Polymorphisms in the microglial marker molecule CX3CR1 affect the blood volume of the human brain

Aim

CX3CR1 , a G‐protein‐coupled receptor, is involved in various inflammatory processes. Two non‐synonymous single nucleotide polymorphisms, V249I (rs3732379) and T280M (rs3732378), are located in the sixth and seventh transmembrane domains of the CX3CR1 protein, respectively. Previous studies have indicated significant associations between T280M and leukocyte functional characteristics, including adhesion, signaling, and chemotaxis, while the function of V249I is unclear. In the brain, microglia are the only proven and widely accepted CX3CR1‐expressing cells. This study aimed to specify whether there were specific brain regions on which these two single nucleotide polymorphisms exert their biological impacts through their functional effects on microglia.

Methods

Associations between the single nucleotide polymorphisms and brain characteristics, including gray and white matter volumes, white matter integrity, resting arterial blood volume, and cerebral blood flow, were evaluated among 1300 healthy Japanese individuals.

Results

The major allele carriers (V249 and T280) were significantly associated with an increased total arterial blood volume of the whole brain, especially around the bilateral precuneus, left posterior cingulate cortex, and left posterior parietal cortex. There were no significant associations between the genotypes and other brain structural indicators.

Conclusion

This finding suggests that the CX3CR1 variants may affect arterial structures in the brain, possibly via interactions between microglia and brain microvascular endothelial cells.

     

Opposite effects of CX3CR1 receptor polymorphisms V249I and T280M on the development of acute coronary syndrome. A possible implication of fractalkine in inflammatory activation

Several lines of evidence suggest that the chemokine fractalkine (FKN) and its receptor CX3CR1 contribute to the accumulation of leukocytes in the atherosclerotic plaque. The M280 allele of the CX3CR1T280M polymorphism modulates leukocyte recruitment and is associated with lower prevalence of cardiovascular disease. The influence of V249I, another CX3CR1 polymorphism, is discussed controversially. We investigated the association of the alleles M280 and I249 of CX3CR1 with coronary artery disease (CAD) and with acute coronary syndrome (ACS). Additionally, we assessed their association with the soluble ligand FKN and inflammatory activation measured by high sensitivity C-reactive protein (hsCRP). The genotypes of the V249I and T280M polymorphisms were determined in 1152 patients with suspected CAD.720 (62.5%) individuals showed significant CAD with an ACS prevalence of 59.3%. Using multivariate regression, we found a harmful influence of I249 (adjusted OR=1.8, P<0.03) and a protective effect of M280 (adjusted OR=0.6, P<0.04) on the occurrence of ACS in patients with CAD. Correspondingly, patients with I249 but without M280 (17%) were at elevated risk of ACS (OR=1.6, P<0.04). During ACS these patients (carrying only I249) had significantly higher circulating concentrations of FKN and high sensitivity C-reactive protein (1.9- and 1.6-fold). We found no association of the I249 or the M280 allele with the occurrence of CAD. In conclusion, I249 and M280 have opposite effects on the occurrence of ACS. The presence of I249 not "balanced" by M280 confers an elevated risk of ACS. A FKN-mediated enhanced inflammatory activation might explain this increased risk.     

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

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

The association of V249I and T280M fractalkine receptor haplotypes with disease course of multiple sclerosis

We investigated the association of CX3CR1 genotypes/haplotypes with MS and performed the prediction analysis of protein sequence variants' effects on CX3CL1/CX3CR1 interaction. We found no association of CX3CR1 with MS susceptibility. Frequency of I(249)T(280) haplotype was significantly lower in SP compared to RR patients (RR>10 years, OR=0.30, 95%CI=0.11-0.79, p=0.01; OR=0.53, 95%CI=0.18-1.56, p=0.2, in SP<10 years vs. RR>10 years). Prediction analysis showed that I249 T280 protein variant would significantly affect CX3CL1/CX3CR1 interaction. Our results suggest that CX3CR1 I???T??? haplotype could have protective effect for switch to SP MS. Further research is warranted to validate and replicate currently observed results.     

Reduced microglial CX3CR1 expression delays neurofibromatosis‐1 glioma formation

Although traditional models of carcinogenesis have largely focused on neoplastic cells, converging data have revealed the importance of non‐neoplastic stromal cells in influencing tumor growth and progression. Leveraging a genetically engineered mouse model of neurofibromatosis type 1 (NF1)‐associated optic glioma, we now demonstrate that stromal microglia express the CX3CR1 chemokine receptor, such that reduced CX3CR1 expression decreases optic nerve microglia. Moreover, genetic reduction of Cx3cr1 expression in Nf1 optic glioma mice delays optic glioma formation. Coupled with previous findings demonstrating that microglia maintain optic glioma growth, these new findings provide a strong preclinical rationale for the development of future stroma‐directed glioma therapies in children. ANN NEUROL 2013;73:303–308     

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

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

CX3CR1基因多态与颈动脉狭窄的相关性

目的 探讨fractalkine受体CX3CR1基因多态V249I和T280M与颈动脉狭窄的相关性.方法 经颈部彩色多普勒超声检查诊断颈动脉狭窄患者318例,通过聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和基因测序确定CX3CR1基因V249I和T280M多态性,并与292例无颈动脉狭窄者比较.结果 CX3CR1基因T280M多态中基因型MM和TM在颈动脉狭窄组明显低于对照组(分别为22.6%和31.2%,OR=0.646,95%CI 0.451～0.928,P=0.017),颈动脉狭窄组M等位基因频率明显低于对照组(分别为13.8%和19.2%,OR=0.677,95%CI 0.499～0.918,P=0.010).CX3CR1基因V249I多态中基因型Ⅱ和Ⅵ在颈动脉狭窄组和对照组差异无统计学意义(分别为39.0%和43.8%,OR=1.012,95%CI 0.731～1.403,P=0.940),颈动脉狭窄组Ⅰ等位基因频率明显低于对照组(分别为28.6%和32.7%,OR=0.842,95%CI 0.660～1.076,P=0.034).颈动脉狭窄组Ⅵ和Ⅱ在稳定斑块组明显增高(OR=0.610,95%CI 0.387～0.962,P=0.033).多因素Logistic回归分析显示MM和TM基因型是颈动脉狭窄的独立危险因素(OR=1.847,95%CI 1.091～3.127,P=0.022).结论 CX3CR1基因T280M多态中MM和TM基因型是颈动脉狭窄的一个独立危险因素,而V249I多态中Ⅱ和Ⅵ基因型与颈动脉粥样硬化斑块稳定性有关.     

缺血性脑血管病患者趋化因子受体CX3CR1基因T280M多态性的研究

目的探讨缺血性脑血管病(ICVD)患者趋化因子受体CX3CR1基因T280M的多态性及其频率。方法采用聚合酶链反应和限制性片段长度多态性方法检测165例ICVD患者(脑梗死85例,腔隙性脑梗死40例,短暂性脑缺血发作40例)与150名健康对照者(正常对照组)CX3CR1基因T280M的多态性,比较两组及ICVD亚组的基因频率。结果正常对照组CX3CR1基因T280M只有TT和TM基因型,ICVD组有TT、TM和MM3种基因型,两组间基因型的差异有统计学意义(P<0.05);ICVD组M等位基因频率(8.7%)明显高于正常对照组(2.3%)(P<0.01);不同ICVD亚组之间基因型及M等位基因频率的差异无统计学意义(均P>0.05)。结论ICVD患者趋化因子受体CX3CR1基因T280M有MM基因型,并且M等位基因的频率明显增高,提示CX3CR1基因T280M多态性可能与ICVD有关。     

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

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

Effects of polymorphisms in chemokine ligands and receptors on susceptibility to coronary artery disease

INTRODUCTION: Atherosclerosis, the underlying disorder of coronary artery disease (CAD), is an inflammatory process involving multiple molecular pathways. Chemokine-mediated mechanisms are potent regulators of atherosclerosis. Genetic variations that alter such signaling pathways could affect susceptibility to CAD. We investigated the effect of 5 common variations of chemokine and chemokine receptor genes (SDF1-3'A, CCR5-delta32, CCR2-64I, CX3CR1-V249I and CX3CR1-T280M) on predisposition to CAD. MATERIALS AND METHODS: The hypothesis was tested by screening the prevalence of the above polymorphisms in 210 angiographically diagnosed CAD patients (152 with history of acute coronary syndromes, 58 with stable disease) in comparison to 165 selected controls with negative coronary angiography. Genotyping was performed by PCR/RFLP analysis. RESULTS: There were no significant differences among cases and controls concerning allelic and genotypic frequencies of SDF1-3'A, CCR5-delta32, CCR2-64I and CX3CR1-V249I variations. A borderline higher allelic frequency of the M280 variant was observed in controls compared to CAD group (adjusted OR=0.65, 95% CI: 0.35-0.99, p=0.05). Subjects carrying at least one copy of the M280 allele were significantly more common in the control group, suggesting an atheroprotective effect of this variant (adjusted OR=0.58, 95% CI: 0.35-0.97, p=0.04). CONCLUSIONS: The study confers additional data in the field of genetic predisposition to CAD: it confirms the atheroprotective effect of the M280 variant in a completely different population and supports the role of the fractalkine-CX3CR1 pathway in atherosclerosis.     

Bone marrow‐derived cells in the central nervous system of a mouse model of amyotrophic lateral sclerosis are associated with blood vessels and express CX3CR1

Amyotrophic lateral sclerosis (ALS) is associated with increased numbers of microglia within the CNS. However, it is unclear to what extent bone marrow (BM)‐derived cells contribute to this microgliosis. We have studied the adoptive transfer of green fluorescent protein (GFP)‐labeled whole BM cells and BM from mice that express GFP only in CX₃CR1+ cells (CX₃CR1^+/GFP) into the CNS of a murine model of ALS having over‐expression of mutant superoxide dismutase (mSOD), and wt littermates. We find that most GFP+ and CX₃CR1^+/GFP cells are found adjacent to the microvasculature within the CNS, both in mSOD and wt mice. GFP+ and CX₃CR1^+/GFP cells within the CNS have a variety of morphologies, including cells with an elongated appearance, weak Iba‐1 immunoreactivity, and often mannose receptor immunoreactivity, indicating that these cells are perivascular microglia. Typically, less than 10% of BM‐derived cells had a stellate‐shape and expressed strong Iba‐1 immunoreactivity, as expected for parenchymal microglia, indicating that BM‐derived cells uncommonly generate parenchymal microglia. Adoptive transfer of BM‐derived cells from CX₃CR1^+/GFP mice revealed that many elongated cells are GFP+, demonstrating that some perivascular cells are derived from BM cells of the CX₃CR1+ lineage. The significantly greater numbers of BM cells in mSOD than in control mice indicate that the presence of these BM cells in the spinal cord is regulated by conditioning stimuli that may include irradiation and inflammatory factors within the CNS. © 2009 Wiley‐Liss, Inc.     

Increased frequency of CD4+T cells expressing fractalkine receptor CX3CR1 in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), but its AIDS susceptible polymorphisms are not associated with the disease

To investigate whether fractalkine receptor CX3CR1 polymorphisms that have been associated with rapid progression to AIDS among HIV-1 positive individuals also affects the risk of human T cell lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP), we compared the allele frequencies of V249I and T280M between 233 HAM/TSP patients and 213 HTLV-1 seropositive asymptomatic carriers (HCs). Although the frequency and absolute number of peripheral blood CX3CR1+CD4+T cells were significantly increased in HAM/TSP patients compared to HCs and uninfected controls independent of HTLV-1 trans-activator protein Tax, we could not observe any association between the two polymorphisms and the risk of HAM/TSP in our cohort.     

Time lapse in vivo microscopy reveals distinct dynamics of microglia‐tumor environment interactions—a new role for the tumor perivascular space as highway for trafficking microglia

Microglial cells are critical for glioma growth and progression. However, only little is known about intratumoral microglial behavior and the dynamic interaction with the tumor. Currently the scarce understanding of microglial appearance in malignant gliomas merely originates from histological studies and in vitro investigations. In order to understand the pattern of microglia activity, motility and migration we designed an intravital study in an orthotopic murine glioma model using CX3CR1‐eGFP^GFP/wt mice. We analysed the dynamics of intratumoral microglia accumulation and activity, as well as microglia/tumor blood vessel interaction by epi‐illumination and 2‐photon laser scanning microscopy. We further investigated cellular and tissue function, including the enzyme activity of intratumoral and microglial NADPH oxidase measured by in vivo fluorescence lifetime imaging. We identified three morphological phenotypes of tumor‐associated microglia cells with entirely different motility patterns. We found that NADPH oxidase activation is highly divergent in these microglia subtypes leading to different production levels of reactive oxygen species (ROS). We observed that microglia motility is highest within the perivascular niche, suggesting relevance of microglia/tumor blood vessel interactions. In line, reduction of tumor blood vessels by antivascular therapy confirmed the relevance of the tumor vessel compartment on microglia biology in brain tumors. In summary, we provide new insights into in vivo microglial behavior, regarding both morphology and function, in malignant gliomas. GLIA 2016;64:1210–1226     

Dopamine receptor 3(DRD3) polymorphism and risk for migraine

Background/objectives: Dopamine has been implicated in the pathogenesis of migraine. We investigated the possible association between the polymorphism 312G>A (rs6280) in the DRD3 gene(essential tremor 1‐ETM1‐ locus, chromosome 3q13) and the risk for migraine and for triggering migraine attacks by alcohol. Methods: We studied the frequency of the DRD3 genotypes and allelic variants in 197 patients with migraine and 282 healthy controls using a polymerase chain reaction and MlsI‐restriction fragment length polymorphisms method. Results: The frequencies of the DRD3 genotypes and DRD3Gly9 were similar in patients with migraine and controls and were unrelated to the age of onset of migraine, gender, family history of migraine and triggering of migraine attacks by alcohol. The frequency of the genotype DRD3Gly9Gly9 was significantly higher in patients with migraine with aura when compared with patients with migraine without aura, but not with controls. Conclusion: DRD3 genotype and allelic variants were not related to the risk for migraine in Caucasian Spanish people.     

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

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

The GH–IGF system in amyotrophic lateral sclerosis: correlations between pituitary GH secretion capacity,insulin‐like growth factors and clinical features

Background and purpose: The growth hormone (GH) and insulin‐like growth factor (IGF) system may be involved in neurodegenerative processes, and some abnormalities have been reported in amyotrophic lateral sclerosis (ALS). Our aim was to investigate the GH–IGF axis in patients with ALS and evaluate correlations between this endocrine system and clinical features. Methods: Serum levels of GH, IGF‐ I, IGF‐ II, insulin, IGF‐binding protein 1 (IGF‐BP1), and IGF‐binding protein 3 (IGF‐BP3) were measured in 25 patients with ALS and 25 age‐, gender‐, and BMI‐matched healthy controls. A GHRH plus arginine test was performed in patients and controls. Clinical status of patients was evaluated with the ALS Functional Rating Scale – Revised (ALSFRS‐R) and upper motor neuron (UMN) score. Results:  GHRH plus arginine test showed GH deficiency (GHD) in 13 (52%) patients with ALS; severe GHD was found in 6 (24%) and partial GHD in 7 (28%) patients. IGF‐I levels were significantly higher in patients with ALS than in healthy controls (182.9 ± 90.8 vs. 139.4 ± 58.1 ng/ml; P = 0.015). IGF‐I levels were higher in patients with ALS with UMN score >10 than those with UMN score <10 (217.8 ± 100.8 vs. 155.5 ± 74.6 ng/ml, P = 0.05). IGF‐II levels were significantly lower in patients with ALS than in healthy controls (720.9 ± 215 vs. 1001.9 ± 475.4 ng/ml; P = 0.03). Conclusions: The results demonstrate an impairment of the GH–IGFs system in ALS. The degenerative process in ALS might lead to a compensatory increase in IGF‐I in an attempt to provide additional support to motor neurons or degenerating muscle fibers. The decrease in IGF‐II levels may also be of pathological significance.     

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

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

Mapping and kinetics of microglia/neuron cell‐to‐cell contacts in the 6‐OHDA murine model of Parkinson's disease

As neuroinflammatory processes are involved in the pathogenesis of Parkinson's disease (PD), we provide several key data describing the time‐course of microglial accumulation in relation with behavioral alterations and neurodegeneration in a murine model of PD induced by intrastriatal injection of 6‐hydroxydopamine (6‐OHDA). Our study argues for a major role of microglia which accumulation is somehow early and transient in spite of the neuronal loss progression. Moreover, we observed less 6‐OHDA‐induced neurodegeneration associated with less inflammatory reaction in DAP‐12 Knock‐In mice. The direct cell‐to‐cell contacts that may support physical interactions between microglia and altered dopaminergic neurons are ill‐defined, while it is currently hypothesized that microglia support an immune‐mediated amplification of neurodegeneration by establishing a molecular cross talk with neurons. Indeed, we sought to map microglia/neuron appositions in substantia nigra (SN) of 6‐OHDA injected C57Bl/6 mice and CX3CR1/^GFP/+ mice. Confocal immunofluorescence analyses followed by 3D reconstitutions reveal close appositions between the soma of TH+ neurons and microglial cell bodies and ramifications. Interestingly, some microglial ramifications penetrated TH⁺ somas and about 40% of GFP⁺ microglial cells in the injured SN harbored TH⁺ intracytoplasmic inclusions. These results suggest a direct cross talk between neurons and microglia that may exert a microphagocytic activity toward TH+ neurons. Altogether, these results obtained in a murine PD model may participate in the understanding of microglial cells' function in neurodegenerative diseases. GLIA 2013;61:1645–1658     

CX3CR1 deficiency induces an early protective inflammatory environment in ischemic mice

The studies on fractalkine and its unique receptor CX3CR1 in neurological disorders yielded contrasting results. We have explored the consequences of CX3CR1 deletion in ischemic (30′ MCAo) mice on: (1) brain infarct size; (2) microglia dynamism and morphology; (3) expression of markers of microglia/macrophages (M/M) activation and polarization. We observed smaller infarcts in cx3cr1^?/? (26.42 ± 7.41 mm³, mean ± sd) compared to wild type (36.29 ± 11.57) and cx3cr1^?/+ (34.49 ± 8.91) mice. We longitudinally analyzed microglia by in vivo two‐photon microscopy before, 1 and 24 h after transient ischemia. Microglia were stationary in both cx3cr1^?/? and cx3cr1^?/+ mice throughout the study. In cx3cr1^?/? mice, they displayed a significantly higher number of ramifications >10 μm at baseline and at 24 h after ischemia compared to cx3cr1^?/+ mice, indicating that CX3CR1 deficiency impaired the development of microglia hypertrophic/amoeboid morphology. At 24 h after ischemia, we performed post mortem quantitative immunohistochemistry for different M/M markers. In cx3cr1^?/? immunoreactivity for CD11b (M/M activation) and for CD68 (associated with phagocytosis) were decreased, while that for CD45^high (macrophage and leukocyte recruitment) was increased. In addition, immunoreactivity for Ym1 (M2 polarization) was enhanced, while that for iNOS (M1) was decreased. Our data show that in cx3cr1^?/? mice protection from ischemia at early time points after injury is associated with a protective inflammatory milieu, characterized by the promotion of M2 polarization markers.     

Association of tumor necrosis factor‐α and matrix metalloproteinase‐3 gene variants with stroke

Background and purpose: There is increasing evidence that the genetic variation in the genes coding for pro‐inflammatory markers and matrix metalloproteinase may play an important role in the pathogenesis of various human diseases including stroke. The aim of this study was to evaluate the association of genetic variants within the genes encoding tumor necrosis factor‐α (TNF‐α) and matrix metalloproteinase‐3 (MMP‐3), with stroke. Methods: Five hundred and twenty‐five ischemic stroke patients and 500 age‐ and sex‐matched controls were included in this study. We analyzed +488 G/A polymorphism in TNF‐α gene and −1612 5A/6A polymorphism in MMP‐3 gene. The genotypes were determined by Amplification Refractory Mutation System PCR. The strength of association between genotypes and stroke was measured by the odds ratio with 95% confidence interval (CI) and chi‐squared analysis. Results: Allelic and genotypic frequencies of TNF‐α G/A polymorphism differed significantly between patients and healthy controls (P < 0.001). A stepwise logistic regression analysis confirmed these findings (P < 0.001). Further, evaluating the association of this polymorphism with stroke subtypes, we found significant association with intracranial large artery atherosclerosis, extracranial large artery atherosclerosis, and stroke of undetermined etiology. As far as MMP‐3−1612 5A/6A polymorphism is concerned, there was no significant difference in genotypic distribution and allelic frequency between the patients and healthy controls (P = 0.5 and 0.9, respectively). We tested the gene–gene interaction between TNF‐α and MMP‐3 genes using the logistic regression model. However, there was no evidence for a gene–gene interaction between TNF‐α and MMP‐3. Conclusion: TNF‐α +488 G/A variant is an important risk factor for ischemic stroke in the South Indians from Andhra Pradesh, whereas MMP‐3−1612 5A/6A polymorphism is not associated with stroke in the same population.