This review focuses on several important functional roles for FKN/CX3CR1 in both health and disease of the central nervous system. It has been posited that FKN is involved in microglial infiltration of the brain during development. Microglia, in turn, are implicated in the developmental synaptic pruning that occurs during brain maturation. The abundance of FKN on mature hippocampal neurons suggests a homeostatic non-inflammatory role in mechanisms of learning and memory. There is substantial evidence describing a role for FKN in hippocampal synaptic plasticity. FKN, on the one hand, appears to prevent excess microglial activation in the absence of injury while promoting activation of microglia and astrocytes during inflammatory episodes. Thus, FKN appears to be neuroprotective in some settings, whereas it contributes to neuronal damage in others. Many progressive neuroinflammatory disorders that are associated with increased microglial activation, such as Alzheimer's disease, show disruption of the FKN/CX3CR1 communication system. Thus, targeting CX3CR1 receptor hyperactivation with specific antagonists in such neuroinflammatory conditions may eventually lead to novel neurotherapeutics.An essential aspect of normal brain function is the bidirectional interaction and communication between neurons and neighbouring glial cells. To this end, the brain has evolved ligand–receptor partnerships that facilitate crosstalk between different cell types. The chemokine, fractalkine (FKN), is expressed on neuronal cells, and its receptor, CX At present, these non-neuronal cell types are broadly categorized into (i) astrocytes, (ii) radial glia, (iii) oligodendrocytes, (iv) ependymal cells and (v) microglia. There exists further subcategories for each group and, more recently, NG2-positive glia are widely considered a distinct cell type –7. The cTogether with astrocytes, microglia can release neuromodulatory chemicals that influence neuronal firing and intracellular signalling. When first described, astrocytes were seen merely as structural scaffolding to support and cushion neuronal cells within the brain, in essence, to fill the gaps between neurons . However3CL1 in the new chemokine nomenclature) [3CR1 receptors on microglia [3CR1 receptors in health and disease of the CNS.Glial cells are now accepted as key neuromodulators at every stage of development and adulthood and, therefore, must possess multiple mechanisms of communication with neuronal cell types. One method in which neurons and microglia are thought to communicate with one another is through neuronally expressed fractalkine  . FKN is icroglia . Therefoicroglia . This ne3.Chemokines are abundantly expressed in many areas of the brain and spinal cord during development . They re3) separating the first two cysteine (C) residues [i protein-coupled CX3CR1; many other chemokine members exhibit more promiscuous binding activity than FKN. The full-length molecule is larger than most other chemokines, containing approximately 373 amino acid residues compared with the more common 70–80 amino acid size range. FKN exists in two distinct forms. The first is an approximately 95 kDa full-length membrane-bound form that possesses a 76-amino acid N-terminal chemokine domain, a 241-amino acid glycosylated mucin-like stalk, an 18-amino acid hydrophobic transmembrane region and a 37-amino acid intracellular C-terminal domain. The second is an approximately 70 kDa soluble form that contains the N-terminal chemokine domain. The extracellular chemokine domain of FKN is proteolytically cleaved from the membrane-bound fraction by the lysosomal cysteine protease, cathepsin S and members of the ADAM  family such as ADAM-10 and ADAM-17 -α-converting enzyme) [3CR1 receptors [There are four distinct subfamilies of chemokines ; categorized based on their amino acid structure. Most chemokines (except the γ subfamily) contain four conserved cysteine residues that form disulfide bonds to produce the defining chemokine domain. FKN is the only member of the δ subfamily of chemokines containing three amino acids (Xresidues . It is a enzyme) –49. The  enzyme) . The soleceptors , whereaseceptors  for micreceptors .et al. [3CR1 mRNA, whereas astrocytes do not. Therefore, neurons and astrocytes expressing FKN can signal to neurons and microglial cell types possessing CX3CR1 receptors. In this way, neurons may regulate microglial proliferation, because exogenous FKN can increase the number of BrdU-labelled microglia [3CR1-containing microglia may potentially be an important factor in many CNS-related pathologies. It is also likely, however, that the sustained high levels of FKN in the brain throughout adulthood serve normal physiological functions in addition to a rapid response mechanism in times of traumatic injury.Unlike most chemokines, FKN is constitutively expressed in the CNS with particularly high levels in hippocampal neurons . Astrocyet al.  also repicroglia . This maicroglia . Thus, c4.3CR1 receptors on neighbouring cells. FKN is constitutively expressed by certain neurons in the CNS but its expression levels can also be increased by several stimulators. For example, the treatment of rat aortic smooth muscle cells (SMCs) with the pro-inflammatory cytokine, TNF-α, induces the expression of FKN and CX3CR1 in a nuclear factor κB (NF-κB)-dependent manner [Cleavage of the extreme N-terminal chemokine domain releases soluble FKN which can function as a signalling molecule and activate CXt manner . Moreovet manner . Whether3CR1 knockout (CX3CR1−/−) mice and stimulated with LPS, microglial cells in these mixed cultures release a reduced amount of TNF-α, nitric oxide (NO) and superoxide [3CR1 receptor is involved in the release of pro-inflammatory substances from activated microglia. Therefore, by disrupting ‘normal’ FKN/CX3CR1 communication, it seems possible to switch the actions of FKN from anti-inflammatory to pro-inflammatory in nature.FKN has been shown to inhibit lipopolysaccharide (LPS)-induced TNF-α release from microglia , suggestperoxide . This su−/− and CX3CR1−/− mice to study whether FKN is neuroprotective or neurodamaging. The use of knockout animals that have had either FKN or CX3CR1 deleted from conception and allowed mature to adulthood with disruption to the FKN/CX3CR1 communication system can respond to FKN treatment differently to wild-type animals following inflammatory and neurodegenerative insults. Therefore, like many cytokines and chemokines studied in recent years, FKN can be either anti-inflammatory or neurodamaging depending on the context. The timing of administration (i.e. pre- or post-insult) and the concentration of FKN will be important factors in determining the response of neurons to potentially neurotoxic injury.By contrast, astrocytes do not constitutively express FKN protein. Astrocytes that are treated with certain pro-inflammatory cytokines such as TNF-α and IL-1β, however, upregulate expression of FKN in a time-dependent manner. Treatment of astrocytes with TNF-α induces FKN expression after 12 h and levels peak at 24 h. IL-1β, on the other hand, increased FKN expression much earlier after 2 h, peaking between 4 and 8 h post-stimulation . It appe5.3CR1, is reportedly expressed on microglia and on neurons [3CR1 is a seven transmembrane domain receptor coupled to Gi and Gz subtypes of G proteins [in vitro. Moreover, microglial IL-1β protein expression is also attenuated in cells treated with FKN and these anti-inflammatory effects are Akt  and PI3-kinase-dependent [The FKN receptor, CX neurons ,45,58–64proteins , activatproteins . In micrependent . FKN alsin vitro cell culture systems to investigate the effects of FKN on neuronal cell types. Treatment of human neurons with FKN induced transient phosphorylation of ERK1/2 within 1 min and Akt within 10 min of exposure [Several studies have also used exposure . The samexposure . This woIt is important to differentiate between FKN's direct effects on neuronal cells versus glial cell-mediated neuromodulatory effects. When hippocampal neurons are cultured with a glial ‘feeder layer’, a pure neuronal cell population can be achieved. Treatment of a pure neuronal hippocampal culture with soluble FKN activates ERK1/2, whereas no JNK or p38 MAPK upregulation occurs. FKN also activates the transcription factor, CREB, in hippocampal neurons . Removal6.et al. [3CR1GFP/GFP mice, in which microglia are fluorescently labelled  and the FKN receptor knocked out (CX3CR1−/−), they quantified the number of PSD-95-labelled dendritic spines on CA1 neurons during the first five weeks of postnatal development. They found that during the second–third weeks, CX3CR1−/− mice possess more synapses than wild-type mice. Interestingly, CX3CR1−/− mice also express reduced numbers of microglia in the CA1 region of the hippocampus during weeks 2–4 of postnatal development. The authors suggest that FKN signalling during development may act as a chemotropic agent to attract microglial cells into the brain. Therefore, knocking out the CX3CR1 receptor would render microglia blind to the attractive FKN cue and lead to reduced numbers of microglia in the brain of CX3CR1−/− mice. If microglia are actively involved in synapse pruning at this developmental stage, then a reduced number of microglial cells could explain the higher density of synapses in the brains of CX3CR1−/− mice [3CR1 signalling is required for proper microglial-recognition of synaptic boutons before and/or during engulfment and phagocytosis.During early postnatal development, neurons forge many more synaptic connections than is necessary for normal adult brain function. Consequently, a portion of these connections is not maintained into adulthood but removed through a process of activity-dependent pruning . More reet al.  report t−/− mice . Howeveret al. [in vitro assays in response to FKN. This inhibitory effect of FKN on neuronal cell migration was dependent on activation of the CX3CR1 receptor, was PTX-sensitive and dependent on PI3-kinase activity. The authors suggest that FKN may inhibit neuronal cell migration by increasing binding and adhesion to laminin, a component of the extracellular matrix [l-lysine-coated surfaces in in vitro assays. Blocking the CX3CR1 receptor and inhibiting Gi protein-coupled responses with PTX diminishes microglial migration in response to FKN [in vivo and that FKN/CX3CR1 signalling promotes the population of CNS tissue with peripherally derived cell types.In contrast to the action on microglia, astrocytes show little or no cell migratory response to FKN. Even activation of astrocytes with TNF-α or IL-1β does not induce a migratory phenotype in response to FKN exposure. Moreover, Lauro et al.  showed tr matrix . Interese to FKN . This su3CR1 signalling is also thought to regulate microglial influx, and population of sites where developing thalamocortical synapses are concentrated (i.e. barrel centres). Hoshiko et al. [3CR1GFP/GFP receptor knockout mice, microglial entry into the barrel centres is delayed by a few days, but is indistinguishable from wild-type mice at postnatal day 9 (P9). The absence of CX3CR1 also delays the maturation of functional glutamate receptors evidenced by the fact that the AMPAR/NMDAR ratio at P9 was significantly lower in CX3CR1−/− than in CX3CR1+/− mice. Moreover, the developmental switch from GluN2B to GluN2A-containing NMDA receptors that is known to occur between the first and second postnatal weeks in the thalamocortical area of the mouse was delayed in CX3CR1−/− mice but this delay was only transient [In the developing barrel field of the somatosensory cortex, FKN/CXo et al.  showed transient . This leet al. [3CR1 expression and microglial cell activation as evidenced by increased MHCII, CD40 mRNA and IL-1β protein levels. Thus, FKN may play a role in the homeostatic suppression of microglial activation. The seemingly natural decline in neuronally expressed FKN in the hippocampus of aged animals [In the adult brain, neuronally derived FKN is thought to maintain microglia in a quiescent state. Lyons et al.  have sho animals  might co7.3CR1 in the regulation of neurotransmission and synaptic plasticity [3CR1 receptors are present on neurons in vivo; even though several papers have reported that CX3CR1 does, indeed, reside on neurons, particularly hippocampal neurons [et al. [in vitro in the absence of any microglial contamination. Heinisch & Kirby [3CR1 localizes to the perinuclear region of serotonergic (5-HT) neurons in the raphe in vivo in addition to exhibiting microglial expression.FKN mRNA and protein levels are abundant in the uninjured adult hippocampus, an observation that prompted some to investigate if FKN plays a physiological role in learning and memory formation. Many recent publications suggest a functional role for both FKN and CXasticity –76. Ther neurons ,59. Meuc [et al.  reported & Kirby  also hav3CR1 receptor. For instance, FKN reduces spontaneous glutamate release and post-synaptic glutamate currents [3CR1−/− mice. Similarly, FKN causes a significant reduction in EPSCs from stimulated CA1 pyramidal neurons [3CR1−/− mice. FKN's depressive actions on EPSCs are dependent on calcium entry into the cell but independent of NMDA receptor activation [As discussed above, there is mounting evidence that FKN exerts direct actions on neurons through the CXcurrents ,45; the currents . FKN alscurrents . Severalcurrents ,74,77. Fcurrents . This de neurons . The abitivation . FKN inhtivation . Taken ttivation .3CR1 receptor because it was absent in CX3CR1−/− mice.Interestingly, there seems to be an important temporal component to the inhibitory actions of FKN on long-term potentiation (LTP). FKN, when present in the circulating perfusate prior to high-frequency stimulation (HFS), inhibits LTP in the CA1 region of acute hippocampal slices. When added just a few minutes after LTP induction, however, FKN has no dampening effect on the maintenance phase of LTP . This inet al. [3R adenosine receptor as the crucial subtype mediating inhibition of LTP by FKN in the CA1. This is in contrast to FKN-mediated neuroprotection which appears to be dependent on adenosine-1 receptor (A1R) activation [3R−/− mice, but that this feature is present in both A1R−/− and/or A2AR−/− mice [3CR1 receptors on microglia which induces adenosine release. This adenosine may, in turn, activate A2AR receptors on microglia (and possibly astrocytes) causing the release of d-serine which acts as a co-agonist at the NMDA receptor, thus potentiating NMDA-mediated fEPSPs. Interestingly, this might explain why FKN inhibits LTP when administered prior to LTP induction but not if added just a few minutes post-HFS. The increase in intracellular calcium that accompanies NMDA fEPSP potentiation by FKN may disrupt LTP induction which has been shown to depend on the duration and amplitude of Ca2+ elevations as well as the metaplastic activation state of the NMDA receptors involved [There is also evidence to suggest that adenosine and adenosine receptors are modulated by FKN activity in the hippocampus. FKN has been shown to cause the release of adenosine from microglia . Exposuret al.  pinpointtivation . In agre−/− mice . More re−/− mice . The autinvolved ,82.3CR1−/− and CX3CR1+/− mice display deficits in motor learning as assessed by standard rotarod training techniques. There is, however, no difference between CX3CR1−/−, CX3CR1+/− and wild-type mice in spontaneous locomotor activity as assessed by the open field test. Similarly, CX3CR1−/− and CX3CR1+/− mice were no more anxious than control mice as assessed by the elevated plus maze [3CR1−/− and CX3CR1+/− mice were assessed for deficits in associative learning and memory. During the training period, CX3CR1−/− and CX3CR1+/− mice displayed similar freezing behaviour to wild-type mice. When the mice were placed back into the chamber 24 h later, however, CX3CR1−/− and CX3CR1+/− mice displayed a reduction in their freezing behaviour. This ‘context-specific’ type of associative memory is thought to be predominantly hippocampal-dependent. Interestingly, when the CX3CR1−/− and CX3CR1+/− mice were placed into a novel environment and exposed to the conditioning stimulus, the animals froze for the same amount of time as their wild-type counterparts [3CR1−/− and CX3CR1+/− mice may display hippocampal-specific deficits in cognition. In agreement with this notion is the fact that CX3CR1−/− and CX3CR1+/− mice perform worse in probe trials of the hidden water maze task—a hippocampal-dependent memory paradigm. Rogers et al. [3CR1−/− and CX3CR1+/− mice. Basal synaptic transmission as well as PPF in the hippocampus was normal. However, CX3CR1−/− and CX3CR1+/− mice showed a reduction in LTP in the CA1 stratum radiatum. This may be explained by the higher basal levels of IL-1β in the hippocampus of CX3CR1−/− and CX3CR1+/− mice compared with wild-type. The origin of this excess IL-1β may be microglial cells, because CX3CR1−/− mice possess more activated microglia in the hippocampus. Treating hippocampal slices with IL-1 receptor antagonist (IL-1RA) rescued the deficits in LTP seen in CX3CR1−/− mice. Moreover, treating CX3CR1−/− mice with IL-1RA reverses the deficits in learning and memory seen in contextual fear-conditioning and water maze spatial memory training. TNF-α levels were also elevated in the cerebellum of CX3CR1−/− mice suggesting an overall increase in inflammatory-associated protein levels in the brains of CX3CR1−/− and CX3CR1+/− mice [CXlus maze . Using sterparts . This ‘cs et al.  also loo+/− mice .3CR1 signalling play several crucial roles in synaptic plasticity, learning and memory and FKN likely contributes to maintaining proper homeostasis of synaptic transmission in the hippocampus  results in an attenuation of neurogenesis. Given the role of hippocampal neurogenesis in learning and memory, reduced neurogenic rate may underlie impairments observed in CX3CR1−/− mice in hippocampal-dependent memory tasks.Hippocampal neurogenesis is often correlated with cognitive function ,84 and ncontrols ,87. Moreet al. [3CR1 receptors. This suggests that FKN may activate CX3CR1 on microglial cells to attenuate IL-1β release to maintain the rate of hippocampal neurogenesis.It is widely accepted that cognitive ability decreases with age and this decline correlates with reduced levels of hippocampal neurogenesis. Interestingly, treatment of older animals with exogenous FKN reverses this age-related decrease in hippocampal neurogenesis . In suppet al.  showed t9.3CR1 co-localization further suggesting that FKN has direct actions on neurons as opposed to indirect actions through microglial-mediated neuromodulation. If the direct actions of FKN on GABA-sensitivity are exclusive to serotonin-containing neurons in the dorsal raphe nucleus, then this mechanism presents a novel drug target for mood-related disorders such as anxiety and depression. However, a recent study by Roseti et al. [A receptor-mediated currents in other brain regions, such as the hippocampus and cortex, and that this action has relevance for disorders such as mesial temporal lobe epilepsy.As well as serving to inhibit over-activation of the hippocampal network under physiological conditions, FKN may also act as a first-line defence response to neuronal injury and neuroinflammation. Pathological levels of glutamate and excitotoxicity can lead to a TACE-mediated increase in soluble FKN, thus limiting neuronal damage ,73,88,89i et al.  suggests10.et al. [3CR1GFP/GFP receptor knockout mouse to study the role of FKN and CX3CR1 signalling in several models of microglial-induced neurotoxicity. CX3CR1−/− mice demonstrated significantly greater microglial activation in response to intraperitoneal (i.p.) administration of LPS. As only microglial cells are labelled with GFP in these CX3CR1−/− mice, Cardona et al. developed an adoptive transfer technique that allowed them inject LPS-activated microglial cells, deficient in CX3CR1 receptors, into the brains of wild-type animals. The ‘behaviours’ of these GFP-labelled microglial cells were compared with those taken from CX3CR1+/− mice. Interestingly, CX3CR1+/− microglial cells migrated far from the site of injection and seemed to preferentially invade white matter tracts of the CNS parenchyma. CX3CR1−/− microglia, however, localized to the site of injection in the frontal cortex of wild-type mice. Moreover, there was significantly more neuronal cell loss surrounding activated GFP+ microglia transplanted from CX3CR1−/− mice than in wild-type brains that received CX3CR1+/− microglial cells. The authors attributed this increased neuronal cell death to an increased production of IL-1β  from CX3CR1−/− microglia upon LPS stimulation. Indeed, when IL-1RA (IL-1 receptor antagonist) was added along with the adoptive transfer microglia, neuronal cell death was reduced significantly and IL-1RA seemed to restore the migratory activity of CX3CR1−/− cells from the site of injection.Microglial cells are the resident macrophages of the brain and are activated in response to neuronal injury . Cardonaet al.  made useet al. [3CR1 signalling in the 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP)-induced neurodegenerative mouse model of Parkinson's disease (PD). Dopaminergic cell death in the substantia nigra pars compacta of MPTP-injected CX3CR1−/− mice was significantly more substantial than that seen in wild-type mice. Moreover, FKN knockout (CX3CL1−/−) mice displayed similar enhancement of neuronal loss to CX3CR1−/− mice indicating that it is the perturbation of FKN-mediated modulation of microglial activity that worsens neuronal cell death, i.e. neuron–microglia crosstalk, rather than a specific loss of either FKN or its receptor. Pabon et al. [Cardona et al.  also evan et al.  showed tn et al. .et al. [in vitro and subsequently added to a hippocampal neuronal culture were neurotoxic and caused 20% neuronal cell death. LPS induced the release of TNF-α from microglia which FKN treatment partially blocked. The addition of a neutralizing antibody against endogenous FKN enhanced neuronal cell death in this co-culture of LPS-activated microglia. These results suggest that tonic activation of CX3CR1 by endogenous FKN may serve as an anti-inflammatory signal that maintains microglial cells in a quiescent state. Mizuno et al. [Activation of microglial cells with LPS can convert the phenotype of microglial cells from a resting state into a phagocytic and neurotoxic form. Zujovic et al.  showed to et al.  showed to et al. .11.et al. [3CL1−/−) in order to determine the role of FKN in ischaemic brain injury. They induced a transient focal cerebral ischaemia (2 h) and allowed a 22 h reperfusion of the middle cerebral artery before sacrificing the mice for histological analysis of tissue injury. Twenty-four per cent of the wild-type mice died in this 22 h period, whereas none of the 20 CX3CL1−/− mice died. Moreover, there was a 28% reduction in the size of the ischaemic infarct in CX3CL1−/− mice correlating well with the reduced mortality rate. This study suggests that FKN expression is detrimental to recovery post-ischaemic injury [et al. [3CL1−/− mice and also CX3CR1−/− mice. They also found that CX3CL1−/− mice possessed a smaller ischaemic infarction following middle cerebral arterial occlusion (MCAO) and interestingly, so did CX3CR1−/− mice. Counterintuitively, Cipriani et al. also showed that administration of exogenous FKN slightly before MCAO to wild-type mice reduced the volume of the subsequent ischaemic infarct. Therefore, FKN is neuroprotective in wild-type mice that receive an ischaemic insult. Exogenous FKN administration had no effect in CX3CR1−/− mice but did, however, increase the size of the infarct seen in CX3CL1−/− mice. Therefore, mice that develop to adulthood with normal levels of FKN present in the brain (i.e. wild-types), respond favourably to FKN pre-treatment post-ischaemia. The story is further complicated, because exogenous FKN treatment is only beneficial at a reasonably narrow concentration range of between 15 and 70 pM; greater than 150 pM FKN becomes harmful in wild-types post-ischaemia. Interestingly, the neuroprotective effects of exogenously applied FKN were lost in A1R−/− knockout mice [Despite the evidence of a neuroprotective role for FKN presented above, there are data suggesting this chemokine is detrimental in some settings. Soriano et al.  generatec injury . Ciprian [et al.  performeout mice .3CR1 signalling in microglia is important for their proper functioning. Under in vitro conditions that mimic an in vivo MCAO injury, i.e. oxygen–glucose deprivation, FKN caused a reduction in TNF-α release from microglia that were cultured from CX3CL1−/− mice. Recently, a neuroprotective role for TNF-α in ischaemic brain injury has been described [3CL1−/− mice. The release of various factors from microglia at the site of injury can have both positive and negative effects on neuronal survival and normal microglia functioning appears disrupted in CX3CL1−/− mice, because FKN does not alter TNF-α release from wild-type microglia. Dénes et al. [3CR1 is crucial for maintaining proper microglial functioning in the CNS. They also showed that the infarct size post-MCAO was significantly reduced in CX3CR1−/− mice compared with wild-types and heterozygotes. Interestingly, they noted increased IL-1β expression in CX3CR1+/− mice compared with knockouts, but the IL-1β was not associated with microglial cell types. Instead, astrocytes in CX3CR1+/− mice produced elevated levels of IL-1β post-MCAO compared with knockout animals. This suggests that astrocytes, under certain conditions, may also express CX3CR1 receptors  mice  were subjected to MCAO followed by a 40 min 8% oxygen challenge. They found that FKN mRNA levels are reduced in the CA3 and CA1 of the hippocampus, in both males and females, 24 h and 3 days post-ischaemia–hypoxia and this reduction in FKN mRNA was evident up to five weeks post-surgery. Twelve weeks post-ischaemia–hypoxia, wild-type and CX3CR1−/− mice were tested for spatial learning ability and cognitive functioning in the T-water maze task. CX3CR1−/− males subjected to neonatal ischaemia–hypoxia showed similar test scores to sham-operated CX3CR1−/− males. Sham-operated CX3CR1−/− females, however, performed better than their ischaemia–hypoxia-subjected counterparts [−/− females post-neonatal ischaemia–hypoxia. Therefore, CX3CR1 signalling seems to play a greater neuroprotective role in female compared with male mice following a neonatal ischaemic-hypoxic event.A more recent study by Pimentel-Coelho et al.  looked aterparts . This ca12.3CR1 signalling partnership may have a role to play in neuroinflammatory and autoimmune diseases of the CNS. MS is perhaps the quintessential autoimmune disorder of the CNS, characterized by inflammation and demyelinating lesions in the spinal cord and brain of affected patients [3CR1 receptor have been found to change in and around the demyelinating lesions that accompany EAE and disease progression. Myelin oligodendrocyte glycoprotein (MOG)-induced EAE in rats causes a marked accumulation of CX3CR1-expressing microglia within brain lesions and sites of inflammation [3CR1 mRNA increased in the periplaque regions of the CNS as well in early-active, late-active and also inactive demyelinated lesions, indicating the infiltration of microglia into these affected areas. Neuronal FKN levels in the same rats remained at control levels, but there was an increase in astrocyte-associated FKN expression at sites of inflammation. This is particularly interesting as it suggests that activated astrocytes may be involved in attracting microglia to the sites of inflammation through the upregulation of FKN on their surface membranes [The FKN/CXpatients ,100. Thepatients ,102. Expammation . Notablyembranes . Neuronaembranes . Howeverembranes . In thiset al. [3CR1−/− mice contributes to more severe EAE in these animals. CX3CR1−/− mice immunized with MOG35–55 peptide displayed earlier onset and more severe EAE symptoms than wild-type animals. The same was true when EAE was induced by adoptive transfer of MOG35–55 reactive T cells. CX3CR1−/− mice induced with EAE showed an accumulation of CD115+Ly6C–CD11c+ dendritic cells in the brain and this correlated with more pronounced demyelination as well as enhanced neuronal damage. CX3CR1−/− mice suffering from EAE also showed over-expression of pro-inflammatory cytokines in CNS tissues. TNF-α levels in EAE-affected CX3CR1−/− mice were higher than their diseased wild-type counterparts. Likewise, IFN-γ mRNA was higher in the cerebellum and spinal cord and IL-17 was more abundant in forebrain and cerebellar regions of CX3CR1−/− mice induced with EAE. By contrast, levels of the anti-inflammatory cytokine, IL-10, was significantly higher in the spinal cord of EAE-wild-type mice compared with diseased CX3CR1−/− animals [3CR1 signalling in regulation of autoimmune responses and the possible role of CX3CR1 in MS in the human population. In particular, polymorphic variants of the CX3CR1 receptor which can affect FKN binding affinity as well as receptor expression have been identified. A study by Stojković et al. [I249/T280 haplotype in patients that display secondary-progressive MS compared with patients in the relapsing–remitting phase of the disease. This genetic study reveals a possible protective effect of the reference I249 allele on secondary-progressive MS when linked with the T280 allele [In a recent study by Garcia et al. , the aut animals . Taken tć et al.  suggests0 allele .13.et al. [3CR1−/− mice, lacking the receptor for FKN, display a distinct repertoire of MDMs that infiltrate the injured spinal cord compared with wild-type mice. CX3CR1−/− mice expressed relatively greater numbers of CCR2+ CNS macrophages post-SCI than wild-type mice, whereas wild-type animals displayed more iNOS+ MDMs in the injured spinal cord. CX3CR1−/− microglia also produced less IL-6 and iNOS mRNA post-SCI. Moreover, functional recovery after SCI in CX3CR1−/− mice was more rapid and sustained, implicating the importance of neuron/microglial signalling in neuronal regeneration. Therefore, in SCIs, microglia expressing the CX3CR1 receptor may release more substances that activate astrocytes and promote glial scar formation in favour of the alternative scenario which would be to promote functional regeneration of neuronal axons. Inhibiting CX3CR1 receptors at the correct temporal window post-SCI may, therefore, serve as a novel drug target to promote neuroregeneration and inhibit microglial activation.Spinal cord injury (SCI) induces a devastating trauma to neuronal cells and results in the destruction and severing of axons, leading to widespread neurodegeneration and inflammation in and around the site of injury . After set al.  have sho14.et al. [3CR1 signalling in the neurotoxic effects of microglia in a triple-transgenic mouse model of AD. This mouse model displays neuronal loss in layer III of the cortex between four and six months of age. Knocking down CX3CR1 in these mice rescued this neuronal loss suggesting that microglia play a role in Aβ-induced neuronal death. There were a greater number of microglia around the areas of neuronal damage and this increase in microglial density preceded neuronal loss indicating that microglia are involved in the elimination process. In triple-transgenic mice that also had the CX3CR1 receptor knocked out, there was no increase in microglial cell densities during this period. Microglial migration velocity to the site of neuronal damage was twofold greater than in healthy areas of the cortex in both transgenic mice and in CX3CR1−/− triple-transgenics. Once neuronal elimination was complete, however, microglial migration to the site of damage halted [3CR1 signalling in microglia during Aβ-induced neuronal stress is detrimental to neuronal survival.AD is a devastating progressive neurodegenerative condition that mainly affects the elderly population and is characterized by alterations in behaviour and cognitive impairment . The claet al.  investige halted . These oet al. [1−40 fibrils into the CA1 region of the hippocampus and measured increased CX3CR1 mRNA and protein levels, whereas FKN protein expression remained unchanged. Injecting siRNA against CX3CR1 along with Aβ1−40 fibrils not only downregulated CX3CR1 protein levels, but also CD11b expression. Moreover, CX3CR1 siRNA attenuates Aβ1−40-induced IL-1β release from microglia. Presumably, this IL-1β release from activated microglia contributes to the impaired LTP measured in the CA1 of rats treated with Aβ1–40 fibrils. Suppression of CX3CR1 signalling using siRNA rescues LTP expression in Aβ1–40-treated rats. Moreover, CX3CR1 siRNA also rescues spatial memory impairments in Aβ1–40-treated rats subjected to the Morris water maze task [Wu et al.  microinjaze task .3CR1 receptor early in AD may prove a fruitful drug-targeting strategy in the future. Duan et al. [It is widely accepted that AD is associated with chronic inflammation which may contribute to disease aetiology and pathogenesis . Targetin et al.  assessedn et al.  which ma15.3CR1 ligand/receptor pair seems to have evolved as a communication link between neurons and microglial cells. Moreover, astrocytes, in the times of inflammation, seem to upregulate FKN which also allows them to talk to microglia and neurons via CX3CR1 receptors. Overall, FKN appears to impart anti-inflammatory effects during neuroinflammatory events, such as stroke. This represents a novel neurotherapeutic target for such conditions. As with all cytokines and chemokines, however, it appears that the concentration and timing of administration will be crucial to maximize neuroprotection and prevent further toxicity to the system. FKN may also have an important role in normal learning and memory because it is expressed at such high levels on hippocampal neurons, in particular. High exogenous levels of FKN appear to have a dampening effect on synaptic transmission and LTP, which is activity-dependent. FKN, therefore, may function to limit over-activation of the hippocampal formation and maintain homeostasis in the neuronal network as a whole. The natural decrease of FKN in the hippocampus with age correlates with cognitive decline seen in older animals. Thus, preventing this downregulation of FKN may also represent a novel therapeutic target in the fight against AD and age-related dementias.The FKN/CX


S100A8 and S100A9, two members of the S100 protein family, have been reported in association with the tumor cell differentiation and tumor progression. Previous study has showed that their expression in stromal cells of colorectal carcinoma (CRC) is associated with tumor size. Here, we investigated the clinical significances of S100A8 and S100A9 in tumor cells of CRC and their underlying molecular mechanisms.in vitro.Expression of S100A8 and S100A9 in colorectal carcinoma and matching distal normal tissues were measured by reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry and western blot. CRC cell lines treated with the recombinant S100A8 and S100A9 proteins were used to analyze the roles and molecular mechanisms of the two proteins in CRC MMP7. β-catenin over-expression in CRC cells by Adβ-catenin increased cell viability and migration. β-catenin knock-down by Adsiβ-catenin reduced cell viability and migration. Furthermore, β-catenin knockdown also partially abolished the promotive effects of recombinant S100A8 and S100A9 proteins on the viability and migration of CRC cells.S100A8 and S100A9 were elevated in more than 50% of CRC tissues and their expression in tumor cells was associated with differentiation, Dukes stage and lymph node metastasis. The CRC cell lines treatment with recombinant S100A8 and S100A9 proteins promoted the viability and migration of CRC cells. Furthermore, the two recombinant proteins also resulted in the increased levels of β-catenin and its target genes c-myc and Our work demonstrated that S100A8 and S100A9 are linked to the CRC progression, and one of the underlying molecular mechanisms is that extracellular S100A8 and S100A9 proteins contribute to colorectal carcinoma cell survival and migration via Wnt/β-catenin pathway. Colorectal carcinoma (CRC) is the third leading cause of cancer-associated death worldwide. The CRC incidence rate has been shown to a dropping over these years due to enhancing CRC screening and removing precancerous polyps, but its high recurrence and poor survival rate remain to post a risk to human health S100A8 and S100A9, two members of the S100 family of calcium-binding proteins, have been originally detected in myeloid cells and known to play roles in innate immune system S100A8 and S100A9 proteins have both intracellular and extracellular functions. Intracellular S100A8 and S100A9 are involved in the calcium sensing, activation of NADPH oxidase and arachidonic acid transport Wnt/β-catenin pathway plays a critical role in CRC development In the present study, the correlation between the expression of S100A8 or S100A9 in CRC cells and the clinicopathological parameters of the patients with CRC was investigated. We found that S100A8 and S100A9 were elevated in CRC, and their expression in tumor cells was associated with the differentiation, Dukes stage and lymph node metastasis. Then the influence of S100A8 and S100A9 in the viability and migration of human colorectal carcinoma cell lines (HCT116 and SW480) and in Wnt/β-catenin pathway was analyzed. We found that the recombinant S100A8 and S100A9 proteins promote the viability and migration of CRC cells and upregulate the activity of Wnt/β-catenin pathway, and that their effect on cell viability and migration was partially mediated by upregulating Wnt/β-catenin pathway. Our study highlights the significance of S100A8 and S100A9 in the progression of CRC, and may provide the potential therapeutic targets in CRC.Colorectal carcinoma and matching distal normal tissues were collected from 42 patients (19 men and 23 women) who had undergone colorectal resection at the First Affiliated Hospital of the Chongqing Medical University. The patients received no chemotherapy, hormonal therapy or radiotherapy before surgery, and the written informed consent was received from all participants. This study was approved by the Ethics Committee of Chongqing Medical University (protocol number 2012-19).2.Human colorectal carcinoma cell lines HCT116 and SW480 were purchased from ATCC . Human embryonic kidney cell line 293 (HEK 293) was purchased from China Center for Type Culture Collection (CCTCC). HCT116, SW480 and HEK293 cells were maintained in modified Eagle's medium (MEM) with 10% fetal bovine serum , in Roswell Park Memorial Institute (RPMI) −1640 with 10% FBS  and in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS , respectively. Cell culture was maintained at 37°C in a humid atmosphere containing 5% COThe specimens were fixed in 10% buffered-formalin, embedded in paraffin blocks and were serially sectioned for hematoxylin/eosin (HE) staining. These sections were viewed twice by two experienced blinded pathologists to verify the diagnosis, histological differentiation, and pathological stage. These tumors were staged according to Dukes staging system into stage A , stage B , stage C  and stage D . Tumor histological differentiation was graded into well (3 cases), moderately (29 cases) and poorly (10 cases). The information of patients' tumor size, pathology and stages are presented in 2O2) in methanol for 15 min to block endogenous peroxidase. And the sections were incubated with the anti-hS100A8 or anti-hS100A9 monoclonal antibody  overnight at 4°C, following incubated with secondary antibody tagged with the peroxidase enzyme  for 30 min at room temperature and were visualized with 0.05% 3,3-diaminobenzidine tetrachloride (DAB) until the desired brown reaction product was obtained. The sections were finally counterstained with hematoxylin. The negative control group was carried out with the same steps as described above except replacing the primary antibody with phosphate buffer solution (PBS).The expression of S100A8 or S100A9 in tissues was examined by IHC. The sections from the formalin fixed, paraffin-embedded tissues were deparaffinized and dehydrated. Then the sections were boiled for 10 min in 0.01 M citrate buffer and incubated with 0.3% hydrogen peroxide , grade 2 (10–25% of positive cells), grade 3 (26–75% of positive cells) and grade 4 (>75% of positive cells). In addition, its staining intensity was also divided into four subgroups . The staining index was obtained by multiplication of proportion and intensity ranging from 0 to 12 E. coli (BL21) following the instructions of calcium chloride transformation. Isopropylthio-β-D-galactoside was used to induce the expression of GST-S100A8 and GST-S100A9. After the bacteria were sonicated, the supernatants were collected, spun and incubated with glutathione-Sepharose 4B beads, and GST-S100A8 and GST-S100A9 on the beads were eluted by elution buffer with reduced glutathione. Finally, the proteins were filtered via 0.22 µm membrane and stored at −80°C. The control protein GST was prepared at the same time. Its plasmid is pGST-Moluc.pGST-Moluc-hS100A8 and pGST-Moluc-hS100A9 have been described previously The recombinant adenovirus carrying β-catenin gene (Adβ-catenin) and β-catenin-siRNA gene (Adsiβ-catenin) and their control (AdGFP or AdRFP) were kindly provided by Professor T.C. He . All were amplified in HEK293 cells before use The recombinant proteins  were subjected to polyacrylamide gel electrophoresis. Following electrophoresis, the gel was placed in a colloidal coomassie staining solution and incubated for 6 hours to overnight. Then distilled water was used to de-stain the gel until the background was transparent. All steps were done on a rotary shaker with slight mixing.MTT  assay was used to assess the cell viability. HCT116 or SW480 cells during log growth stage were seeded in 96-well plates (1000 cells/well) with MEM or RPMI1640 containing 10% FBS and incubated overnight. Then the cells in different groups were treated with and without GST, GST-hS100A8 and GST-hS100A9 in MEM or RPMI-1640 containing 1% FBS for 24, 48, 72, 96 and 120 h, then the MTT reagent  was added (20 µl/well) and incubated for 4 h at 37°C, 100 ml dimethyl sulfoxide was added to dissolve formazan product for 10 min at room temperature. Finally, the absorbance was daily measured for the following five days at 492 nm using a microplate reader. Each condition was done in quintuplicate, and the overall experiment was repeated thrice.2 incubator. After two weeks, the cells were stained by crystal violet and clones were counted. The colony-forming rate was obtained as: (colony number/seeded cell number)×100%. The experiment was repeated thrice.HCT116 or SW480 cells during log growth stage were collected and seeded in 6-well plates (100 cells/well), which were treated with and without GST, GST-hS100A8 and GST-hS100A9 at 37°C in a 5% CO5) were seeded in the upper chamber and suspended in 400 µl serum-free MEM or RPMI1640 culture medium in duplicate. The bottom chamber was filled with 500 µl of MEM or RPMI1640 containing 20% FBS as a chemoattractant. After incubation with GST, GST-hS100A8 and GST-hS100A9 for 24 h, the transmembrane cells were dried for 5 min, then fixed with methanol and stained with hematoxylin-eosin for 10min. Finally the transmembrane cells were counted with microscopy at 100×. Mean values were obtained from five randomly selected fields for each well. The experiment was repeated thrice.A chamber of non-type I-collagen-coated 24-well culture inserts  was used for the transwell migration assay. Cells . Nuclear protein and cytoplasmic protein were extracted using Nuclear-Cytosol Extraction Kit  according to manufacturer's instructions. Proteins were quantitated by BCA (bicinchoninic acid) assay. The proteins of the lysate were separated in 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto the PVDF membranes. Then the membranes were blocked with 5% bovine serum albumin at room temperature for 2 h and incubated with rabbit anti-β-catenin polyclonal antibody , or mouse anti-S100A8 monoclonal antibody , or mouse anti-S100A9 monoclonal antibody , or mouse anti-β-actin monclonal antibody , or goat anti-Histone antibody  at 4°C overnight. After washed with TBS supplemented with 0.1% Tween 20 (3×5min), the membranes were incubated with goat anti-rabbit IgG serum , goat anti-mouse IgG serum  or rabbit anti-goat IgG serum  for 2 h at room temperature and enhanced chemoluminescence  in substrate for 5 to 20 min. The results were recorded by using the Bio-Rad Electrophoresis Documentation  and Quantity One Vesion 4.5.0.2O2 for 5 min and incubated with rabbit anti-β-catenin polyclonal antibody  overnight at 4°C, following incubated for 30 min with secondary antibody tagged with the peroxidase enzyme , and then visualized with 0.05% DAB until the desired brown reaction product was obtained. Finally, all sections were dehydrated, cleared and mounted in a neutral gum under cover slips. The negative control group was carried out with the same steps as described above except replacing the primary antibody with PBS. The staining of samples was observed under an inverted phase contrast microscope and representative images were captured .The cells were plated and cultured onto cleaned-up cover slips. After treated with and without GST (10 µg/ml), GST-hS100A8 (10 µg/ml) and GST-hS100A9 (10 µg/ml) for 48h, the cells were treated with 0.03% HMMP7 were detected by RT-PCR. GAPDH was used as an endogenous control. The PCR primers . Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was carried out as described by using the Takara RNA PCR kit. Primers were also synthesized by Invitrogen. The mRNA levels of S100A8, S100A9, c-myc, and mers see  were desTo detect the effects of β-catenin on the biological roles of S100A8 and S100A9 in CRC cell lines, the cells were grown and infected with Adsiβ-catenin or Adβ-catenin for 36 h and then treated with and without GST, GST-hS100A8 or GST-hS100A9 subsequent 24 and 72 h. After incubation for the subsequent 24 h, the cell migration capability was analyzed by transwell migration assay, and after 72 h, the cell viability was analyzed by MTT assay. The overall experiment was repeated thrice.All the values and figures in the text were presented as mean ± SEM (standard error of the mean). One-way ANOVA followed by the S-N-K test was used for the analyses of semi-quantitative RT-PCR, colony formation, cell viability, transwell migration and western blot assay. Chi-square test and Yates-corrected chi-squared test were used to analyze the relationship between the expression of S100A8 or S100A9 in tumors and various clinicopathological parameters. All the statistical analyses were performed using GraphPad Prism 5 . Significant probability values were indicated as p<0.05*, p<0.01**, p<0.001***.We performed immunohistochemical (IHC) staining to detect the expression of S100A8 and S100A9 in sections of 42 pairs of samples  and found that S100A8 and S100A9 were elevated in tumor cells of CRC tissues compared with the matching distal normal tissues . This waTo further investigate whether the expression of S100A8 and S100A9 in tumor cells was associated with disease progression, we analyzed their expression against the clinicopathological parameters, including gender, age, tumor location, tumor size, histological differentiation, Dukes stage and lymph node metastasis . Our datExtracellular S100A8 and S100A9 represent danger signals and trigger cellular responses in the dose-dependent manner in tumors. To further evaluate the influence of extracellular S100A8 or S100A9 on the viability and migration of CRC cells, we prepared recombinant proteins GST, GST-hS100A8 and GST-hS100A9 to treat cells, and GST was used as the control for GST-hS100A8 and GST-hS100A9. Their purities were all over 90% , while the proteins at 40, 80 and 120 µg/ml had no effect on the cell viability . The simAfter the HCT116 cells were treated with GST-hS100A8 (10 µg/ml) and GST-hS100A9 (10 µg/ml) for 24 h, the number of migratory cells was significantly increased by 1.53-fold (p<0.001) and 1.67-fold (p<0.001) by transwell migration assay respectively, compared to the cells treated with GST protein . The simIt is well known that aberrant activation of the canonical Wnt/β-catenin pathway plays a critical role in the development of CRC. To test whether S100A8 and S100A9 can regulate the Wnt/β-catenin pathway, we first investigated the influence of S100A8 and S100A9 on β-catenin level, which is a key effector of this signaling pathway.After HCT116 and SW480 cells were treated with recombinant GST-hS100A8 (10 µg/ml) or GST-hS100A9 (10 µg/ml) for 36 h, we detected β-catenin levels in the whole and nuclear lysates. We found that the levels of total β-catenin and nuclear β-catenin were increased in HCT116 cell line . The simMMP7, which are two classic target genes of the pathway MMP7 was not detected in the cells  and β-catenin level of HCT116 and SW480 cell lines were very similar, the subsequent investigations were performed only with SW480 cell line. We transduced the cells with Adβ-catenin and Adsiβ-catenin for over-expression and knock-down of β-catenin level, respectively and after 36 h, β-catenin level was increased by 59.4% and reduced by 56.9%, as compared to the cells infected with AdGFP (control for Adβ-catenin) and AdRFP (control for Adsiβ-catenin), respectively . At the We also found that β-catenin over-expression increased the number of migratory cells (p<0.001), conversely β-catenin knock-down reduced the number of migratory cells (p<0.001). Furthermore, β-catenin knock-down also partially abolished the cell migration led by treatment of S100A8 (p<0.01) or S100A9 (p<0.01) . It suggS100 proteins participate in numerous functions including protein phosphorylation, enzymatic activation, calcium homeostasis, and interaction with cytoskeletal components Extracellular S100A8/A9 proteins secreted from cells function as danger associated molecular pattern ligands for cell surface receptors, activating signaling cascades and triggering cellular responses MMP7, the target genes of the pathway, in the cell lines. All suggest that extracellular S100A8 and S100A9 can upregulate Wnt/β-catenin pathway. Based on the previous studies showed that β-catenin upregulation was involved in the proliferation and migration of colon cancer cells S100 proteins have common structural features and display sequence homology Extracellular S100A8 and S100A9 in tumor microenvironment may mainly exert their biological roles in CRC progression, and their secretion to tumor microenvironment may be affected by a variety of factors including tumor cells, stromal cells, and other composition of the tumor microenvironment. In addition, there are still some unanswered questions about why S100A8 and S100A9 could exert the promotive effects on activation of Wnt/β-catenin pathway in CRC. We speculate that these promotive effects on Wnt/β-catenin pathway might be via some cell surface receptors. Two pattern recognition receptors, Toll-Like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE), have shown to be involved in S100A8/A9-mediated pathologic effects such as infection, autoimmunity and cancer In conclusion, this data demonstrates that expression of S100A8 and S100A9 in tumor cells is associated with differentiation, Dukes stage and lymph node metastasis in CRC. Furthermore, our data also show that extracellular S100A8 and S100A9 could promote viability and migration of colorectal cancer cell lines HCT116 and SW480 through upregulation of Wnt/β-catenin pathway. Take together, our results provide important information regarding the roles of S100A8 and S100A9 in malignancy and should be paid much attention considering that S100A8 and S100A9 has been increasingly recognized as tumor markers and also as new molecular targets for developing cancer therapeutics.Figure S1Identification of GST-S100A8 and GST-S100A9 by SDS-PAGE and Western blotting. (A) Recombinant human GST-S100A8 was about 37 kDa, GST-S100A9 was about 40 kDa and GST was about 26 kDa; their purities were all >90% (by Quantity One Software after SDS-PAGE). (B) GST-S100A8 and GST-S100A9 was recognized by anti-S100A8 and anti-S100A9 antibodies through western blotting. Left lane 1, GST protein; left lane 2, GST-hS100A8 protein. Right lane 1, GST protein; right lane 2, GST-hS100A9 protein. kDa, kilodalton.(TIF)Click here for additional data file.Figure S2Immunofluorescence analysis of β-catenin expression in S100A8- and S100A9-treated SW480 cells. SW480 cells were treated with and without recombinant S100A8 or S100A9 protein (10 µg/ml) for 36 h before being stained by the antibody against β-catenin, and then FITC-labeled secondary antibody were applied (green fluorescence). The nucleus was counterstained with DAPI (blue). The images were visualized under a laser scanning confocal microscope. The representative images are shown in the graph. The intense fluorescence for β-catenin level is in nucleus after treatment with GST-hS100A8 and GST-hS100A9. Red scale bars = 100 µm.(TIF)Click here for additional data file.Figure S3The effect of recombinant S100A8 and S100A9 proteins on mRNA expression of c-myc and MMP7 in CRC cells. The mRNA expression of c-myc and MMP7 was detected using RT-PCR. GAPDH was used as an internal reference control. The relative mRNA expression of c-myc or MMP7 is quantified by c-myc/GAPDH or MMP7/GAPDH densitometric ratios and is shown in the graph. * p<0.05, ** p<0.01 and **** p<0.001, all vs. GST control.(TIF)Click here for additional data file.Table S1Primers used in RT-PCR.(DOC)Click here for additional data file.Materials and Methods S1(DOC)Click here for additional data file.


GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage.Granule-bound starch synthase (GBSS) is responsible for amylose synthesis, but the role of  Starch is the main carbohydrate consumed for human nutrition, and is a major component of cereals, tubers, legumes, and fruits. Starch consists of a mixture of two different carbohydrates, namely amylose (20%–30%) and amylopectin (70%–80%) GBSSI gene expression in the endosperm of wheat GBSSI, driven by the CaMV 35S promoter, was introduced into sweet potato by Agrobacterium tumefaciens-mediated transformation. One of the resulting 26 transgenic plants had an absence of amylose in the tuberous roots GBSS gene expression in sweet potato, starch has been produced with varying amylose-amylopectin ratios. Low-amylose starch may be used in the food industry, while high-amylose starch is useful in the candy industry and for synthesizing plastics GBSS proteins are crucial in regulating the formation of amylose. Inhibition of GBSSI expression by RNAi interference resulted in an amylose-free transgenic sweet potato GBSS genes in eudicots belong to the GBSSII family and their expression was consistent with the pattern observed for GBSSII genes in monocots. This suggests that the GBSSI subfamily has been lost during the evolutionary process leading to eudicots GBSSI genes has been characterized from maize GBSSII genes were isolated from rice GBSSI and GBSSII genes in banana.GBSS proteins are also known as waxy proteins Musa spp.) is the main staple food of the tropics. Its fruits are vital for food security in many tropical and subtropical counties and banana is also among the most popular fruits in industrialized countries http://banana-genome.cirad.fr/). This genome sequence database provides unique opportunities for the genome-wide investigation of genes involved in banana fruit starch synthesis. In this study, we investigated the changes in amylose content, GBSS enzyme activity and GBSS protein from preharvest to postharvest of banana (Musa acuminata L. AAA group cv. Brazilian) fruit. Six MaGBSS genes were cloned, and their sequence characteristics, chromosomal location, and expression patterns were studied in different tissues and at different stages of fruit development or storage. The number, size, and shape of starch granules in banana fruit during development and storage were also observed.Banana , 1851 bp (accession No. KF512021), 675 bp (accession No. KF512022), 1845 bp (accession No. KF512023), 2265 bp (accession No. KF512024), and 906 bp (accession No. KF512025), respectively. Genes encoding MaGBSSI-1, MaGBSSI-2, and MaGBSSI-4 contain 13 exons and use TGA as stop codon. MaGBSSI-3 consists of 6 exons and uses TAG as stop codon. MaGBSSII-1 and MaGBSSII-2 contain 10 exons and 1 exon, respectively, and use TAA as stop codon (MaGBSS family members were BLASTed against the banana genome sequence database (http://banana-genome.cirad.fr/). MaGBSSI-1, MaGBSSI-2, and MaGBSSI-3 are dispersed along chromosome 3. MaGBSSII-1, MaGBSSII-2, and MaGBSSI-4 are located on chromosomes 4, 8, and 9, respectively (Six genes encoding op codon . The sixectively .http://expasy.org/compute.pi/), which was consistent with the GBSS protein detected using SDS-PAGE and western blotting analyses   . A phylo2278470) .MaGBSSI and MaGBSSII in different banana tissues  were higher than the later stages (from 30 d to 60 d). In contrast, MaGBSSI-3 was weakly expressed at the early stages but was highly upregulated (approximately 50-fold) at 50 d of development  of starch granule-filling, and MaGBSSI-3 might be involved in the later stages (30–60 d) of starch granule-filling during the development of banana fruit  of fruit ripening and was barely detectable during the later stages (10 d–30 d) of fruit storage  and the number of granules decreased significantly thereafter. Fewer granules were observed at 5 d of storage and granules could not be detected by 10 d of storage . These rAmylose content is an important factor contributing to the yield and quality of banana fruit. Slack and Wainwright In our study, a GBSS protein purified from banana fruit was approximately 55.0 kDa . This moMaGBSS genes were sequenced and characterized from banana fruit. MaGBSSI-1, MaGBSSI-2, MdGBSSII-1 and MaGBSSII-2 each have 13 exons and three conserved regions   obtained from a SSH cDNA MaGBSSI-3 locus contains 6 exons and is characterized by 5 amino acid substitutions  within Box 2 , maize (NM001111531), wheat (AB019624), and barley (SBU23945) contain 13, 13, 11, and 11 exons, respectively. These results suggest that MaGBSSI might be the specific gene in banana fruit.In this report, six , and 3) . The strin Box 2 . This geGBSSI family members are located on chromosomes 7AS, 7DS, and 4AL GBSSII genes are located on chromosomes 2B, 2D, and on the long arm of chromosome 2A MaGBSSI-1, MaGBSSI-2 and MaGBSSI-3 were found on banana chromosome 1 and MaGBSSI-4 is located on chromosome 9. Genes encoding banana MaGBSSII-1 and MaGBSSII-2 are located on chromosomes 4 and 8, respectively  are expressed in the early stage (0–30 d) of starch granule formation, and are similar to the GBSSII-2 gene in peach fruit MaGBSSI-1 and MaGBSSI-2 in the early stages of banana fruit development was approximately 5-fold higher than that of MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2. This result suggests that MaGBSSI-1 and MaGBSSI-2 might play an important role in early starch accumulation in banana fruit. The second group consists of a single, late-expressing gene (MaGBSSI-3), which is expressed in the later stage (30–60 d) of starch granule formation during the development of banana fruit  and have more regular shapes. In potato tubers, SSIII activity also alters granule shape MaGBSSI-3 expression during the development of banana fruit  have an effect on granule number and form na fruit , suggestMaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2 were 1851 bp, 1851 bp, 675 bp, 1845 bp, 2265 bp, and 906 bp, respectively. Among the six MaGBSS genes, only MaGBSSI-3 was highly expressed in reproductive tissues and at the late developmental stages of banana fruit. Scanning electron microscopy analysis showed that the level of MaGBSSI-3 expression is correlated with the quantity and size of starch granules in banana fruit during development and storage. These results suggest that up-regulated expression of MaGBSSI-3 might be a key factor regulating amylose metabolism by affecting the variation of GBSS levels and the quantity and size of starch granules in banana fruit. Further work is required to elucidate how the different expression levels of the MaGBSSI-3 gene at different times could result in changes of the quality and size of starch granules of banana fruit.In conclusion, amylose content, GBSS enzyme activity, and GBSS protein levels gradually increased during banana fruit development, but they decreased during banana fruit storage. Full-length cDNAs encoding M. acuminata L. AAA group cv. Brazilian) fruits were obtained from the banana plantation at the Institute of Tropical Bioscience and Biotechnology . Root, stem, leaf, bract, flower, peel, and pulp tissues were collected separately using tweezers and frozen immediately in liquid nitrogen, then stored at −80°C until further analysis. Pulps of 0 d, 10 d, 20 d, 30 d, 40 d, 50 d, and 60 d after emergence from the pseudostem were collected, immediately frozen in liquid nitrogen and stored at −80°C for expression analysis.Banana 2 in a boiling water bath for 10 min, and then centrifuged for 4 min at 4,000 rpm. The supernatant was transferred to a 20 mL volumetric flask, and the residue was extracted two times with 80% Ca(NO3)2, yielding a combined extract volume of 20 mL. All experiments were repeated three times. The total starch content was detected following the method of Yang et al. Banana pulp was immersed in 0.5% sodium bisulfite solution for 10 min to prevent browning, and then dried at 40°C for 24 h. Pulp was ground and centrifuged. The residue was suspended in 5 mL 80% Ca for western blot analysis. Membranes were probed with rabbit anti-GBSS polyclonal antibody  diluted 1∶1000 in PBS-Tween 20 (PBST) plus 3% BSA, followed by alkaline phosphatase (AP)-conjugated anti-rabbit IgG secondary antibody  diluted 1∶1000. Positive signals on the membranes were detected by a 5-bromo-4chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) solution .GBSS protein samples were mixed with SDS-PAGE sample buffer, boiled 5 min, and separated on 12% polyacrylamide gels. Proteins were transferred to Hybond™ –N18 primer and a Reverse Transcriptase M-MLV Kit according to the manufacturer's instructions .Total RNA was extracted from various tissues using the plant RNAout Kit . The integrity and concentration of isolated RNAs were examined by agarose gel electrophoresis and spectrophotometry . First-strand cDNA was synthesized in a 20.0 µL reaction mixture using 1.0 µg of total RNA, an Oligo(dT)GBSS homologues in banana were identified using a BLAST-based method. Primer pairs were designed to amplify each gene and their sequences are presented in E. coli DH5α competent cells. Positive recombinant plasmids were selected by the white/blue selection method and verified by colony PCR. Target DNA was then confirmed by restriction enzyme digestion and sequence determination. Full-length cDNA sequences encoding MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2 were submitted to GenBank.PCR products were purified using an agarose gel DNA Purification Kit  and inserted into the pMD19-T vector . Ligated products were transformed into GBSS coding sequences were BLASTed against the banana genome sequence database (http://banana-genome.cirad.fr/) to recover their corresponding genomic DNA sequences. Exon lengths were calculated by alignment of genomic DNA sequences with cDNA sequences, and introns were determined according to the “GC-AG” rule Banana GBSSI or GBSSII cDNA sequences with other homologues in the GenBank database was performed using the BLAST program (E<0.001). The deduced amino acid sequences were aligned using the computer program Clustal W, and a homology tree was constructed with neighbor-joining method using MEGA software . The number for each interior branch is the percent bootstrap values calculated from 1,000 replicates.Similarity of the full-length banana GBSS genes were quantified by Q-RTPCR using an iQ5 real-time PCR detection system  and a SYBR ExScript RT-PCR Kit . Each reaction of 25.0 µL contained 12.5 µL SYBR® Premix ExTaq™ , 1.0 µL of each primer (5.0 µM), 8.5 µL ddH2O, and 2.0 µL cDNA (40 ng). An actin gene (accession No. EF672732) was used as an internal reference control.Specific primer pairs were designed using Primer 5.0 software and their sequences are listed in −1 for melting curve analysis. Reaction mixtures lacking cDNA templates were run as negative controls to rule out contaminating DNA. All analyses were repeated three times using biological replicates. Relative expression levels of each gene were calculated using the 2CT−ΔΔ method The amplification program consisted of one cycle of 95°C for 1 min, followed by 40 cycles of 95°C for 10 s, 57°C for 15 s, and 72°C for 30 s. Melting curve analysis was performed at the end of 40 cycles to ensure proper amplification of target fragments. Fluorescence readings were collected from 60°C to 90°C at a heating rate of 0.5°C sGBSS genes during different stages of development and storage of banana fruit, pulp was isolated after 0 d, 30 d, and 60 d of development and from the fruit 0 d, 5 d, and 10 d following storage.Based on the expression of The samples were fixed in stubs using double-faced tape and coated with a 10 nm-thick platinum layer in a Bal-tec MEDo020 Coating system . The samples were analyzed in an FEI Quanta 600 FEG Scanning Electron Microscope . SEM observations were performed in the secondary electron mode operating at 15 kV.Figure S1GBSS in banana. A, Structural organization of banana GBSS genes. Solid boxes indicate exons, and bold lines represent introns. B, Chromosomal localization of banana GBSS genes.Sequence motifs and chromosomal localization of six genes encoding (TIF)Click here for additional data file.Figure S2Multiple amino acid alignment of the complete coding sequences of MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2 proteins from banana.(DOC)Click here for additional data file.Table S1Primers used in this study.(DOC)Click here for additional data file.


Attenuation of Wnt/β-catenin activity by tankyrase inhibitors is an appealing strategy in treatment of OS. The efficacy of the tankyrase inhibitor JW74 was evaluated in three OS cell lines  both at the molecular and functional level. At the molecular level, JW74 induces stabilization of AXIN2, a key component of the β-catenin destruction complex, resulting in reduced levels of nuclear β-catenin. At the functional level, JW74 induces reduced cell growth in all three tested cell lines, in part due to a delay in cell cycle progression and in part due to an induction of caspase-3-mediated apoptosis. Furthermore, JW74 induces differentiation in U2OS cells, which under standard conditions are resistant to osteogenic differentiation. JW74 also enhances differentiation of OS cell lines, which do not harbor a differentiation block. Interestingly, microRNAs (miRNAs) of the let-7 family, which are known tumor suppressors and inducers of differentiation, are significantly upregulated following treatment with JW74. We demonstrate for the first time that tankyrase inhibition triggers reduced cell growth and differentiation of OS cells. This may in part be due to an induction of let-7 miRNA. The presented data open for novel therapeutic strategies in the treatment of malignant OS.Wnt/ As a result, β-catenin accumulates in the cytoplasm and subsequently translocates to the nucleus where it regulates transcription of Wnt/β-catenin target genes, in part by binding to transcription factor T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) The Wnt/β-catenin levels are tightly controlled by the cytoplasmic destruction complex (DC), which consists of the rate-limiting proteins AXIN1/2, the adenomatous polyposis coli protein (APC), casein kinase (CK1)α, and glycogen synthase kinase 3 (GSK3)β and additional associated proteins including TRF-1-interacting ankyrin-related ADP-ribose polymerase 1 or 2  β-catenin associates with the DC, is phosphorylated by CK1-α and GSK3ββ-catenin levels can be attenuated by TNKS In the absence of Wnt signaling, β-catenin signaling, considerable efforts have been made to identify drugs that inhibit Wnt/β-catenin signaling either by blocking Wnt secretion β-catenin binding to its transcription factor targets β-catenin signaling Due to the biological relevance of Wnt/β-catenin signaling is implicated in normal osteoblast differentiation and aberrant Wnt/β-catenin signaling disrupts normal bone development β-catenin have not been observed in OS, but instead increased β-catenin activity has been linked to increased expression of Wnt receptors or an inhibition or loss of expression of secreted inhibitors β-catenin may have therapeutic potential for OS patients Osteosarcoma (OS) is the most common primary malignant bone cancer μmol/L were done immediately before use.The cell lines U2OS, SaOS-2 (both from American type culture collection [ATCC]), and KPD μmol/L) for 24, 48, or 72 h. Cell lysates were generated by incubating in 200 mL lysis buffer  on ice for 10 min, followed by a short sonication. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting was performed using primary antibodies; AXIN2 (76G6) , Tankyrase-1/2 (H-350) , LaminB1 , active β-catenin ABC , total β-catenin (610154) , and ACTIN (Santa Cruz Biotechnology). Antibodies were visualized using secondary horseradish peroxidase-conjugated antibodies  and enhanced chemiluminescent substrate .One hundred fifty thousand cells grown overnight in six-well plates were treated with 0.1% DMSO (control) or JW74 (10–0.5 μ–10 μmol/L). Luciferase and Renilla activity were determined using Dual-Glo Luciferase Assay System .Transfection of 2000 U2OS cells plated in 96-well plates was done the following day with reporter plasmid pTA-Luc-STF and control plasmid-expressing Renilla, as in −ΔΔCt method normalizing to PGK1 or RNU44 for mRNA and miRNA analyses, respectively. PGK1 or RNU44 were used as housekeeping genes, due to their unchanged expression during treatment Isolation of total RNA and cDNA synthesis were performed using Cell-to-Ct kit for mRNA or miRNA , following the manufacturer's protocol. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with primers and master mix from Ambion, using cDNA from 100 to 500 cells/well. The detection limit was set to cycle threshold value = 36. Relative quantifications were calculated with the 2μmol/L JW74. Two million treated cells were stained with 2 μg/mL Hoechst 33342 and 20 μL/test of PE-mouse anti-human Ki-67 , as described previously Three hundred thousand cells in T25 flasks were attached overnight and treated for 72 h with DMSO (control) or 5 μmol/L). Proliferation rates based on cell confluence were determined by live cell imaging , as described previously Two to three thousand cells attached overnight in 96-well plates were treated with culturing medium containing 0.1% DMSO (control) or JW74  (Essens Bioscience) was included in the medium (1:1000 dilution), enabling quantitative measurement of Caspase-3 activity by fluorescence live cell imaging in the IncuCyte. Data show total number of cells with high Caspase-3 activity in each well 52 h post treatment-start. Annexin V assay was performed using the Alexa Fluor 488 annexin V and propidium iodide (PI) kit for flow cytometry . 100,000 U2OS cells were plated in six-well plates and incubated with DMSO or 10 μmol/L) only; (3) 0.1% DMSO in combination with a differentiation cocktail , or (4) differentiation cocktail combined with JW74 (10 μmol/L). Cells were not passaged during the experiment (maximum 24 days), but medium and supplements were changed twice per week. Osteogenic differentiation was determined quantitatively, using alkaline phosphatase (ALP) activity as a marker. The ALP assay kit (Abcam) was performed as recommended by manufacturer. Data are presented relative to total protein concentration. Degree of osteogenic differentiation was also assessed by alizarin red staining .Thirty thousand cells attached overnight in 24-well plates were incubated in culturing medium supplemented with one of four combinations: (1) 0.1% DMSO (control); (2) JW74 (10 P = > 0.05), Student's t-test was performed. If the Shapiro–Wilk test failed (P < 0.05), Mann–Whitney rank sum test was applied. P < 0.05 was regarded as a statistically significant difference.Statistical analyses were performed using SigmaPlot 11 . For comparisons of two groups, normal distributions of datasets were first analyzed with the Shapiro–Wilk test. When the Shapiro–Wilk test passed  were strongly reduced in a dose-dependent manner . We also performed flow cytometry to determined the expression of the proliferation marker Ki-67 in U2OS following 48 h treatment with DMSO or 10 μmol/L JW74. Ki-67 expression was reduced from 97.5% in DMSO-treated cells to 86.7% in JW74-treated cells (data not shown).Having shown that JW74 exerts molecular effects on key mediators of the canonical Wnt signaling pathway, we next wanted to evaluate the functional effects of tankyrase inhibition. We first studied the proliferative capacity of OS cells during short-term in vitro treatment with JW74. For this purpose, we used the a live cell imaging machine (IncuCyte), which captures cellular images every second hour throughout the duration of the experiment enabling us to determine the effect of the drug on cell confluence over time. The time lapse experiment clearly showed that tankyrase inhibition had a dose-dependent growth-limiting effect on U2OS, KPD, and SaOS-2 cells A. In addμmol/L)  C.μmol/L JW74 for 72 h and found an increased number of cells in the G1-phase (45.5–54.8%) and a decreased number of cells in S-phase (27.4–24.0%) and G2/M (22.2–16.2%) compared to control-treated cells .We subsequently performed flow cytometric cell cycle analyses of Hoechst-stained U2OS cells treated with 5 ed cells D, indicaμmol/L JW74 alone, compared to control-treated cells (DMSO)  A. The chs (DMSO) A. We alss (DMSO) B. Intrigc-MYC expression, we hypothesized that microRNA (miRNA) let-7 levels might be elevated following JW74 treatment. miRNA let-7 is a master regulator of differentiation let-7 orthologs evaluated .The majority of OS tumors are poorly differentiated and induction of differentiation may be an interesting therapeutic strategy, as cells may become more susceptible to treatment upon induced differentiation SOX2 expression in U2OS cells. Thus, mechanisms involving SOX2 do not seem responsible for the observed differentiation in our system.It has also been shown that SOX2 plays a key role in maintaining OS cells in an undifferentiated state, being essential for self-renewal and acting as an antagonist of the Wnt pathway let-7 are tumor suppressors and key regulators of differentiation let-7 orthologs following incubation with JW74. To our knowledge, neither tankyrase nor the Wnt/β-catenin signaling pathway has to date been directly linked with the let-7 systems. As we observed reduced C-MYC levels following JW74 incubation, regulation of let-7 through C-MYC is a possibility. However, further work is required to elucidate the links between tankyrase inhibition and increased let-7 levels. Interestingly, β-catenin has been described as a regulator of other miRNAs, including miR-15, miR-16, miR-375, and miR-122a β-catenin regulate these miRNAs are not known.The miRNA family let-7 orthologs in response to JW74 treatment is of particular importance in the light of therapeutic attempts to reduce the proliferative capacity and trigger differentiation of poorly differentiated cancer cells through increased let-7 levels. Let-7 replacement therapy has shown great potential as a novel cancer therapeutic in xenograft models, where the tumor regresses following introduction of let-7The significant upregulation of multiple


Although neuroinflammation has been studied extensively in animal models of cerebral ischemia, their contrasting functions are still not completely understood. A major participant in neuroinflammation is microglia and microglial activation usually regulated by the chemokine CX3CL1  and its receptor, CX3CR1. Here, we examined the involvement of CX3CR1 on ischemia-induced chronic neuroinflammation and cognitive function using small interfering RNA (siRNA). Forty adult male Wistar rats were included in the study and received either ischemia or sham surgery then were randomized to receive either CX3CR1 siRNA or scrambled RNA as control starting at 7 days after reperfusion. Behavioral testing commenced 28 days after siRNA delivery and all rats were euthanized after behavioral testing. Our data showed that: (i) transient global cerebral ischemia significantly decreased fractalkine/CX3CR1 signaling in the hippocampus; (ii) inhibition of CX3CR1 function exacerbated the ischemia-induced chronic increase in microglial activation and pro-inflammatory cytokine levels; (iii) inhibition of CX3CR1 function worsened ischemia-induced chronic cognitive impairment; (iv) inhibition of CX3CR1 function in sham rats resulted in increased IL-1β expression and impaired behavioral performance. However, no significant effect of CX3CR1 on ischemia-induced neurodegeneration was seen. The present study provides important insight to understanding the involvement of CX3CR1 in chronic neuroinflammation and cognitive impairment. Ischemic brain injury is, in part, an inflammatory disease. The pathophysiology of cerebral ischemia includes a sequence of events that involves the actions of several central and peripheral cell types as well as a large number of inflammatory molecules ,2. For eThe question whether neuroinflammation is destructive and/or beneficial following ischemic injury has been debated for a long time but the answers remain equivocal. A major participant in the neuroinflammatory process is microglia since these cells are among the first to display very early activation in ischemic injury and are capable of contributing to neuronal damage by expressing potentially harmful molecules, such as interleukin-1  (IL-1), tumor necrosis factor-Î± (TNF-α), and reactive oxygen species . Howevercx3cr1 gene is substituted with the gene for the green fluorescent protein (GFP) producing a defective receptor for fractalkine [Among the molecules that regulate microglial activation is the chemokine CX3CL1 , which is constitutively expressed by normal neurons. CX3CL1 signals to microglia by binding to only one receptor, CX3CR1, whose function is to inhibit microglial activity ,9. In thctalkine . Using tctalkine , Parkinsctalkine , and Alzctalkine  report tctalkine . Howeverctalkine -18. ThesMale Wistar rats  were obtained from Harlan Laboratories . Animals were housed in pairs in a pathogen-free vivarium under controlled condition  and a 14:10-h light:dark cycle was maintained. All animals were housed in the same room so that temperature, humidity, and lighting conditions were similar for all groups. Animals had free access to food and water delivered through an automated and filtered system. Animals were also handled daily throughout the study so that they could get acclimated to the research personnel thereby decreasing stress. Experiments started 1 week after arrival of the animals from the breeder and all protocols in this study were approved by the Institutional Animal Care and Use Committee and in accordance with the National Institutes of Health guidelines.n = 0) were excluded from the study. In addition, observing for sluggishness, extreme aversion to being touched, and weight loss assessed pain level. Animals were not given postoperative analgesia but were euthanized immediately when persistent pain was observed (n = 0). Sham-operated animals were subjected to the same anesthesia and surgery that consisted of a neck incision without carotid manipulation and an incision behind the occipital bone without cauterization of the vertebral arteries. All efforts were made to minimize animal distress and reduce the number of rats used.The four-vessel occlusion method was used to induce transient global cerebral ischemia as described previously . Brieflyn = 10 sham and n = 10 ischemia) or scrambled RNA (n = 10 sham and n =10 ischemia). The drug was delivered for 28 days followed by behavioral testing  using the coordinates according to Paxinos and Watson  taking iRats were tested in three versions of the water maze and the tub was filled with tepid water (22  ± 2°C) and made opaque by the addition of powdered milk. In the cued learning task, the pool was divided into four quadrants of equal surface area and the starting locations for testing were assigned north, south, east, and west . The behavioral testing walls were already painted white so just the other distal (to the water maze) visual cues in the rooms were removed during the habituation training and actual testing. The cued spatial learning and memory  task is sensitive to hippocampal dysfunction . The day+) from the water (located in the southeast quadrant); whereas the other one painted black was floating (P-) and not able to offer sufficient buoyancy to support the rat (located in the southwest quadrant). Both visible goals were placed 10 cm above the water level. For this task, path taken to reach the correct goal was recorded as well as search errors based on the choices of P+ compared to P- since the aim was to train the rats to avoid P-. Search errors represent the number of P- choices made during the daily trials.The rats were tested in the discrimination-learning test after 2 days of rest following the cued learning and memory task. The discrimination-learning task is also sensitive to non-hippocampal dysfunction . Rats weThis task consisted of a series of paired sample and test trials using cylindrical objects as visible cues that are suspended directly above the submerged platform in the water maze. At the beginning of each sample trial, either a black or white cylinder was suspended directly above the submerged platform in the water maze. In subsequent test trials, both cylinders were present but the cylinder not present during the preceding sample trial was suspended over the platform and served as a cue for the location of the goal. Thus, if on a given sample trial, the black cylinder cued the platform, and then on the succeeding test trial, the white cylinder was used to cue the platform. The black and white cylinders were selected as sample stimuli for each pair of trials according to a semi-random schedule that ensured each cylinder served as the sample stimulus on 50% of the trials over the phase of the experiment. For each test trial, the platform was moved to another quadrant with the non-sample cylinder located directly above it while the sample cylinder was moved to a different quadrant. Based on a random schedule, the position of the submerged platform was changed after each sample and test trial to eliminate the use of spatial cues. All quadrants were used equally for locating cues in the sample and test trials and the platform was positioned randomly. The day before testing, rats were allowed a habituation swim for 10 s without the submerged platform.At the beginning of each sample trial, the rat was placed in the pool at the same location (in the center of the south-east quadrant), facing the wall of the pool, and allowed to swim to the submerged platform under the sample cylinder. The rat was allowed to remain on the platform for 10 s. If the rat failed to find the platform within 60 s, it was picked up and placed on the platform for 10 s then removed and placed under a heat lamp while the platform is moved and the cylinders put in position for the test trial. The heat lamp allowed for the rats to get dry before the test trial. Getting the cylinders and platform ready for the test trial took approximately 10 s. Rats received four pairs of sample and test trials per day for 4 days and the paired sessions were conducted with intervals of 60, 90, 120, or 180 s delay between the sample and test trials . The order of the delays varied each day according to a random schedule.2 inhalation and the brains were removed and cut in half sagitally. Half of the brain was immediately placed in liquid nitrogen until processed and used for western blot, real-time polymerase chain reaction, and ELISA. The other half was used for Fluoro-Jade staining and immunohistochemistry. For immunohistochemistry and Fluoro-Jade, brains were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3 overnight then cryoprotected before sectioning.The day after the completion of behavioral testing, all rats were euthanized using COThe fixed brains were sectioned at 30 μm thickness using a cryostat and the free-floating method was used as described previously . BrieflyTo assess CX3CR1 expression, 0.5 g of frozen hippocampal tissue from each rat was used for western blot. Briefly, tissues were homogenized in extraction buffer , 150 mM NaCl, 5 mM EDTA, protease inhibitor mixture , and 100 mM phenylmethylsulfonyl fluoride (Sigma Aldrich) then the homogenates were centrifuged at 10,000 × g for 10 min, and the supernatants were collected. Total protein concentration was determined using the BCA-Protein assay . The supernatant was boiled for 5 min at 95°C, and stored at −20°C until use.For western blot, equal amounts of protein (40 μg) from each rat were loaded and separated by SDS-PAGE gel electrophoresis as previously described . BrieflyThe Quantikine Rat Specific ELISA kit  was used to determine IL-1β, TNF-α, IL-4, and IL-10 protein expression. ELISA was performed according to the manufacturer’s instructions. Briefly, 0.5 g of frozen hippocampal tissue from each rat was homogenized with a glass homogenizer in 1 mL buffer containing 1 mmol/L phenylmethylsulfonyl fluoride, 1 mg/L pepstatin A, 1 mg/L aprotinin, and 1 mg/L leupeptin in PBS (pH 7.2) and centrifuged at 12,000 × g for 20 min at 4°C. The supernatant was collected and total protein was determined. Standards, controls, and samples (50 μL) were pipetted into a 96-well plate precoated with polyclonal antibodies specific for IL-1β, TNF-α, IL-4, and IL-10, then incubated at room temperature for 2 h on an orbital plate shaker (approximately 250 rpm) then washed 5×  before adding the conjugate. After several washes, the chromogen (tetramethylbenzidine) was added to each well and incubated for an additional 30 min. Color reaction was stopped by an equal volume of stop solution and reaction was read in a microplate reader  at a wavelength of 450 nm (650-nm reference wavelength). The color change was proportional to the concentration of the cytokines measured and all samples measured within the range of the standard curve. Assays were sensitive to 1.5 pg/mL of IL-1β, 5 pg/mL of TNF-α, 5 pg/mL of IL-4, and 10 pg/mL of IL-10; and inter- and intra-assay coefficients of variation were <10%.T) method was used [To examine mRNA expression of CX3CR1, RT-PCR was used. After 0.2 g of frozen hippocampal tissue was homogenized in Trizol , total RNA was extracted using an RNeasy extraction kit  according to the manufacturer’s protocol. From the RNA, cDNA was synthesized with CX3CR1 primers  and GAPDH  primers  at 50°C using the SuperScript III First-Strand Synthesis  following the manufacturer’s instructions. After reverse transcription, the cDNA was diluted and the sample was amplified by RT-PCR  using SYBR Green master mix or TaqMan Universal PCR Master Mix  and the following cycling parameters were used: initial denaturation and enzyme activation at 95°C, followed by 45 cycles of denaturation at 95°C, and extension at 72°C. Copy numbers of CX3CR1 transcripts were normalized against those of GAPDH (housekeeping gene) transcripts for each sample. All reactions were performed in triplicate. For quantitative analysis of target gene mRNA, the comparative threshold cycle  = 7.34, P <0.05) compared to the sham groups  = 12.11, P <0.05) and protein expression  = 11.04, P <0.05) in the hippocampus of rats that received the CX3CR1 siRNA  = 10.91, P <0.05) and  = 12.31, P <0.05), respectively, in comparison to the sham animals  = 7.55, P <0.05). Conversely, level of ED1 positive cells was significantly higher in the ischemia CX3CR1 deficient rats compared to the ischemia control group  = 8.22, P <0.05).We examined the effects of CX3CR1 signaling on microglial activation and the expression of pro- and anti-inflammatory cytokines on ischemia-induced chronic neuroinflammation. For this purpose, ischemic and sham rats were given either CX3CR1 siRNA or scrambled RNA as control continuously via osmotic pump starting at 7 days after reperfusion and behavioral testing commenced 28 days after. We then examined hippocampal tissues for microglial expression and levels of IL-1β, TNF-α, IL-4, and IL-10. Our results indicated that ischemic rats had significantly increased number of immunoreactve cells that express the microglial markers OX-42 and ED1  = 7.81, P <0.05) compared to the ischemia animals  = 8.33, P <0.05) compared to the ischemic group with deficient CX3CR1 function  = 11.62, P <0.05) was evident in the ischemic rats with deficient CX3CR1 function , hypertrophic and bushy (activated), and ameboid (highly activated). Ramified microglia have small cell bodies, and thin, long and highly branched processes. In contrast, hypertrophic and bushy microglia have larger cell bodies, with thicker and shorter processes that form thick bundles around an enlarged cell body whereas ameboid microglia have a large cell body with almost absent processes Figure C. Quanti,36 = 7.8 = 8.10, P <0.05) and TNF-α  = 10.93, P <0.05) were significantly elevated in the ischemic rats given the CX3CR1 siRNA when compared to the ischemic control group  = 7.44, P <0.05) and IL-10  = 8.13, P <0.05) were assessed, we found significantly increased expression in the ischemic control group compared to the rats with ischemic rats with deficient CX3CR1 function  = 2.93, P = 0.21) between the ischemia control rats and the ischemia group with deficient CX3CR1 function  = 7.82, P <0.05) and CX3CR1  = 7.95, P <0.05) where both ischemic groups had increased mean swim latency and frequently took the indirect route to reach the goal compared to the sham control animals. Post-hoc comparisons showed that the ischemic rats given the CX3CR1 siRNA were more impaired in performing the cued learning task compared to the ischemia control group  = 3.14, P = 0.19) was seen between ischemia and CX3CR1. These results suggest that CX3CR1 deficiency in both physiologic and pathologic conditions can induce cognitive impairment on hippocampal-dependent tasks.We also aimed at evaluating the role of CX3CR1 signaling on ischemia-induced cognitive impairment using three versions of the water maze test with varying difficulty and habituation test was given before starting each behavioral task. In the first version, the cued learning task was used wherein rats were trained to locate the goal using a visible cue. Analysis of behavioral performance in the cued learning test showed significant main effects of ischemia  = 7.90, P <0.05) was seen in the ischemic groups because they most often took the path to the incorrect goal compared to the sham control group while the ischemic group given CX3CR1 siRNA had the worst performance overall. Post-hoc comparison showed that performance of sham rats given CX3CR1 siRNA in the discrimination learning task was significantly impaired when compared to the sham control group but not significantly different from the ischemia control animals. No interaction effect  = 3.01, P = 0.17) was seen between ischemia and CX3CR1  in most of the trials compared to the sham control group but ischemic animals given CX3CR1 siRNA made the most errors in  = 7.19, P <0.05) identifying the correct goal  = 4.30, P = 0.10) was seen between ischemia and CX3CR1. Together, these results suggest that deficiency in CX3CR1 signaling can result in cognitive impairment in the discrimination learning task for both intact and injured animals.On the second version of the water maze task, rats were tested in discrimination learning where they need to distinguish the difference between the correct ncy  = 8.02, P <0.05) as well as a significant main effect of CX3CR1  = 8.75, P <0.05) in that ischemic rats demonstrate significantly increased mean swim latency exacerbated by CX3CR1 deficiency; and silencing of CX3CR1 function in the sham animals also resulted in longer mean swim latency  = 9.71, P <0.05) and CX3CR1  = 10.23, P <0.05) were seen in error rate wherein ischemic rats and sham animals with CX3CR1 deficiency made more errors in the delayed non-matching-to-sample task when compared to the sham control group; however, ischemic rats that received the CX3CR1 siRNA showed the worst performance overall  = 3.23, P = 0.14) was seen between ischemia and CX3CR1. These results suggest that CX3CR1 deficiency in both intact and injured animals can result in impaired performance in the non-hippocampal-dependent cognitive task.In the third version of the water maze test, rats were tested in the delayed non-matching-to-sample task consisting of a sample and test trials and a time delay in between trials. Our results showed significant main effect of ischemia  transient global cerebral ischemia significantly decreased fractalkine/CX3CR1 signaling in the hippocampus; (ii) inhibition of CX3CR1 function exacerbates the ischemia-induced chronic increased in microglial activation and pro-inflammatory cytokine levels; (iii) inhibition of CX3CR1 function worsens ischemia-induced chronic cognitive impairment; and (iv) inhibition of CX3CR1 function in sham rats resulted in increased IL-1β expression and impaired behavioral performance. However, our results show no significant effects of CX3CR1 deficiency on ischemia-induced neurodegeneration in the hippocampus, which are at odds with previous reports using the focal cerebral ischemia injury model where deficiency in CX3CR1 signaling in knockout mice decreased cortical neurodegeneration -18,29. BOur data show that both fractalkine and CX3CR1 expression are significantly decreased in the hippocampus during the post-recovery period following transient global cerebral ischemia. These results are in contrast to earlier reports that the ischemia-induced downregulation of fractalkine/CX3CR1 expression occurs within 24 h of injury followed by an upregulation at 48 h to 7 days after ischemic injury . The conIn the current study we also show that transient global cerebral ischemic can induce persistent neuroinflammation evidenced by elevated IL-1β and TNF-α weeks after injury and these findings are consistent with previous reports . One of Microglial activation is not a univalent state, and at least two activated phenotypes, ‘classically activated’  or ‘alternatively activated’ , have been identified. Inflammatory microglia (M1) release a number of proinflammatory cytokines such as TNF-α and IL-1β, reactive oxygen species, and nitrous oxide . While MOur results also show that CX3CR1 deficiency worsens the behavioral impairment induced by ischemic injury. Silencing the expression of CX3CR1 exacerbates the learning and memory deficits seen in the performance of ischemic rats in the cued learning, discrimination learning, and delayed non-matching-to-sample tasks. Likewise, silencing the expression of CX3CR1 in sham rats resulted in impaired behavioral performance. The enhanced behavioral deficit induced by CX3CR1 deficiency is accompanied by increased proinflammatory cytokines and a change in microglial morphology from ramified to bushy phenotype. Our findings on the ischemic animals are consistent with previous reports that show CX3CR1 deficiency intensifies microglial activation, increases IL-1β expression, and worsens the behavioral deficits in the transgenic mouse model of Alzheimer’s disease . SimilarThe present study provides important insight to understanding the involvement of CX3CR1 in the chronic neuroinflammation and cognitive impairment as a consequence of transient global cerebral ischemia. Although there is substantial literature in the area of fractalkine/CX3CR1 signaling in the brain, no consensus as to the role of this signaling pathway as either neuroprotective or neurodegenerative has been agreed upon. Furthermore studies delineating the involvement of fractalkine/CX3CR1 during the acute phase and long-term recovery period after ischemic injury are necessary to address the complexity of this signaling pathway in neuropathology. As well, studies on the role of fractalkine/CX3CR1 in neuropathology must include assessment of behavioral outcomes to truly understand the role and potential therapeutic value of this unique chemokine/receptor duo.The authors do not have any conflict of interest to declare.TLB conceived of the project, received the funding, and prepared the manuscript. JW performed the behavioral testing and experimental protocols. MW performed the surgeries and helped with the experimental protocols. All authors read and approved the final manuscript.