BACKGROUND
neuroinflammation within the brain is a common feature of neurodegeneration and one of the hallmarks of these changes is the activation of microglia.
microglia are cns  mononuclear phagocytes that under normal conditions are surveillant but are activated in response to various brain insults  <cit> .
activation can result in phagocytosis and the release of substances such as neurotrophic factors and cytokines/chemokines  <cit> .
while some of these functions are likely to be beneficial, it has been proposed that an excess of particularly proinflammatory cytokines may be damaging and that this immune response may contribute to disease pathogenesis.
neurons express the chemokine cx3cl <dig>  in a membrane bound or soluble form.
its receptor within the cns, cx3cr <dig>  is expressed solely on microglia  <cit> .
cx3cl <dig> is thought to act as a chemoattractant recruiting microglia to sites of injury and the normal signalling between cx3cl <dig> and cx3cr <dig> is thought to provide part of the neuronal-microglial cross talk that maintain microglia in their resting state  <cit> .
the role of cx3cr <dig> has been extensively studied in many models of neurodegeneration and brain injury through the use of cx3cr <dig> knockout mice, although often with conflicting results.
in a triple transgenic  model of alzheimer’s disease  it was shown that loss of cx3cr <dig> resulted in the increase of microglial migration and prevented neuronal loss while levels of aβ deposition remained unaltered  <cit> .
the effect of cx3cl1/cx3cr <dig> signalling seen in the 3xtg model contrasts with the findings reported for both the appps <dig> and r <dig>  models of ad where loss of cx3cr <dig> altered microglial activation and reduced aβ deposition  <cit> .
knockout of cx3cr <dig> in a toxin-induced model of parkinson’s disease  and a genetic model of amyotrophic lateral sclerosis   worsened the phenotype, in both cases producing more neuronal loss and, for the als model, accelerated deterioration of hind limb grip function and reduced survival were also seen  <cit> .
in related experiments with a toxin-induced model of pd in rats, treatment with the ligand cx3cl <dig> was neuroprotective with reduced microglial activation  <cit> .
in addition, in a model of tauopathy, a humanised mapt transgenic mouse also lacking cx3cr <dig> showed altered microglial activation, enhanced tau phosphorylation and aggregation as well as poorer spatial working memory  <cit> .
in contrast, smaller infarcts and better functional recovery was seen in the absence of cx3cr <dig> in a model of ischaemia  <cit> .
prion diseases or transmissible spongiform encephalopathies  are progressive neurodegenerative diseases that include creuzfeldt-jakob disease  in humans, bovine spongiform encephalopathy  in cattle and scrapie in sheep  <cit> .
in common with many other neurodegenerative diseases, a key feature of prion diseases is the accumulation of aggregates of an abnormally folded protein.
in prion diseases these misfolded protein aggregates propagate by the conversion of normal cellular prion protein  to abnormal isoforms, designated prpsc and constitute lethal infectious agents.
prion infection is accompanied by spongiform change and neuronal loss in the brain.
in contrast to other infectious diseases little if any evidence of inflammation is seen in the periphery.
however, even before significant neuronal loss occurs, there is widespread activation of microglia, glia and astrocytes in the brain  <cit> .
proinflammatory cytokines increase during disease progression, however, this is greatly reduced relative to that seen during bacterial or viral infections  <cit> .
in wild type mice infected with a mouse-adapted scrapie prion strain  cx3cl <dig> is reported to be upregulated in astrocytes but there is no change in surviving neurons and cx3cr <dig> expression is also upregulated on microglia  <cit> .
analysis of hamster brains infected with the prion strain 263k showed that cx3cl <dig> is downregulated during disease progression  <cit> .
these data, together with the prominence of activated microglia early in prion disease and the parallels with other neurodegenerative disorders, suggest that cx3cl1/cx3cr <dig> signalling may play an important role in prion disease pathogenesis.
to evaluate this hypothesis we challenged cx3cr <dig> knockout mice with three different prion strains  <cit> .
our results show that for two mouse-adapted scrapie strains  and a mouse-passaged bse strain , cx3cr <dig> deficiency shortens the incubation time thereby suggesting that cx3cl1/cx3cr <dig> signalling is partially protective in prion disease.
methods
animals
cx3cr1+/- mice  on a balb/c background were obtained as embryos from the european mouse mutant archive, resurrected and bred to homozygosity  <cit> .
wild type balb/colahsd mice were purchased from harlan, uk ltd .
prion transmission
inocula for three different prion strains were made from the brains of terminally sick mice as 1%  homogenates in sterile d-pbs.
chandler/rml  and me <dig>  are two different mouse-adapted strains of scrapie and mrc <dig>  is a mouse-passaged strain of bse  <cit> .
following anaesthesia with isofluorane/o <dig>  female mice were inoculated intra-cerebrally with  <dig> μl of inocula into the right parietal lobe as previously described  <cit> .
mice were examined daily for clinical signs of prion disease and were culled once a definitive diagnosis had been made or earlier if showing any signs of distress or excessive weight loss.
diagnostic signs of clinical prion disease include ataxia, impaired righting reflex and a sustained hunched posture  <cit> .
incubation time was defined as the number of days from inoculation to confirmed diagnosis.
the kaplan-meier log-rank test was used to analyse survival data using the statistical package spss .
animals were housed, maintained and cared for in accordance with institutional, uk and international regulations and standards on animal welfare and conform to arrive guidelines  <cit> .
ethical approval was granted by the medical research council prion unit animal research ethics committee and carried out under uk home office licence ppl70/ <dig>
neuropathology and immunohistochemistry
mouse brains were fixed in 10% buffered formal saline  and prion-infected tissue was treated in 98% formic acid for one hour to remove infectivity.
tissues were paraffin wax embedded, sagitally sectioned and stained as previously described  <cit> .
haematoxylin and eosin  stained sections were assessed to determine the relative levels and distribution of spongiosis and neuronal loss.
prion deposition was evaluated with the anti-prp monoclonal antibody icsm <dig>  and gliosis was determined with an anti-glial fibrillary acid protein  antibody .
microglia were visualised by staining with an anti-iba <dig> polyclonal antibody .
real-time rt-pcr
rna was extracted using a trizol plus rna purification kit  from half brains taken from terminally sick prion infected mice and uninfected controls .
cdna was generated using a quantitect reverse transcription kit .
contaminating genomic dna was removed during the extraction with a dnase i digestion and this was confirmed by including a no reverse-transcription control for each sample.
real-time rt-pcr reactions were carried out using taqman gene expression master mix enzyme  on a  <dig> fast real-time pcr system .
gene specific taqman gene expression assays  were used for cxcl <dig>  il-12b, il-1b, arg- <dig> and cx3cl <dig>  each reaction was duplexed independently with two endogenous controls .
all reactions were carried out in triplicate.
statistical tests were carried out using graphpad instat .
RESULTS
transmission of prions to cx3cr1-/- mice
cx3cr <dig> knockout mice were generated by replacing the cx3cr <dig> gene with the green fluorescent protein  reporter gene  <cit> .
the gene targeting results in the loss of the wild type cx3cr <dig> mrna transcript and the generation of a chimeric transcript of an untranslated cx3cr <dig> exon spliced to the gfp exon.
the mice have a normal lifespan with no visible abnormal phenotype.
the presence of gfp has not been reported to interfere with microglial function and microglia are still able to become activated in the absence of cx3cr <dig>  <cit> .
to investigate the role of cx3cl1/cx3cr <dig> neuronal-glial cross talk in prion disease we inoculated female cx3cr1-/- mice and balb/c wild type controls intracerebrally with three different mouse-adapted prion strains.
chandler/rml and me <dig> are two distinct scrapie-derived prion strains and mrc <dig> was generated from the passage of cattle bse in mice  <cit> .
each of these prion strains transmits readily to wild type mice and produce a distinctive incubation time and patterns of prpsc distribution, gliosis and spongiosis  <cit> .
onset of diagnostic signs of prion disease was significantly earlier in cx3cr1-/- mice relative to controls for all three prion strains .
for chandler/rml prions the mean incubation time  was reduced by 4% from 150 ±  <dig> in cx3cr1+/+ to 144 ±  <dig> in cx3cr1-/- mice  and for me <dig> the mean incubation time was reduced by 7% from 178 ±  <dig> in cx3cr1+/+ to 168 ±  <dig> in cx3cr1-/- mice .
for mrc <dig> the mean incubation time was reduced by 7% from 206 ±  <dig> in cx3cr1+/+ to 200 ±  <dig> in cx3cr1-/- mice .
neuropathology
brain sections from terminally sick mice from each group were evaluated for evidence of neuropathological changes.
overall, for chandler/rml inoculated mice, the patterns of spongiosis, gliosis and prp distribution were the same for both wild type and cx3cr1-/- groups.
even in the absence of cx3cl1/cx3cr <dig> signalling, microglia show extensive proliferation and based on morphology appear to be activated and similarly distributed to that seen in the controls .
similarly, for me <dig> and mrc <dig> inoculated animals, no differences were seen in the patterns of spongiosis, gliosis, prp and microglia distribution between cx3cr1-/- and cx3cr1+/+ groups .
neuropathological differences are seen between the three prion strains, however, these are characteristic of the strains and do not appear to have been influenced by loss of cx3cr <dig>
prion strains
prion strains in mice are characterised by their incubation time, neuropathology and the biochemical properties of prpsc <cit> .
to evaluate the effect of cx3cr <dig> knockout on the faithful maintenance of strain characteristics we examined the brains of infected animals by western blotting using the anti-prp antibody icsm <dig>  no differences were seen in prpsc type between cx3cr1-/- and cx3cr1+/+ animals .
cytokine mrna expression
based on immunohistochemical staining with iba <dig>  we saw no differences in microglial proliferation and distribution between cx3cr <dig> knockout and wild type mice.
the activation status of microglia may be inferred by the relative abundance of various cytokines and chemokines in the brain.
in other models of neurodegeneration in the absence of cx3cr <dig>  alterations in the cytokine/chemokine environment has been observed  <cit> .
we therefore measured the mrna levels, by real time rt-pcr, of various cytokines/chemokines in the brains of terminally sick mice for each of our three prion strains and in uninfected controls for both wild type and cx3cr <dig> knockout mice.
firstly, we looked at levels of cytokines/chemokines known to be induced in prion disease   <cit> .
cxcl <dig> expression was significantly increased in all three prion strains relative to uninfected controls with 5-fold increases for chandler/rml and me <dig> and a 15-fold increase for mrc <dig> .
no significant differences were seen between knockout and wild type groups .
il-12b expression was also significantly increased in all prion-infected groups .
however, a 2-fold increase was seen in cx3cr1-/- relative to cx3cr1+/+ mice  but only with the me <dig> prion strain .
il-1b expression was significantly increased in all prion-infected groups , however, no significant differences were seen between cx3cr1-/- and wild type controls .
microglia may be activated through either a classical  or alternative  pathway  <cit> .
cxcl <dig> is a marker of the m <dig> pathway and arg- <dig> is a marker of the m <dig> pathway.
as described, cxcl <dig> mrna expression is induced equally in both cx3cr <dig> knockout and wild type mice with all prion strains, however, no differences were observed for arg- <dig> mrna expression by mouse genotype or prion infection .
this suggests that the m <dig> microglia activation pathway is triggered by prion infection and this is not altered by loss of cx3cr <dig>  we also looked at cx3cl <dig>  mrna expression and saw no significant differences between cx3cr <dig> knockout and wild type mice or between infected and uninfected groups .
discussion
we have shown that cx3cr <dig> deficiency results in an acceleration in the onset of the clinical signs of prion disease, as illustrated by shorter incubation times, with three different prion strains.
these data suggest that in wild type animals intact cx3cl1/cx3cr <dig> signalling is partially protective and may act to somewhat restrain the microglial response.
the difference in incubation time observed in knockout mice is quantitatively modest but highly statistically significant with a reduction of 4% for chandler/rml and 7% for both me <dig> and mrc <dig> prion strains and no overall differences observed in microglial activation either by neuropathology or cytokine/chemokine expression in terminally sick mice.
these data suggest that cx3cl1/cx3cr <dig> signalling plays only a minor role in modulating microglial activation in prion disease.
although microglial activation was unaltered in our study it is worth noting that for both neuropathological examination and cytokine/chemokine production, we looked only at brains from terminally sick animals.
it is possible that microglia are activated earlier in the absence of cx3cr <dig> but by end stage disease microglia are activated and distributed as seen in wild type mice.
if microglia are activated earlier in cx3cr1-/- mice the earlier exposure to neurotoxic factors may contribute to the earlier onset of clinical disease.
we observed a 2-fold increase in il-12b expression in cx3cr1-/- relative to cx3cr1+/+ mice  but only with the me <dig> prion strain which suggests a prion strain specific effect.
given that this was not observed with other proinflammatory cytokines or with the other prion strains it is difficult to assess the relevance of this finding.
the role of cx3cl1/cx3cr <dig> signalling in neurodegeneration is variable, highly dependent on the model used and the phenotype measured and probably reflects the nature of the neuroinflammatory response triggered by each disease process.
our data suggest that for prion disease the neuronal-microglia cross talk provided by this interaction is partially protective but is not sufficient to override other pathways of microglial activation or neuronal loss.
the protective role of cx3cl1/cx3cr <dig> is also seen in models of ad , als , tauopathy and pd  <cit> .
this suggests that different neurodegenerative diseases share common pathways that ultimately contribute to neuronal death and that the relative importance of each pathway is governed by the nature of the original insult.
CONCLUSIONS
we have demonstrated that loss of cx3cr <dig> results in an acceleration of onset of prion disease with three distinct prion strains  suggesting that intact cx3cl1/cx3cr <dig> signalling is partially protective in prion disease.
this occurs in the context of unchanged microglial activation, as assessed by cellular distribution and cytokine profiles.
the role of cx3cl1/cx3cr <dig> signalling in different models of neurodegeneration is highly variable suggesting that its influence is dependent on the precise cytokine environment created by each disease process.
abbreviations
ad: alzheimer’s disease; als: amyotrophic lateral sclerosis; bfs: buffered formal saline; bse: bovine spongiform encephalopathy; cjd: creuzfeldt-jakob disease; cns: central nervous system; d-pbs: dulbecco’s phosphate buffered saline; gfap: glial fibrillary acid protein; gfp: green fluorescent protein; h&e: haematoxylin and eosin; pd: parkinson’s disease; prpc: normal cellular prion protein; prpsc: abnormal disease associated isoform of prion protein; rt-pcr: reverse transcriptase-polymerase chain reaction; tse: transmissible spongiform encephalopathy.
competing interests
jc is a director and shareholder of d-gen limited, an academic spin-out company in the field of prion diagnosis, decontamination and therapeutics.
d-gen markets the monoclonal antibody icsm <dig> used in this study.
authors’ contributions
sl planned the experiments; sa, gj, and sl carried out the experimental work.
sb and sl analysed the data; sl and jc wrote the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
methods-western blotting and additional figure legends.
click here for file
additional file 2: figure s1
western blots of prpsc from infected mouse brains.
click here for file
additional file 3: figure s2
cx3cl <dig> mrna expression.
click here for file
acknowledgements
we are grateful to jackie linehan and her staff for histology and huda al-doujaily and jonathan wadsworth for preparation of inocula.
we also thank biological services facility staff for animal care and ray young and richard newton for preparation of figures.
this work was funded by the uk medical research council .

 neuroinflammation within the brain is a common feature of neurodegeneration and one of the hallmarks of these changes is the activation of microglia. microglia are cns mononuclear phagocytes that under normal conditions are surveillant but are activated in response to various brain insults. activation can result in phagocytosis and the release of substances such as neurotrophic factors and cytokines/chemokines. while some of these functions are likely to be beneficial, it has been proposed that an excess of particularly proinflammatory cytokines may be damaging and that this immune response may contribute to disease pathogenesis. neurons express the chemokine cx3cl in a membrane bound or soluble form. its receptor within the cns, cx3cr is expressed solely on microglia. cx3cl is thought to act as a chemoattractant recruiting microglia to sites of injury and the normal signalling between cx3cl and cx3cr is thought to provide part of the neuronal-microglial cross talk that maintain microglia in their resting state. the role of cx3cr has been extensively studied in many models of neurodegeneration and brain injury through the use of cx3cr knockout mice, although often with conflicting results. in a triple transgenic model of alzheimer’s disease it was shown that loss of cx3cr resulted in the increase of microglial migration and prevented neuronal loss while levels of aβ deposition remained unaltered. the effect of cx3cl1/cx3cr signalling seen in the 3xtg model contrasts with the findings reported for both the appps and r models of ad where loss of cx3cr altered microglial activation and reduced aβ deposition. knockout of cx3cr in a toxin-induced model of parkinson’s disease and a genetic model of amyotrophic lateral sclerosis worsened the phenotype, in both cases producing more neuronal loss and, for the als model, accelerated deterioration of hind limb grip function and reduced survival were also seen. in related experiments with a toxin-induced model of pd in rats, treatment with the ligand cx3cl was neuroprotective with reduced microglial activation. in addition, in a model of tauopathy, a humanised mapt transgenic mouse also lacking cx3cr showed altered microglial activation, enhanced tau phosphorylation and aggregation as well as poorer spatial working memory. in contrast, smaller infarcts and better functional recovery was seen in the absence of cx3cr in a model of ischaemia. prion diseases or transmissible spongiform encephalopathies are progressive neurodegenerative diseases that include creuzfeldt-jakob disease in humans, bovine spongiform encephalopathy in cattle and scrapie in sheep. in common with many other neurodegenerative diseases, a key feature of prion diseases is the accumulation of aggregates of an abnormally folded protein. in prion diseases these misfolded protein aggregates propagate by the conversion of normal cellular prion protein to abnormal isoforms, designated prpsc and constitute lethal infectious agents. prion infection is accompanied by spongiform change and neuronal loss in the brain. in contrast to other infectious diseases little if any evidence of inflammation is seen in the periphery. however, even before significant neuronal loss occurs, there is widespread activation of microglia, glia and astrocytes in the brain. proinflammatory cytokines increase during disease progression, however, this is greatly reduced relative to that seen during bacterial or viral infections. in wild type mice infected with a mouse-adapted scrapie prion strain cx3cl is reported to be upregulated in astrocytes but there is no change in surviving neurons and cx3cr expression is also upregulated on microglia. analysis of hamster brains infected with the prion strain 263k showed that cx3cl is downregulated during disease progression. these data, together with the prominence of activated microglia early in prion disease and the parallels with other neurodegenerative disorders, suggest that cx3cl1/cx3cr signalling may play an important role in prion disease pathogenesis. to evaluate this hypothesis we challenged cx3cr knockout mice with three different prion strains. our results show that for two mouse-adapted scrapie strains and a mouse-passaged bse strain, cx3cr deficiency shortens the incubation time thereby suggesting that cx3cl1/cx3cr signalling is partially protective in prion disease. methods animals cx3cr1+/- mice on a balb/c background were obtained as embryos from the european mouse mutant archive, resurrected and bred to homozygosity. wild type balb/colahsd mice were purchased from harlan, uk ltd. prion transmission inocula for three different prion strains were made from the brains of terminally sick mice as 1% homogenates in sterile d-pbs. chandler/rml and me are two different mouse-adapted strains of scrapie and mrc is a mouse-passaged strain of bse. following anaesthesia with isofluorane/o female mice were inoculated intra-cerebrally with μl of inocula into the right parietal lobe as previously described. mice were examined daily for clinical signs of prion disease and were culled once a definitive diagnosis had been made or earlier if showing any signs of distress or excessive weight loss. diagnostic signs of clinical prion disease include ataxia, impaired righting reflex and a sustained hunched posture. incubation time was defined as the number of days from inoculation to confirmed diagnosis. the kaplan-meier log-rank test was used to analyse survival data using the statistical package spss. animals were housed, maintained and cared for in accordance with institutional, uk and international regulations and standards on animal welfare and conform to arrive guidelines. ethical approval was granted by the medical research council prion unit animal research ethics committee and carried out under uk home office licence ppl70/ neuropathology and immunohistochemistry mouse brains were fixed in 10% buffered formal saline and prion-infected tissue was treated in 98% formic acid for one hour to remove infectivity. tissues were paraffin wax embedded, sagitally sectioned and stained as previously described. haematoxylin and eosin stained sections were assessed to determine the relative levels and distribution of spongiosis and neuronal loss. prion deposition was evaluated with the anti-prp monoclonal antibody icsm and gliosis was determined with an anti-glial fibrillary acid protein antibody. microglia were visualised by staining with an anti-iba polyclonal antibody. real-time rt-pcr rna was extracted using a trizol plus rna purification kit from half brains taken from terminally sick prion infected mice and uninfected controls. cdna was generated using a quantitect reverse transcription kit. contaminating genomic dna was removed during the extraction with a dnase i digestion and this was confirmed by including a no reverse-transcription control for each sample. real-time rt-pcr reactions were carried out using taqman gene expression master mix enzyme on a fast real-time pcr system. gene specific taqman gene expression assays were used for cxcl il-12b, il-1b, arg- and cx3cl each reaction was duplexed independently with two endogenous controls. all reactions were carried out in triplicate. statistical tests were carried out using graphpad instat.  transmission of prions to cx3cr1-/- mice cx3cr knockout mice were generated by replacing the cx3cr gene with the green fluorescent protein reporter gene. the gene targeting results in the loss of the wild type cx3cr mrna transcript and the generation of a chimeric transcript of an untranslated cx3cr exon spliced to the gfp exon. the mice have a normal lifespan with no visible abnormal phenotype. the presence of gfp has not been reported to interfere with microglial function and microglia are still able to become activated in the absence of cx3cr. to investigate the role of cx3cl1/cx3cr neuronal-glial cross talk in prion disease we inoculated female cx3cr1-/- mice and balb/c wild type controls intracerebrally with three different mouse-adapted prion strains. chandler/rml and me are two distinct scrapie-derived prion strains and mrc was generated from the passage of cattle bse in mice. each of these prion strains transmits readily to wild type mice and produce a distinctive incubation time and patterns of prpsc distribution, gliosis and spongiosis. onset of diagnostic signs of prion disease was significantly earlier in cx3cr1-/- mice relative to controls for all three prion strains. for chandler/rml prions the mean incubation time was reduced by 4% from 150 ±  in cx3cr1+/+ to 144 ±  in cx3cr1-/- mice and for me the mean incubation time was reduced by 7% from 178 ±  in cx3cr1+/+ to 168 ±  in cx3cr1-/- mice. for mrc the mean incubation time was reduced by 7% from 206 ±  in cx3cr1+/+ to 200 ±  in cx3cr1-/- mice. neuropathology brain sections from terminally sick mice from each group were evaluated for evidence of neuropathological changes. overall, for chandler/rml inoculated mice, the patterns of spongiosis, gliosis and prp distribution were the same for both wild type and cx3cr1-/- groups. even in the absence of cx3cl1/cx3cr signalling, microglia show extensive proliferation and based on morphology appear to be activated and similarly distributed to that seen in the controls. similarly, for me and mrc inoculated animals, no differences were seen in the patterns of spongiosis, gliosis, prp and microglia distribution between cx3cr1-/- and cx3cr1+/+ groups. neuropathological differences are seen between the three prion strains, however, these are characteristic of the strains and do not appear to have been influenced by loss of cx3cr prion strains prion strains in mice are characterised by their incubation time, neuropathology and the biochemical properties of prpsc. to evaluate the effect of cx3cr knockout on the faithful maintenance of strain characteristics we examined the brains of infected animals by western blotting using the anti-prp antibody icsm no differences were seen in prpsc type between cx3cr1-/- and cx3cr1+/+ animals. cytokine mrna expression based on immunohistochemical staining with iba we saw no differences in microglial proliferation and distribution between cx3cr knockout and wild type mice. the activation status of microglia may be inferred by the relative abundance of various cytokines and chemokines in the brain. in other models of neurodegeneration in the absence of cx3cr alterations in the cytokine/chemokine environment has been observed. we therefore measured the mrna levels, by real time rt-pcr, of various cytokines/chemokines in the brains of terminally sick mice for each of our three prion strains and in uninfected controls for both wild type and cx3cr knockout mice. firstly, we looked at levels of cytokines/chemokines known to be induced in prion disease. cxcl expression was significantly increased in all three prion strains relative to uninfected controls with 5-fold increases for chandler/rml and me and a 15-fold increase for mrc. no significant differences were seen between knockout and wild type groups. il-12b expression was also significantly increased in all prion-infected groups. however, a 2-fold increase was seen in cx3cr1-/- relative to cx3cr1+/+ mice but only with the me prion strain. il-1b expression was significantly increased in all prion-infected groups, however, no significant differences were seen between cx3cr1-/- and wild type controls. microglia may be activated through either a classical or alternative pathway. cxcl is a marker of the m pathway and arg- is a marker of the m pathway. as described, cxcl mrna expression is induced equally in both cx3cr knockout and wild type mice with all prion strains, however, no differences were observed for arg- mrna expression by mouse genotype or prion infection. this suggests that the m microglia activation pathway is triggered by prion infection and this is not altered by loss of cx3cr we also looked at cx3cl mrna expression and saw no significant differences between cx3cr knockout and wild type mice or between infected and uninfected groups. discussion we have shown that cx3cr deficiency results in an acceleration in the onset of the clinical signs of prion disease, as illustrated by shorter incubation times, with three different prion strains. these data suggest that in wild type animals intact cx3cl1/cx3cr signalling is partially protective and may act to somewhat restrain the microglial response. the difference in incubation time observed in knockout mice is quantitatively modest but highly statistically significant with a reduction of 4% for chandler/rml and 7% for both me and mrc prion strains and no overall differences observed in microglial activation either by neuropathology or cytokine/chemokine expression in terminally sick mice. these data suggest that cx3cl1/cx3cr signalling plays only a minor role in modulating microglial activation in prion disease. although microglial activation was unaltered in our study it is worth noting that for both neuropathological examination and cytokine/chemokine production, we looked only at brains from terminally sick animals. it is possible that microglia are activated earlier in the absence of cx3cr but by end stage disease microglia are activated and distributed as seen in wild type mice. if microglia are activated earlier in cx3cr1-/- mice the earlier exposure to neurotoxic factors may contribute to the earlier onset of clinical disease. we observed a 2-fold increase in il-12b expression in cx3cr1-/- relative to cx3cr1+/+ mice but only with the me prion strain which suggests a prion strain specific effect. given that this was not observed with other proinflammatory cytokines or with the other prion strains it is difficult to assess the relevance of this finding. the role of cx3cl1/cx3cr signalling in neurodegeneration is variable, highly dependent on the model used and the phenotype measured and probably reflects the nature of the neuroinflammatory response triggered by each disease process. our data suggest that for prion disease the neuronal-microglia cross talk provided by this interaction is partially protective but is not sufficient to override other pathways of microglial activation or neuronal loss. the protective role of cx3cl1/cx3cr is also seen in models of ad, als, tauopathy and pd. this suggests that different neurodegenerative diseases share common pathways that ultimately contribute to neuronal death and that the relative importance of each pathway is governed by the nature of the original insult.  we have demonstrated that loss of cx3cr results in an acceleration of onset of prion disease with three distinct prion strains suggesting that intact cx3cl1/cx3cr signalling is partially protective in prion disease. this occurs in the context of unchanged microglial activation, as assessed by cellular distribution and cytokine profiles. the role of cx3cl1/cx3cr signalling in different models of neurodegeneration is highly variable suggesting that its influence is dependent on the precise cytokine environment created by each disease process. abbreviations ad: alzheimer’s disease; als: amyotrophic lateral sclerosis; bfs: buffered formal saline; bse: bovine spongiform encephalopathy; cjd: creuzfeldt-jakob disease; cns: central nervous system; d-pbs: dulbecco’s phosphate buffered saline; gfap: glial fibrillary acid protein; gfp: green fluorescent protein; h&e: haematoxylin and eosin; pd: parkinson’s disease; prpc: normal cellular prion protein; prpsc: abnormal disease associated isoform of prion protein; rt-pcr: reverse transcriptase-polymerase chain reaction; tse: transmissible spongiform encephalopathy. competing interests jc is a director and shareholder of d-gen limited, an academic spin-out company in the field of prion diagnosis, decontamination and therapeutics. d-gen markets the monoclonal antibody icsm used in this study. authors’ contributions sl planned the experiments; sa, gj, and sl carried out the experimental work. sb and sl analysed the data; sl and jc wrote the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 methods-western blotting and additional figure legends. click here for file additional file 2: figure s1 western blots of prpsc from infected mouse brains. click here for file additional file 3: figure s2 cx3cl mrna expression. click here for file acknowledgements we are grateful to jackie linehan and her staff for histology and huda al-doujaily and jonathan wadsworth for preparation of inocula. we also thank biological services facility staff for animal care and ray young and richard newton for preparation of figures. this work was funded by the uk medical research council.
microglia are resident mononuclear phagocytes of the brain that become activated in response to insults including neurodegenerative diseases such as alzheimer’s disease, parkinson’s disease and prion disease. in prion disease, microglial proliferation has been suggested to contribute to overall disease progression, however, in different mouse models of neurodegeneration, loss of cx3cr has been shown to either worsen or improve the phenotype depending on the paradigm. to investigate the role of cx3cl1/cx3cr signalling in prion disease we infected cx3cr null mice with three different strains of prions. our data suggest a protective role for cx3cl1/cx3cr cross-talk in prion disease. following challenge with chandler/rml, me and mrc prion strains, cx3cr knockout mice showed highly significant reductions in incubation time. in the central nervous system the chemokine cx3cl is expressed by neurons and its exclusive receptor cx3cr is expressed solely on microglia.
 microglia are resident mononuclear phagocytes of the brain that become activated in response to insults including neurodegenerative diseases such as alzheimer’s disease, parkinson’s disease and prion disease. in the central nervous system the chemokine cx3cl is expressed by neurons and its exclusive receptor cx3cr is expressed solely on microglia. cx3cl1/cx3cr signalling is thought to maintain microglia in their resting state and disrupting this equilibrium may allow microglia to become activated. in prion disease, microglial proliferation has been suggested to contribute to overall disease progression, however, in different mouse models of neurodegeneration, loss of cx3cr has been shown to either worsen or improve the phenotype depending on the paradigm.  to investigate the role of cx3cl1/cx3cr signalling in prion disease we infected cx3cr null mice with three different strains of prions. following challenge with chandler/rml, me and mrc prion strains, cx3cr knockout mice showed highly significant reductions in incubation time. no differences were seen in the pattern and localisation of activated microglia in the brain or in the mrna expression levels of chemokines/cytokines.  our data suggest a protective role for cx3cl1/cx3cr cross-talk in prion disease. prion diseaseincubation timecx3cr1microgliacytokineschemokines 