confocal imaging
of TH-IR neurons and GFP+ microglia) compared to Cx3cr1+/– mice (inset
in j). Panels a–k: same magnification, scale bars 200 mm.
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microglial neurotoxicity following systemic inflammation. We proposethat roles of CX3CR1 in microglial activation will differ depending onthe nature and chronicity of the activating stimulus. For example,Cx3cr1–/– mice show a normal level of microglial activation afterfacial nerve axotomy5 and after laser-induced injury9. Furthermore,Cx3cl1–/– mice show relative protection from cerebral ischemia25, andintrathecal injection of CX3CL1 enhances nociception26,27, possibly byactivating microglia.
The molecular foundations of microglial neurotoxicity have beenobscure. Here, we identify CX3CR1 as the first selective regulator ofmicroglial neurotoxicity in vivo. Mice deficient for CD200, a neuronal
glycoprotein whose receptor, CD200R, is expressed by all myeloidcells, show aberrant microglial physiology including morphologicalactivation of microglia in the resting CNS and accelerated responseto facial nerve transection28,29. None of these attributes of alteredmicroglial function are observed in Cx3cr1–/– mice5,7,30, indicatingdifferent functions for CD200/CD200R and CX3CL1/CX3CR1 inregulating microglia.
We propose that tonic neuronal release of CX3CL1 provides specificrestraint of microglia in the healthy CNS, where the blood-brain barrieris intact. In support of this concept, we found 4300 pg of solubleCX3CL1 per milligram of aqueous extracts of the adult mouse brain
Table 1 Stereological counts of neurons in the SNpc
Saline MPTP 2d MPTP 7d
Wild-type Cx3cr1–/– Cx3cl1–/– Wild-type Cx3cr1–/– Wild-type Cx3cr1+/– Cx3cl1+/– Cx3cr1–/– Cx3cl1–/–
n 4 4 4 7 7 7 3 3 7 7
TH 11,549 ± 612 12,088 ± 752 11,958 ± 851 6,550 ± 640 2,907 ± 551 7,403 ± 632 7,086 ± 900 7,616 ± 1,761 3,564 ± 615 3,728 ± 604
Nissl 15,081 ± 730 13,851 ± 968 16,678 ± 2,506 8,448 ± 1,031 4,295 ± 741 11,309 ± 880 10,258 ± 943 12,829 ± 2,514 6,073 ± 906 6,791 ± 735
SNpc neurons (mean ± s.e.m.) were counted by stereology. P o 0.001 when comparing wild-type mice (saline-treated versus MPTP-treated), and when comparing MPTP-treated groups(wild-type versus Cx3cr1–/– or Cx3cl1–/–) at day 2 or day 7 after MPTP administration (ANOVA with Newman-Keuls test).
Cx3cr1+/+ Cx3cr1+/–
SOD1G93A
Cx3cr1–/– Cx3cr1–/–
Cx3cr1+/–
Cx3cr1–/–
0
10
20
30
40
50
60
70
80
90
0.09
Num
ber
of n
euro
ns p
er s
ectio
n
0
14 15 16 17Age (weeks)
18 19 20
10
20
3040
506070
8090
100
Male SOD1G93A
Hin
dlim
b st
reng
th (
gram
s)
0
20
40
60
80
100
Per
cent
age
at te
rmin
al s
tage
0.030.02
i j k
a b c d
e f g h
0 50 100Age (d)
150 200
Cx3cr1–/–Male SOD1G93A
Cx3cr1+/–Male SOD1G93A
Cx3cr1–/–Female SOD1G93A
Cx3cr1+/–Female SOD1G93A
Figure 6 Microglial activation, neuron loss, hindlimb grip strength and survival in SOD1G93A/Cx3cr1 mice. (a–d) IBA-1 immunohistochemistry shows a
progression of resting microglial cells in nontransgenic Cx3cr1–/– spinal cord (a) to microglial activation in SOD1G93A/Cx3cr1+/+ (b), SOD1G93A/Cx3cr1+/– (c)and SOD1G93A/Cx3cr1–/– (d) mice. (e–h) Nissl staining shows healthy-appearing neurons in nontransgenic spinal cord (e, arrows). In contrast, healthy-
appearing neurons in SOD1G93A transgenic mice were fewer in CX3CR1-deficient mice. Many surviving cells showed chromatin condensation (f,g, arrowheads).
No healthy-appearing neurons were observed in the ventral horns of the lumbar spinal cords of SOD1G93A/Cx3cr1–/– mice, and the remaining neuronal nuclei
showed abnormally condensed chromatin (h, arrowheads). (i) Quantitation showed significantly (P ¼ 0.03) more remaining neuronal cells in SOD1G93A/
Cx3cr1+/– mice than in SOD1G93A/Cx3cr1–/– mice, and much higher neuronal counts in healthy nontransgenic Cx3cr1–/– controls. (j) Graphs of hindlimb grip
strength measurements showing that male SOD1G93A/Cx3cr1–/– mice lost grip strength more rapidly between weeks 15–18 than did SOD1G93A/Cx3cr1+/–
males. (k) Kaplan-Meier survival curves revealed that male SOD1G93A/Cx3cr1–/– mice died sooner than any of the other groups. Scale bars in a–h, 50 mm.
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(data not shown), and others have reported the presence in the CNS ofADAM10, the catalyst of constitutive CX3CL1 release31. When theblood-brain barrier is disrupted, the roles of CX3CL1/CX3CR1 may bequite different: as noted above, CX3CL1-deficient mice are moderatelyprotected from focal ischemic stroke25 and mice lacking CX3CR1develop increased disease severity after the induction of experimentalautoimmune encephalomyelitis (EAE)6, due at least in part to thedeficient recruitment of regulatory natural killer cells7.
The activities of CX3CL1/CX3CR1 in various tissues are remarkablyvaried. Recent evidence from animal models implicates CX3CL1 as amediator of atherosclerosis32, and an allele of CX3CR1 that exertsimpaired adhesive function and blunted signaling is associated withdominantly inherited, reduced risk for atherosclerotic end points33.Based on these and other findings, efforts to develop pharmacologicalinhibitors of CX3CR1 are in progress34. Our current findings suggestthat impaired CX3CR1 function in the CNS may worsen neurodegen-eration. Therefore, agents designed to block CX3CR1 might show en-hanced safety profiles if they were excluded from the CNS. Further, thedominantly acting atheroprotective CX3CR1I249/M280 variant may meritevaluation as a risk factor for susceptibility or severity in neurodegen-erative disorders. Finally, the signaling by which CX3CR1 regulatesmicroglial neurotoxicity in vivo can now be further addressed, as such aneffort may lead to new therapeutic strategies for neuroprotection.
METHODSMice. Cx3cl1+/+, Cx3cl1–/–, Cx3cr1–/–, Cx3cr1+/– and Cx3cr1+/+ mice were
generated from heterozygous breeding pairs, backcrossed for more than
10 generations to C57BL/6 (refs. 2,5). Experimental protocols were performed
in accordance with US National Institutes of Health guidelines on animal care
and were approved by the Cleveland Clinic Animal Care and Use Committee.
Lineage marker analyses of the CX3CR1-GFP+ population in the CNS.
Cx3cr1+/– mice were perfused and 30-mm sections were stained with rabbit
polyclonal antibody to IBA-1 (anti–IBA-1), antibody to NG2 (anti-NG2) or
antibody to GFAP (anti-GFAP), followed by Cy3-conjugated secondary anti-
bodies (mounted in FluorSave, Calbiochem). For IBA-1 and GFAP stainings,
sections were incubated overnight at 4 1C. For NG2 staining, sections were
incubated with primary antibody (gift from W. Stallcup) at room temperature
(22 1C) for 5 h, followed by incubation at 4 1C for 48 h. Sections were imaged
by confocal microscopy, and projections of 20-mm z-stacks at 40� magnifica-
tion are shown.
Induction and analysis of systemic inflammation by lipopolyssacharide
administration. Mice were injected daily (i.p.) for 4 d with LPS (Sigma;
20 mg in 100 ml phosphate-buffered saline, PBS) or mock-injected with PBS.
Four hours after final injection, mice were killed and peritoneal cells were
aspirated with Hank’s Balanced Salt Solution(HBSS), washed and resuspended
in TRIZOL (Invitrogen). RNA was extracted and subjected to RPA with32P-labeled antisense riboprobes (BD Pharmingen) with results for each
cytokine normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
signals in two independent experiments. Sections from LPS- or saline-injected
mice were stained with rabbit polyclonal anti–IBA-1 or antibody to annexin V
(Novus Biologicals), counterstained with 0.5% cresyl violet (Sigma), and
positive cells in the dentate gyrus were counted at 40� magnification in
6–8 matched 30-mm sagittal sections. Results are presented as annexin
V–positive cells per mm2, with areas measured from 10� images using Image
J.1.34vi software (National Institutes of Health).
Isolation of microglial cells, RNase protection and flow cytometry. After
perfusion in HBSS without Ca2+ and Mg2+, brains were collected in 10 ml
HBSS per brain, containing 0.05% collagenase D (Roche), 0.1 mg ml–1 N-tosyl-
L-leucine chloromethyl ketone (TLCK, Sigma), 10 mg ml–1 dispase (Roche) and
10 mM HEPES buffer (Invitrogen). Brain tissues were dispersed with a glass
dounce homogenizer, and cells were separated over discontinuous per-
coll gradients (Supplementary Methods online). Cells were washed and
resuspended in TRIZOL for RNA extraction. RNA was assessed using a
Bioanalyzer (Affymetrix). Representative cytokines were determined by RPA
as described for peritoneal cells. For flow cytometry, cells were diluted in PBS
(4 � 105 cells ml–1), and GFP+ cells were acquired on an LSR (BD Immuno-
cytometry), gated by forward and side light-scattering properties and analyzed
with WinList software (Verity Software).
Adoptive transfer of microglia. For intracranial microinjections, microglia
were resuspended at 8 � 106 cells ml–1 (Supplementary Methods) and trans-
ferred within 30 min of isolation. Recipient mice (4–5 weeks old) were
anesthetized with ketamine (30 mg per kg body weight) and xylazine (4 mg
per kg body weight) and injected intracranially with a 60-ml cell suspension
(B5 � 105 cells). Using 1-ml syringes, 25-gauge 5/8’’ needles were inserted to a
2-mm depth in the frontal cortex with handmade needle-cap adaptors. C57BL/
6 recipients received Cx3cr1+/–, or Cx3cr1–/– microglia alone or with 3 ng of
IL-1RA or human albumin carrier protein. Il1r1–/– mice received Cx3cr1–/–
microglia. Sections were analyzed by confocal microscopy (GFP/NeuN-Cy5/
TUNEL-rhodamine). Microglia and apoptotic neurons were counted in four
high-power fields (HPF) per section (4 sections per mouse, n ¼ 4 mice per
group), and results are shown as number of cells per HPF. For stereotactic
placement of microglia, recipient mice 8–10 weeks old were anesthetized with
ketamine (200 mg per kg body weight) and xylazine (10 mg per kg body
weight), and heads were secured in a stereotaxic head frame (Kopf Instru-
ments). A 10-ml Hamilton syringe with a 29-gauge needle was inserted into the
left motor cortex through a small hole drilled through the skull. Cells (2–3 �105 cells in 10 ml) were injected at a flow rate of 1 ml min–1 at the following
coordinates anteroposterior, –0.12 mm; lateral, 1.7 mm; dorsoventral, 2 mm.
After completion, the needle was left in place for 5 min, then withdrawn at
0.2 mm min–1. Brains were removed 36 h after transfer, sectioned serially from
the level of the injection site to a depth of 1,000 mm and double-stained for
NeuN and TUNEL or annexin V.
Administration of MPTP and analysis of effects. Male mice, 8–10 weeks old
and weighing 22–28 g, were injected i.p. four times at 1-h intervals with saline
or MPTP (Sigma; 10 mg per kg body weight) and killed 2 d or 7 d after
injection. Brains were serially sectioned through the substantia nigra and
stained with antibody to TH (Chemicon) followed by Cy3-conjugated or
biotinylated secondary antibodies; this was followed by diaminobenzidine
(DAB) substrate development and cresyl violet counterstaining. Confocal
images of TH+ neurons and GFP+ microglia were obtained in 25-mm z-stacks.
We counted the number of TH-IR and Nissl-stained SNpc neurons in seven
mice per group, using the optical fractionator method35,36 (Supplementary
Methods). The total number of SNpc neurons was calculated as the product of
neuron (TH-IR or Nissl-stained) densities and the volume of the SNpc (ref. 36).
Generation and analysis of SOD1G93A/Cx3cr1 mice. Male G93A-SOD1
mutation (SOD1G93A) transgenic mice20 from Jackson Laboratories were bred
with Cx3cr1–/– females. F1 nontransgenic/Cx3cr1–/– or nontransgenic/Cx3cr1+/–
females were bred with SOD1G93A/Cx3cr1+/– males to produce F2 SOD1G93A/
Cx3cr1–/–, SOD1G93A/Cx3cr1+/–, SOD1G93A/Cx3cr1+/+ and nontransgenic/
Cx3cr1–/– mice.
Limb strength was assessed between the ages of 7 and 23 weeks (n ¼ 6–20
per group) as described37. Survival was recorded for each SOD1G93A/Cx3cr1–/–
and SOD1G93A/Cx3cr1+/– mouse, with the terminal stage defined by righting
response Z 20 s.
Histopathology was analyzed at 133 d in terminal SOD1G93A mice and in
four age-matched nontransgenic SOD1G93A/Cx3cr1–/– littermates. Motor neu-
rons in lumbar spinal cords were counted in Nissl-stained sections, and
microglial cells were visualized by IBA-1 immunohistochemistry. The area
occupied by IBA-1 immunoreactivity was obtained using Image J. 1.34vi
software, in six images acquired at 20� from three spinal cords per mouse
(n ¼ 5 mice per group).
Statistics. We used analysis of variance (ANOVA) to analyze numbers of
annexin V–positive cells after LPS administration, neuronal apoptosis in the
adoptive transfer experiments and motor neuron counts and microglial area in
spinal cords of SOD transgenic mice. Cytokine analyses by RPA were compared
using Student’s t-test. The numbers of TH-IR and Nissl-stained neurons in the
NATURE NEUROSCIENCE VOLUME 9 [ NUMBER 7 [ JULY 2006 923
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SNpc were determined by stereology and compared by ANOVA with Newman-
Keuls post-test. Behavior data (body weight, forelimb grip strength and
hindlimb grip strength) were collected for males and females in three groups
of mice, SOD1G93A/Cx3cr1+/–, SOD1G93A/Cx3cr1–/– and nontransgenic
Cx3cr1–/–, with separate analyses of data from weeks 9 to 14 and weeks 15 to
20. We used a random coefficient mixed model to estimate the effects of
genotype, gender and age (Supplementary Methods).
Note: Supplementary information is available on the Nature Neuroscience website.
ACKNOWLEDGMENTSWe acknowledge B. Trapp (Cleveland Clinic, Cleveland) for IBA-1 antibodies,W. Stallcup (Burnham Institute, La Jolla, California) for NG-2 antibodies,C. Canasto (Mount Sinai School of Medicine, New York) for technical assistancewith CX3CL1 mice, R. Zhang (Mass Spectrometry Core II, Cleveland Clinic) forassistance with MPP+ measurements, C. Shemo (Flow Cytometry Core, ClevelandClinic) for assistance with flow cytometry, and J. Drazba (Lerner Research InstituteImaging Core, Cleveland Clinic) for assistance with confocal microscopy. R.H.Miller (Case Medical School, Cleveland) provided helpful comments about themanuscript. This work was supported by the US National Institute of Health(NS32151), the Charles A. Dana Foundation, the National Multiple SclerosisSociety (fellowship FG1528-A-1 to A.C.), the Robert Packard Foundation forALS Research at Johns Hopkins University and the Boye Foundation.
AUTHOR CONTRIBUTIONSA.E.C. performed the experimental design of the LPS and MPTP models, andcarried out the microglia isolation, tissue staining and microglial transferexperiments. E.P.P. and V.K. carried out the experiments with SODG93A
transgenic mice and assisted with manuscript preparation. M.E.S. and S.M.C.assisted in the maintenance of the mouse colony, genotyping, histopathologicalstaining and neuronal counting. I.M.D. assisted in the development of thestereotaxic protocol. D.H. collaborated in the colocalization of lineage markerswith the GFP reporter. G.K. assisted with the confocal analyses and imaging.S.D. assisted with stereology methods. R.D. collaborated in the analysis of thegene expression data from nuclease protection assays. J.-C.L. performed thestatistical analyses for all experiments. D.N.C., S.J., S.A.L. and D.R.L. generatedthe highly inbred receptor- and ligand-deficient mouse strains, and assisted withthe experimental design and manuscript preparation. R.M.R. provided the basisfor the development of the experimental designs. A.E.C. and R.M.R. analyzedthe data, interpreted the results and prepared the manuscript.
COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.
Published online at http://www.nature.com/natureneuroscience
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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