Microglial CX3CR1 promotes adult neurogenesis by inhibiting Sirt 1/p65 signaling independent of CX3CL1
© The Author(s). 2016
Received: 11 August 2016
Accepted: 10 September 2016
Published: 17 September 2016
Homo and heterozygote cx3cr1 mutant mice, which harbor a green fluorescent protein (EGFP) in their cx3cr1 loci, represent a widely used animal model to study microglia and peripheral myeloid cells. Here we report that microglia in the dentate gyrus (DG) of cx3cr1 −/− mice displayed elevated microglial sirtuin 1 (SIRT1) expression levels and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 activation, despite unaltered morphology when compared to cx3cr1 +/− or cx3cr1 +/+ controls. This phenotype was restricted to the DG and accompanied by reduced adult neurogenesis in cx3cr1 −/− mice. Remarkably, adult neurogenesis was not affected by the lack of the CX3CR1-ligand, fractalkine (CX3CL1). Mechanistically, pharmacological activation of SIRT1 improved adult neurogenesis in the DG together with an enhanced performance of cx3cr1 −/− mice in a hippocampus-dependent learning and memory task. The reverse condition was induced when SIRT1 was inhibited in cx3cr1 −/− mice, causing reduced adult neurogenesis and lowered hippocampal cognitive abilities. In conclusion, our data indicate that deletion of CX3CR1 from microglia under resting conditions modifies brain areas with elevated cellular turnover independent of CX3CL1.
KeywordsMicroglia Water maze Morphometry Adult neurogenesis Sirtuin 1 NF-kB p65 acetylation
CX3CR1 is a seven transmembrane domain receptor coupled to Gi and Gz subtypes of G proteins, activation of which is linked to several intracellular second messengers like phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) . CX3CR1 is prominently expressed by monocytes, subsets of NK and dendritic cells, and the brain microglia . Surprisingly little is known on which intracellular signaling pathways in microglia are affected by the lack of CX3CR1. Microglial cells are fundamentally distinct from other brain cells in that they are derived from primitive myeloid progenitors that arise during embryogenesis [3–5]. They represent the resident phagocytic cells in the brain, taking part in immune-mediated defense mechanisms and clearing cell debris . Microglial cells are constantly surveying their surroundings and are implicated in synaptic pruning, during both development and throughout adulthood, and therefore believed to play a role in regulating homeostatic neuronal synaptic plasticity [7, 8]. Neurons and microglia are thought to communicate with one another through neuronal expression of the CX3CR1 ligand CX3CL1 (or fractalkine). CX3CL1 is expressed at the cell membrane of selected neurons and binds to and activates CX3CR1 receptors on microglia [9, 10]. CX3CL1 exists in two distinct forms: a full-length membrane-bound form and a shed form that contains the N-terminal chemokine domain. The shed chemokine domain of CX3CL1 acts, when cleaved, as a signaling molecule and can bind microglial-expressed CX3CR1 receptors , whereas its membrane-bound mucin stalk can serve as a cell adhesion molecule . CX3CL1 is abundantly expressed in the granular cell layer of the rat dentate gyrus (DG), where addition of recombinant CX3CL1 reverses age-related decline in adult neurogenesis . Both, cx3cr1 −/− and cx3cr1 +/− mice display reduced hippocampal neurogenesis compared with wild-type controls [13, 14]. However, it is not clear to what extent CX3CL1 is mandatory for proliferation and neurogenesis. In addition to the already mentioned reduced hippocampal neurogenesis, cx3cr1 −/− mice were reported to exhibit excessive hippocampal IL-1β expression and either enhanced  or attenuated long term potentiation (LTP)  resulting in improved  or impaired cognitive functions [13, 14]. It is important to mention that Maggi et al. were using exclusively female mice (3 months of age). Rogers et al. and Bachstetter et al. performed all experiments with male mice, 3 months and 4 months of age, respectively. In addition to these conflicting results, little is known about the intracellular signaling cascades activated by CX3CR1 deficiency, which might impact synaptic plasticity and cognition. One of these pathways could include the NF-kB signaling pathway, which may trigger microglial activation and induce the release of inflammatory factors including IL-1β, as seen following irradiation , during normal aging  or neurodegeneration . Along these lines, sirtuin 1 (SIRT1), a member of the sirtuin family, might be modulated in microglia by the lack of CX3CR1 because it can suppress inflammatory responses by inhibiting NF-kB signaling [19, 20].
Here we investigated the consequences of the microglial CX3CR1 deletion on cell morphology, activation of the NF-kB signaling pathway, the expression of SIRT1, interference with neuroblasts, immature neurons and cognition. Our findings indicate that, under brain homeostasis, hippocampal microglia from cx3cr1 −/− mice are very similar, if not identical, to microglia from adult wild-type animals in contrast to the situation in newborns or during development. However, hippocampal cx3cr1 −/− microglia show activation of SIRT1 and the NF-kB pathway in areas of adult neurogenesis. We found that manipulation of SIRT1 activation in cx3cr1 −/− mice directly impacts cognitive performance, while the same treatment had no detectable effect on cognition in wild-type littermates.
Materials and methods
This article does not contain any studies with human participants performed by any of the authors.
For all animal experiments “Principles of laboratory animal care” (NIH publication No. 86–23, revised 1985) were followed. All experiments were approved by the Federal Ministry for Nature, Environment and Consumers’ Protection of the states of Baden-Württemberg (G12/71 and G11/50), and were carried out in accordance to the respective national, federal, and institutional regulations. Adult, 8–12 weeks old, male mice were used for the experiments. Mice were group housed up to five per cage with 12 h light/dark cycle with lights on at 6 a.m. Food and water were available ad libitum. cx3cr1 gfp/gfp on a C57BL/6 J background were obtained from the Jackson Laboratory. cx3cl1 −/− mice were described previously . In all experiments, wild type littermates were used as controls.
Morris Water Maze (MWM) test
The MWM was used to measure spatial learning and memory . The apparatus consisted of a black plastic pool with a diameter of 120 cm. A black escape-platform (square, 10 × 10 cm) was located 1.0 cm below (hidden) the water surface. The temperature of the water was kept constant throughout the experiment (20 ± 0.5 °C), and a 10 min recovery period was allowed between the training trials. The training consisted of 6 consecutive days of testing, with four trials per day. If the mouse failed to find the escape platform within the maximum time (60 s), the animal was placed on the platform for 10 s by the experimenter. During the first 6 days of testing, the mice were trained with a hidden platform. The platform location was kept constant, and the starting position varied between four constant locations at the pool rim. Mice were placed in the water with their nose pointing toward the wall at one of the starting points in a random manner. On the 7th day, the platform was removed, and the mice were allowed to swim for 60 s to determine their search bias. On testing day 8, mice were trained to find a visible platform, which had a 10 cm high pole with a white flag and was changed every trial to a new position. Timing of the latency to find the visible platform was started and ended by the experimenter. A computer running the BIOBSERVE software (BIOBSERVE) analyzed all variables of the MWM test. All behavioral experiments were carried out in a double-blind fashion and mice were tested in random order.
EX527 (cat. no. 2780) was purchased from Tocris Bioscience (Bristol, UK), dissolved in 4 % DMSO/10 % cyclodextrin in PBS and administered intraperitoneally (200 μl, i.p.) at a concentration of 250 ng/μL once daily for 21 days . Resveratrol (cat. no. R5010, Sigma-Aldrich, St. Louis, USA) was dissolved in PBS and i.p. injected once daily over a period of 20 days at a concentration of 2 μg/μL .
Adult mice received two intraperitoneal (i.p.) BrdU-injections (Sigma-Aldrich, St. Louis, MO, USA, 50 mg/kg in 0.9 % NaCl, 8 h apart) over 3 days . Animals were perfused 3 weeks after the last BrdU injection.
Histology was performed as described previously [26, 27]. Mice were perfused intracardially with ice-cold PBS, pH 7.4, and 4 % paraformaldehyde/PBS, pH 7.4. Brains were removed, postfixed overnight in the same fixative, and paraffin embedded or frozen at −20 °C. Coronal sections of 20 μm thickness were cut, deparaffinized in xylene, and blocked with 10 % goat serum/PBS plus 0.2 % Triton X-100. 30 μm thick free-floating coronal brain sections were used from perfusion-fixed frozen brain. Sections were incubated with primary antibody against: Iba-1 (24 h, dilution 1:500 at 4 °C, cat. no. 019-19741, Wako, Osaka, Japan)  to detect microglia, NeuN (24 h, dilution 1:200 at 4 °C, cat. no. MAB377, Merck Millipore, Darmstadt, Germany)  to detect neurons, DCX (48 h, dilution 1:250 at 4 °C, C-18, sc-8066, Santa Cruz Biotechnology, Dallas, USA)  to detect neuroblasts and immature neurons, BrdU (24 h, dilution 1:200 at 4 °C, cat. no. OBT0030, ABD, Serotec, Raleigh, NC, USA)  to detect BrdU incorporation, Lamp2 (24 h, dilution 1:250 at 4 °C, cat. no. ab13524, Abcam, Cambridge, UK)  to label lysosomes and late endosomes, Ki67 (24 h, dilution 1:800 at 4 °C, cat. no. ab15580, Abcam, Cambridge, UK)  to detect proliferating cells, CD11b (48 h, dilution 1:200 at 4 °C, cat. no. ab8878, Abcam, Cambridge, UK)  to label ramified parenchymal microglia, and activated caspase-3 (24 h, dilution 1:100 at 4 °C, cat. no.9661, Cell Signaling Technology, Danvers, MA, USA) , which is involved in the activation cascade of caspases responsible for apoptosis execution. As secondary antibodies we used: Alexa-Fluor-647–conjugated secondary antibody (cat. no. A31573, Life technologies, Waltham, USA) incubated for 2 h, dilution 1:500, at room temperature, Alexa-Fluor-568–conjugated secondary antibody (cat. no. A11011, Life technologies) was incubated at a dilution of 1:500 for 2 h at 20–22 °C, and Alexa Fluor 488–conjugated secondary antibody (cat. no. A11008, Life technologies), which was added with a dilution of 1:500 overnight at 4 °C. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, cat. no. 236276, Boehringer). Images were either taken using the BZ-9000 Biorevo microscope (Keyence, Neu-Isenburg, Germany) or the Olympus FluoView 1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). Integrated fluorescence intensities were measured using Image J 1.4 software .
Cells were counted using the optical fractionator, a method for unbiased stereological analysis. This method was performed using a computer-assisted image analysis system, consisting of a Leica DMRB/DIC fluorescence microscope equipped with a computer-controlled motorized stage, a video camera, and the Stereo Investigator software (MicroBrightField, Williston, VT). The number of positive cells throughout the rostrocaudal extent of the dentate gyrus was counted with a coded one-in-16 series for frozen sections (40 μm). We used a modified version of the optical fractionator method as reported previously [35–37]. The total numbers of positive cells were multiplied by 16 and reported as total number of cells per dentate gyrus. For immunocytochemical analysis of paraffin sections (30 μm), serial coronal sections were collected spanning the rostrocaudal extent of the hippocampus. For quantification, every 12th section was selected. Every positive cell was counted on these sections, and, to obtain the relative total number of cells in the dentate gyrus, these counts were multiplied by 12 to account for the sampling frequency . In control experiments we could not detect significant differences in cell numbers, when samples were quantified by both quantitation methods.
Three-dimensional reconstruction of microglia
40-μm coronal cryo sections from adult brain tissue were stained with anti-Iba-1 (cat. no. 019-19741, Wako)  for 48 h (dilution 1:500 at 4 °C), followed by staining with an Alexa Fluor 488–conjugated secondary antibody (cat. no. A11008, Life technologies), which was added with a dilution of 1:500 overnight at 4 °C. Nuclei were counterstained with DAPI. Imaging was performed on an Olympus Fluoview 1000 confocal laser scanning microscope (Olympus) using a 20× 0.95 NA objective. Z stacks were imaged with 1.14-μm steps in z direction, 1024 × 1024 pixel resolution were recorded and analyzed using IMARIS software (Bitplane). Four cortical cells were reconstructed per analyzed mouse.
Microdissection of microglia was performed using a Zeiss PALM MicroBeam as described previously, with modification . Fast immunochemistry of serial sections was performed with CD11b antibodies (Serotec). Immunostained sections were counterstained with DAPI to facilitate the identification of individual cells. RNA was isolated with the Rneasy Micro Plus Kit (Qiagen), and reverse transcription (RT), preamplification, and real-time PCR were performed using Applied Biosystems reagents according to the manufacturer’s recommendations. The primer pairs were used as described previously .
Microglia isolation and flow cytometry
Adult microglia cells were harvested from dissected hippocampal tissue using density gradient separation and were prepared as described before . In short, samples were stained for CD11b and CD45 (eBioscience, BD Pharmingen) . Cell suspensions were acquired on a FACS Canto II (Becton Dickinson) or cell populations were sorted with a MoFlo Astrios (Beckman Coulter) and further processed. Data were analyzed using FlowJo software (Tree Star).
Gene expression analysis
FACS-sorted microglial cell populations were collected directly in cell lysis buffer and subsequently RNA was isolated with the Arcturus Pico Pure RNA Isolation Kit (Life Technologies) according to the manufacturer’s protocol. Reverse transcription and real-time PCR analysis were performed using high capacity RNA-to-cDNA-Kit and Gene Expression Master Mix reagents (Applied Biosystems) according to the manufacturer’s recommendations. RT-PCRs were analyzed with a LightCycler 480 (Roche) and carried out as described previously [26, 38]. The following primers were used: HDAC1: forward 5′-TCACCATGGCGATGGCGTGG-3′, reverse 5′-TGCCGTCTCGCAGTGGGTAGT-3′; HDAC2: forward 5′-ACAGACCCCAAAGGAGCCAAGT-3′, reverse 5′-GCCAATGTCCTCAAACAGGGAAGG-3′; HDAC3: forward 5′-CCTCTGACTTCCTTCTGGGTTCCCC-3′, reverse 5′-TCACACCCTCTCCTCCTTGCCA-3′; HDAC4: forward 5′-TCGGGCACAGTCCTCCCCAG-3′, reverse 5′-TGCGTCCACGGATGCACTCA-3′; HDAC 5: forward 5′-CCTGTCCCGTCCGTCTGTCTGTT-3′, reverse 5′-GCCATCTGCCGACTCGTTGGGAGA-3′; HDAC6: forward 5′-CTGGCGGACTAGAAAGAGCCTTTCC-3′, reverse 5′-GGGGTGACTGGGGATTGTGCC-3′; HDAC7: forward 5′-CCCACATCAGATAACCCAACCACAG-3′, reverse 5′-CTGGAGGGCAGGGGAGCCTTA-3′; HDAC8: forward 5′-CTCGCGGACGGTTGGAAGTGG-3′, reverse 5′-AGTGGACCATACTGGCCCGTT-3′; HDAC9: forward 5′-AGCTTCTCGTGGCTGGTGGA-3′, reverse 5′-CGATTCAGGGGTCGGTGGCG-3′; HDAC10: forward 5′-TTGCTGCAGGTGGCTGCTCC-3′, reverse 5′-CTCGGGCCATGGTTCGCTGG-3′; HDAC11: forward 5′-CAGCCCAGCGGGCATTGTGA-3′, reverse 5′-TCTGTGCCGAGACGCAGGGA-3′; Sirt1: forward 5′-AGCTGGGGTTTCTGTCTCCTGTGG-3′, reverse 5′-ACGGCTGGAACTGTCCGGGAT-3′; Sirt2: forward 5′-CTCGGCCTCTTCTTGTTTCCGCT-3′, reverse 5′-CGAGTCTGAATCGGTCCGGCTC-3′; Sirt3: forward 5′-CGCTTGACCCTCTAGGCGCC-3′, reverse 5′-CCTTCTCCCACCTGTAACACTCCCG-3′; Sirt4: forward 5′-ACGGATGCATGCACAGAGTCCTG-3′, reverse 5′-GAACACGTCGCCGTCGGGAG-3′; GAPDH: forward 5′-TCCTGCACCACCAACTGCTTAGCC-3′, reverse 5′-GTTCAGCTCTGGGATGACCTTGCC-3′.
Mice underwent diffusion tensor imaging (DTI) examination at 1562 × 250 μm3 spatial resolution with a cryogenic cooled resonator (CCR) at ultrahigh field (7 T) as described previously [39, 40]. Diffusion images were acquired along 30 gradient directions plus 5 references without diffusion encoding with a total acquisition time of 35 min. Fractional anisotropy (FA) maps were statistically compared by whole brain-based spatial statistics (WBSS) at the group level vs. wt controls.
Sirtuin 1 activity assay
To quantify sirtuin 1 (Sirt1) activity, nuclear extracts from sorted microglia (pooled from hippocampi of three mice per group) were prepared. Nuclear extracts were used to measure deacetylase activity of an acetylated histone using Epigenase Universal SIRT Activity/Inhibition Assay Kit (Epigentek, Farmingdale, NY) .
Statistical analysis was performed using GraphPad Prism (GraphPad Software, Version 6.0, La Jolla, USA). In general, chosen sample sizes are similar to those reported in previous publications . All data were tested for normality applying the Kolmogorov-Smirnov test. If normality was given, an unpaired t test was applied. If the data did not meet the criteria of normality, the Mann–Whitney U test was applied. To test for effects of treatment or genotype a two-factorial analysis of variance (ANOVA) with post hoc Bonferroni test or Tukey-Kramer HSD test was applied. Differences were considered significant when p < 0.05. Number of animals per group is given as “n”. To obtain unbiased data, experimental mice were all processed together by technicians and cell quantifications were performed blinded by two scientists independently and separately.
Efficient adult hippocampal neurogenesis relies on the presence of CX3CR1, but is independent on the presence of CX3CR1
Cx3cr1 −/− microglia show increased sirtuin 1 and NF-kB signaling locally in the dentate gyrus
Activation of sirtuin 1 restores cognitive performance and neurogenesis in cx3cr1 −/− mice
With the present study we show that, under resting conditions, SIRT1 and the NF-kB pathway are activated in cx3cr1 −/− microglia residing within the murine DG area. This activation seems to be restricted to the DG and was largely diminished in the hippocampal CA1 region. As a result of pharmacological SIRT1 activation, impaired adult neurogenesis and lowered hippocampal cognitive performance was restored in cx3cr1 −/− mice.
We hypothesize that the NF-kB signaling pathway and SIRT1 enzyme in microglia interact to maintain cellular homeostasis in vivo. Since the fractalkine receptor CX3CR1 inhibits cAMP signaling including the cAMP-dependent protein kinase A (PKA) via coupling to a Gi-protein coupled receptor , deletion of CX3CR1 from microglia might facilitate activation of PKA and subsequently NF-kB activation . Stimuli causing PKA activation appear to be restricted to certain brain regions with e.g. enhanced cellular turnover like the DG because only marginal acp65 signals were detected outside the DG as seen in the CA1 area. In response to increased acetylation of p65 in cx3cr1 −/− microglia, SIRT1 activity is amplified, most likely to counteract excessive NF-kB signaling. Activation of SIRT1 can induce deacetylation of the RelA/p65 component of the NF-kB complex. The deacetylation of Lys310 inhibits the transactivation capacity of RelA/p65 subunit and consequently suppresses the transcription of the NF-kB-dependent gene expression . However, in cx3cr1 −/− microglia SIRT1 activity, although elevated, appears to be insufficient to prevent NF-kB-dependent gene expression  as indicated by elevated protein levels of IL-1β in the hippocampus  or by increased CXCL10, TNF-α and IL-1β mRNA expression in microglia micro-dissected from DG of cx3cr1 −/− mice (Fig. 4). Only additional SIRT1 activation can effectively counteract activation of the NF-kB pathway. Interestingly, in wild-type mice where no microglial NF-kB activation was detectable, activation of SIRT1 had no effect on adult neurogenesis or performance in the water maze test (data not shown). Previous studies have shown that especially IL-1β can impair adult neurogenesis by decreasing proliferation in the DG of cx3cr1 −/− mice . A similar mechanism was observed in wild-type mice under stressed conditions when elevated IL-1β reduced hippocampal neurogenesis and administration of an IL-1 receptor antagonist restored the neurogenesis rate following stress exposure . Notably, despite the pro-inflammatory activation status of cx3cr1 −/− microglia, no morphological changes could be detected in comparison to cx3cr1 +/+ microglia, confirming previous findings within an independent set of experiments . During development in the hippocampus, microglial morphology seems to depend, at least partially, on the fractalkine receptor considering that at P8 a small population of cx3cr1 −/+ cells was typified by a very large surface area. This group of cells was absent in brains of cx3cr1 −/− mice . However, one report based on immunohistochemistry indicates that under normal physiological conditions genetic cx3cr1 deficiency is associated with microglial alterations, including increased cell number and enlargement of the soma . While the receptor for CX3CL1, CX3CR1, is highly expressed on microglia, CX3CL1 is constitutively expressed at high levels on healthy neurons. CX3CL1 is expressed as a transmembrane protein that can be cleaved in a soluble form, consisting of the extracellular N-terminal chemokine domain, by the activity of the lysosomal cysteine protease cathepsin S (CatS) or by members of the disintegrin and metalloproteinase (ADAM) family, ADAM-10 and ADAM-17 . The anatomical expression of CX3CL1 on neurons and CX3CR1 on microglia suggests that neurons may maintain microglia in a surveilling/ramified state through a repressive CX3CL1 signal . From this point of view it is unexpected to find intact proliferation of neural stem/progenitor cells and adult neurogenesis in CX3CL1-deficient mice while the same process is impaired in mice lacking CX3CR1. If binding of CX3CL1 elicits a tonic inhibitory signal, which maintains microglia in a quiescent state, its absence should result in activated or pro-inflammatory microglia with negative impact on neurogenesis and neurodevelopment. While there is a consensus among studies for a neuroprotective role of CX3CL1 signaling in vitro, some in vivo studies even suggest a neurotoxic role of CX3CL1 as seen in animal models for Alzheimer’s- and Parkinson’s disease. Here, CX3CL1 can act as a repressor of microglial phagocytic activity and cause overall microglia activation [57, 58]. A further remarkable mode of action was observed in lung endothelial cells, which respond to stimuli and produce CX3CL1. This leads to the endothelial attachment of the subset mononuclear leukocytes that express the sole CX3CL1 receptor, CX3CR1 . Following a challenge with lipopolysaccharides (LPS), cx3cl1 −/− mice exhibit reduced expression of CX3CR1 and impaired NF-kB signaling in lung tissue when compared to wt controls . CX3CL1 is a molecule that may have various activities, with either no, beneficial or destructive potential, likely depending on the activation state of its main target cells. In support of our findings cx3cl1 −/− mice do not have histologic abnormalities in any major organs (including the brain), hematopoietic lineages in blood and lymphoid tissue are essentially normal, and they do not exhibit any overt behavioral abnormalities . There is also the possibility of an alternative CX3CR1 ligand, which might act similar to CX3CL1. In humans eotaxin-3/CC chemokine ligand 26 was recently reported to be a functional ligand for CX3CR1 ; in mice, the CCL26 gene, however, may be a pseudogene since no cDNA or expressed sequence tag (EST) has been reported . Similar to previous reports we found that hippocampal neurogenesis was decreased in mice that lack CX3CR1 . These mice display significant deficits in cognitive functions and LTP induction due to increased action of IL-1β . There are two reports indicating that cx3cr1 −/− mice have improved hippocampal cognitive abilities compared to wild-type controls [15, 54] with either enhanced  or impaired  generation of neuronal precursors. The reason for this inconsistency is presently unclear.
Our findings indicate that in the SGZ of the DG area in cx3cr1 −/− mice the number of DCX+ cells is reduced, independent of the CX3CR1 ligand CX3CL1. Enhanced microglial NF-kB-dependent gene expression in the DG results in elevated levels of chemokines such as IL-1β and consequently in the inhibition of neurogenesis and spatial cognitive function. Manipulation of SIRT1 activity interferes with NF-kB signaling, adult neurogenesis and ultimately hippocampal learning and memory.
Enhanced green fluorescent protein
Fluorescence activated cell sorting
Morris water maze
Nuclear factor kappa-light-chain-enhancer of activated B cells
We thank Maria Oberle, Margarethe Ditter and Christine El Gaz for excellent technical assistance.
MP was supported by the BMBF-funded competence network of multiple sclerosis (KKNMS), the competence network of neurodegenerative disorders (KNDD), the DFG (SFB 992, FOR1336, PR 577/8-3, Reinhart-Koselleck grant), and the Fritz-Thyssen Foundation. TB was supported by the Marie Curie Integration Grant project N° 248033- MBfUSEDIT submitted under the Call FP7-PEOPLE-2009-RG and the DFG (BL 1153/1-1).
Availability of data and materials
TB, MP, SJ, SL designed experiments and performed analyses and drafted the manuscript; SS, RPM, MS and AS performed immunohistochemistry, western blot analyses and interpreted data; TB, AS supervised and interpreted behavioral studies; AM, KB performed reconstruction of cells; LAH and DVE contributed with MRI experiments and analysis; DE and OS performed molecular experimental work, analyzed and discussed data. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- Al-Aoukaty A, Rolstad B, Giaid A, Maghazachi AA. MIP-3alpha, MIP-3beta and fractalkine induce the locomotion and the mobilization of intracellular calcium, and activate the heterotrimeric G proteins in human natural killer cells. Immunology. 1998;95:618–24.PubMedPubMed CentralView ArticleGoogle Scholar
- Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, Sher A, Littman DR. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol. 2000;20:4106–14.PubMedPubMed CentralView ArticleGoogle Scholar
- Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330:841–5. doi:10.1126/science.1194637.PubMedPubMed CentralView ArticleGoogle Scholar
- Kierdorf K, Erny D, Goldmann T, Sander V, Schulz C, Perdiguero EG, Wieghofer P, Heinrich A, Riemke P, Holscher C, et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat Neurosci. 2013;16:273–80. doi:10.1038/nn.3318.PubMedView ArticleGoogle Scholar
- Schulz C, Gomez Perdiguero E, Chorro L, Szabo-Rogers H, Cagnard N, Kierdorf K, Prinz M, Wu B, Jacobsen SE, Pollard JW, et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science. 2012;336:86–90. doi:10.1126/science.1219179.PubMedView ArticleGoogle Scholar
- Shemer A, Erny D, Jung S, Prinz M. Microglia plasticity during health and disease: an immunological perspective. Trends Immunol. 2015;36:614–24. doi:10.1016/j.it.2015.08.003.PubMedView ArticleGoogle Scholar
- Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P, Giustetto M, Ferreira TA, Guiducci E, Dumas L, et al. Synaptic pruning by microglia is necessary for normal brain development. Science. 2011;333:1456–8. doi:10.1126/science.1202529.PubMedView ArticleGoogle Scholar
- Schafer DP, Lehrman EK, Kautzman AG, Koyama R, Mardinly AR, Yamasaki R, Ransohoff RM, Greenberg ME, Barres BA, Stevens B. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron. 2012;74:691–705. doi:10.1016/j.neuron.2012.03.026.PubMedPubMed CentralView ArticleGoogle Scholar
- Hatori K, Nagai A, Heisel R, Ryu JK, Kim SU. Fractalkine and fractalkine receptors in human neurons and glial cells. J Neurosci Res. 2002;69:418–26. doi:10.1002/jnr.10304.PubMedView ArticleGoogle Scholar
- Kim KW, Vallon-Eberhard A, Zigmond E, Farache J, Shezen E, Shakhar G, Ludwig A, Lira SA, Jung S. In vivo structure/function and expression analysis of the CX3C chemokine fractalkine. Blood. 2011;118:e156–67. doi:10.1182/blood-2011-04-348946.PubMedPubMed CentralView ArticleGoogle Scholar
- Chapman GA, Moores K, Harrison D, Campbell CA, Stewart BR, Strijbos PJ. Fractalkine cleavage from neuronal membranes represents an acute event in the inflammatory response to excitotoxic brain damage. J Neurosci. 2000;20:RC87.PubMedGoogle Scholar
- Haskell CA, Cleary MD, Charo IF. Molecular uncoupling of fractalkine-mediated cell adhesion and signal transduction. Rapid flow arrest of CX3CR1-expressing cells is independent of G-protein activation. J Biol Chem. 1999;274:10053–8.PubMedView ArticleGoogle Scholar
- Bachstetter AD, Morganti JM, Jernberg J, Schlunk A, Mitchell SH, Brewster KW, Hudson CE, Cole MJ, Harrison JK, Bickford PC, et al. Fractalkine and CX 3 CR1 regulate hippocampal neurogenesis in adult and aged rats. Neurobiol Aging. 2011;32:2030–44. doi:10.1016/j.neurobiolaging.2009.11.022.PubMedView ArticleGoogle Scholar
- Rogers JT, Morganti JM, Bachstetter AD, Hudson CE, Peters MM, Grimmig BA, Weeber EJ, Bickford PC, Gemma C. CX3CR1 deficiency leads to impairment of hippocampal cognitive function and synaptic plasticity. J Neurosci. 2011;31:16241–50. doi:10.1523/JNEUROSCI.3667-11.201131/45/16241.PubMedView ArticleGoogle Scholar
- Maggi L, Scianni M, Branchi I, D’Andrea I, Lauro C, Limatola C. CX(3)CR1 deficiency alters hippocampal-dependent plasticity phenomena blunting the effects of enriched environment. Front Cell Neurosci. 2011;5:22. doi:10.3389/fncel.2011.00022.PubMedPubMed CentralView ArticleGoogle Scholar
- Xue J, Dong JH, Huang GD, Qu XF, Wu G, Dong XR. NF-kappaB signaling modulates radiationinduced microglial activation. Oncol Rep. 2014;31:2555–60. doi:10.3892/or.2014.3144.PubMedGoogle Scholar
- Hickman SE, Kingery ND, Ohsumi TK, Borowsky ML, Wang LC, Means TK, El Khoury J. The microglial sensome revealed by direct RNA sequencing. Nat Neurosci. 2013;16:1896–905. doi:10.1038/nn.3554.PubMedPubMed CentralView ArticleGoogle Scholar
- Benzing WC, Wujek JR, Ward EK, Shaffer D, Ashe KH, Younkin SG, Brunden KR. Evidence for glial-mediated inflammation in aged APP(SW) transgenic mice. Neurobiol Aging. 1999;20:581–9.PubMedView ArticleGoogle Scholar
- Chen J, Zhou Y, Mueller-Steiner S, Chen LF, Kwon H, Yi S, Mucke L, Gan L. SIRT1 protects against microglia-dependent amyloid-beta toxicity through inhibiting NF-kappaB signaling. J Biol Chem. 2005;280:40364–74. doi:10.1074/jbc.M509329200.PubMedView ArticleGoogle Scholar
- Yeung F, Hoberg JE, Ramsey CS, Keller MD, Jones DR, Frye RA, Mayo MW. Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J. 2004;23:2369–80. doi:10.1038/sj.emboj.7600244.PubMedPubMed CentralView ArticleGoogle Scholar
- Cook DN, Chen SC, Sullivan LM, Manfra DJ, Wiekowski MT, Prosser DM, Vassileva G, Lira SA. Generation and analysis of mice lacking the chemokine fractalkine. Mol Cell Biol. 2001;21:3159–65. doi:10.1128/MCB.21.9.3159-3165.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods. 1984;11:47–60.PubMedView ArticleGoogle Scholar
- Kumar R, Hunt CR, Gupta A, Nannepaga S, Pandita RK, Shay JW, Bachoo R, Ludwig T, Burns DK, Pandita TK. Purkinje cell-specific males absent on the first (mMof) gene deletion results in an ataxia-telangiectasia-like neurological phenotype and backward walking in mice. Proc Natl Acad Sci U S A. 2011;108:3636–41. doi:10.1073/pnas.1016524108.PubMedPubMed CentralView ArticleGoogle Scholar
- Park HR, Kong KH, Yu BP, Mattson MP, Lee J. Resveratrol inhibits the proliferation of neural progenitor cells and hippocampal neurogenesis. J Biol Chem. 2012;287:42588–600. doi:10.1074/jbc.M112.406413.PubMedPubMed CentralView ArticleGoogle Scholar
- Darcy MJ, Trouche S, Jin SX, Feig LA. Age-dependent role for Ras-GRF1 in the late stages of adult neurogenesis in the dentate gyrus. Hippocampus. 2014;24:315–25. doi:10.1002/hipo.22225.PubMedPubMed CentralView ArticleGoogle Scholar
- Dann A, Poeck H, Croxford AL, Gaupp S, Kierdorf K, Knust M, Pfeifer D, Maihoefer C, Endres S, Kalinke U, et al. Cytosolic RIG-I-like helicases act as negative regulators of sterile inflammation in the CNS. Nat Neurosci. 2012;15:98–106. doi:10.1038/nn.2964.View ArticleGoogle Scholar
- Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch UK, Mack M, Heikenwalder M, Bruck W, Priller J, Prinz M. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat Neurosci. 2007;10:1544–53. doi:10.1038/nn2015.PubMedView ArticleGoogle Scholar
- Goldmann T, Wieghofer P, Muller PF, Wolf Y, Varol D, Yona S, Brendecke SM, Kierdorf K, Staszewski O, Datta M, et al. A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation. Nat Neurosci. 2013;16:1618–26. doi:10.1038/nn.3531.PubMedView ArticleGoogle Scholar
- Raasch J, Zeller N, van Loo G, Merkler D, Mildner A, Erny D, Knobeloch KP, Bethea JR, Waisman A, Knust M, et al. IkappaB kinase 2 determines oligodendrocyte loss by non-cell-autonomous activation of NF-kappaB in the central nervous system. Brain. 2011;134:1184–98. doi:10.1093/brain/awq359.PubMedPubMed CentralView ArticleGoogle Scholar
- Fatt MP, Cancino GI, Miller FD, Kaplan DR. p63 and p73 coordinate p53 function to determine the balance between survival, cell death, and senescence in adult neural precursor cells. Cell Death Differ. 2014;21:1546–59. doi:10.1038/cdd.2014.61.PubMedPubMed CentralView ArticleGoogle Scholar
- Chal J, Oginuma M, Al Tanoury Z, Gobert B, Sumara O, Hick A, Bousson F, Zidouni Y, Mursch C, Moncuquet P, et al. Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nat Biotechnol. 2015;33:962–9. doi:10.1038/nbt.3297.PubMedView ArticleGoogle Scholar
- Fulmer CG, VonDran MW, Stillman AA, Huang Y, Hempstead BL, Dreyfus CF. Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination. J Neurosci. 2014;34:8186–96. doi:10.1523/JNEUROSCI.4267-13.2014.PubMedPubMed CentralView ArticleGoogle Scholar
- Farioli-Vecchioli S, Micheli L, Saraulli D, Ceccarelli M, Cannas S, Scardigli R, Leonardi L, Cina I, Costanzi M, Ciotti MT, et al. Btg1 is required to maintain the pool of stem and progenitor cells of the dentate gyrus and subventricular zone. Front Neurosci. 2012;6:124. doi:10.3389/fnins.2012.00124.PubMedPubMed CentralView ArticleGoogle Scholar
- Nistico R, Florenzano F, Mango D, Ferraina C, Grilli M, Di Prisco S, Nobili A, Saccucci S, D’Amelio M, Morbin M, et al. Presynaptic c-Jun N-terminal Kinase 2 regulates NMDA receptor-dependent glutamate release. Sci Rep. 2015;5:9035. doi:10.1038/srep09035.PubMedPubMed CentralView ArticleGoogle Scholar
- Gould E, Beylin A, Tanapat P, Reeves A, Shors TJ. Learning enhances adult neurogenesis in the hippocampal formation. Nat Neurosci. 1999;2:260–5. doi:10.1038/6365.PubMedView ArticleGoogle Scholar
- Mildner A, Schlevogt B, Kierdorf K, Bottcher C, Erny D, Kummer MP, Quinn M, Bruck W, Bechmann I, Heneka MT, et al. Distinct and non-redundant roles of microglia and myeloid subsets in mouse models of Alzheimer’s disease. J Neurosci. 2011;31:11159–71. doi:10.1523/JNEUROSCI.6209-10.2011.PubMedView ArticleGoogle Scholar
- West MJ, Slomianka L, Gundersen HJ. Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator. Anat Rec. 1991;231:482–97. doi:10.1002/ar.1092310411.PubMedView ArticleGoogle Scholar
- Goldmann T, Zeller N, Raasch J, Kierdorf K, Frenzel K, Ketscher L, Basters A, Staszewski O, Brendecke SM, Spiess A, et al. USP18 lack in microglia causes destructive interferonopathy of the mouse brain. EMBO J. 2015;34:1612–29. doi:10.15252/embj.201490791.PubMedPubMed CentralView ArticleGoogle Scholar
- Harsan LA, David C, Reisert M, Schnell S, Hennig J, von Elverfeldt D, Staiger JF. Mapping remodeling of thalamocortical projections in the living reeler mouse brain by diffusion tractography. Proc Natl Acad Sci U S A. 2013;110:E1797–806. doi:10.1073/pnas.1218330110.PubMedPubMed CentralView ArticleGoogle Scholar
- Harsan LA, Paul D, Schnell S, Kreher BW, Hennig J, Staiger JF, von Elverfeldt D. In vivo diffusion tensor magnetic resonance imaging and fiber tracking of the mouse brain. NMR Biomed. 2010;23:884–96. doi:10.1002/nbm.1496.PubMedView ArticleGoogle Scholar
- Kumar P, Periyasamy R, Das S, Neerukonda S, Mani I, Pandey KN. All-trans retinoic acid and sodium butyrate enhance natriuretic peptide receptor a gene transcription: role of histone modification. Mol Pharmacol. 2014;85:946–57. doi:10.1124/mol.114.092221.PubMedPubMed CentralView ArticleGoogle Scholar
- Brown JP, Couillard-Despres S, Cooper-Kuhn CM, Winkler J, Aigner L, Kuhn HG. Transient expression of doublecortin during adult neurogenesis. J Comp Neurol. 2003;467:1–10. doi:10.1002/cne.10874.PubMedView ArticleGoogle Scholar
- Combadiere C, Salzwedel K, Smith ED, Tiffany HL, Berger EA, Murphy PM. Identification of CX3CR1. A chemotactic receptor for the human CX3C chemokine fractalkine and a fusion coreceptor for HIV-1. J Biol Chem. 1998;273:23799–804.PubMedView ArticleGoogle Scholar
- Sengupta N, Seto E. Regulation of histone deacetylase activities. J Cell Biochem. 2004;93:57–67. doi:10.1002/jcb.20179.PubMedView ArticleGoogle Scholar
- Zupkovitz G, Tischler J, Posch M, Sadzak I, Ramsauer K, Egger G, Grausenburger R, Schweifer N, Chiocca S, Decker T, et al. Negative and positive regulation of gene expression by mouse histone deacetylase 1. Mol Cell Biol. 2006;26:7913–28. doi:10.1128/MCB.01220-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen LF, Mu Y, Greene WC. Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-kappaB. EMBO J. 2002;21:6539–48.PubMedPubMed CentralView ArticleGoogle Scholar
- Cao D, Wang M, Qiu X, Liu D, Jiang H, Yang N, Xu RM. Structural basis for allosteric, substrate-dependent stimulation of SIRT1 activity by resveratrol. Genes Dev. 2015;29:1316–25. doi:10.1101/gad.265462.115.PubMedPubMed CentralView ArticleGoogle Scholar
- Solomon JM, Pasupuleti R, Xu L, McDonagh T, Curtis R, DiStefano PS, Huber LJ. Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage. Mol Cell Biol. 2006;26:28–38. doi:10.1128/MCB.26.1.28-38.2006.PubMedPubMed CentralView ArticleGoogle Scholar
- Min KJ, Yang MS, Jou I, Joe EH. Protein kinase A mediates microglial activation induced by plasminogen and gangliosides. Exp Mol Med. 2004;36:461–7. doi:10.1038/emm.2004.58.PubMedView ArticleGoogle Scholar
- Liu TF, Yoza BK, El Gazzar M, Vachharajani VT, McCall CE. NAD + −dependent SIRT1 deacetylase participates in epigenetic reprogramming during endotoxin tolerance. J Biol Chem. 2011;286:9856–64. doi:10.1074/jbc.M110.196790.PubMedPubMed CentralView ArticleGoogle Scholar
- Koo JW, Duman RS. IL-1beta is an essential mediator of the antineurogenic and anhedonic effects of stress. Proc Natl Acad Sci U S A. 2008;105:751–6. doi:10.1073/pnas.0708092105.PubMedPubMed CentralView ArticleGoogle Scholar
- Hellwig S, Brioschi S, Dieni S, Frings L, Masuch A, Blank T, Biber K. Altered microglia morphology and higher resilience to stress-induced depression-like behavior in CX3CR1-deficient mice. Brain Behav Immun. 2015. Doi 10.1016/j.bbi.2015.11.008
- Pagani F, Paolicelli RC, Murana E, Cortese B, Di Angelantonio S, Zurolo E, Guiducci E, Ferreira TA, Garofalo S, Catalano M, et al. Defective microglial development in the hippocampus of Cx3cr1 deficient mice. Front Cell Neurosci. 2015;9:111. doi:10.3389/fncel.2015.00111.PubMedPubMed CentralView ArticleGoogle Scholar
- Reshef R, Kreisel T, Beroukhim Kay D, Yirmiya R. Microglia and their CX3CR1 signaling are involved in hippocampal- but not olfactory bulb-related memory and neurogenesis. Brain Behav Immun. 2014;41:239–50. doi:10.1016/j.bbi.2014.04.009.PubMedView ArticleGoogle Scholar
- Hundhausen C, Misztela D, Berkhout TA, Broadway N, Saftig P, Reiss K, Hartmann D, Fahrenholz F, Postina R, Matthews V, et al. The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion. Blood. 2003;102:1186–95. doi:10.1182/blood-2002-12-3775.PubMedView ArticleGoogle Scholar
- Ransohoff RM, Liu L, Cardona AE. Chemokines and chemokine receptors: multipurpose players in neuroinflammation. Int Rev Neurobiol. 2007;82:187–204. doi:10.1016/S0074-7742(07)82010-1.PubMedView ArticleGoogle Scholar
- Liu Z, Condello C, Schain A, Harb R, Grutzendler J. CX3CR1 in microglia regulates brain amyloid deposition through selective protofibrillar amyloid-beta phagocytosis. J Neurosci. 2010;30:17091–101. doi:10.1523/JNEUROSCI.4403-10.2010.PubMedPubMed CentralView ArticleGoogle Scholar
- Shan S, Hong-Min T, Yi F, Jun-Peng G, Yue F, Yan-Hong T, Yun-Ke Y, Wen-Wei L, Xiang-Yu W, Jun M, et al. New evidences for fractalkine/CX3CL1 involved in substantia nigral microglial activation and behavioral changes in a rat model of Parkinson’s disease. Neurobiol Aging. 2011;32:443–58. doi:10.1016/j.neurobiolaging.2009.03.004.PubMedView ArticleGoogle Scholar
- Cambien B, Pomeranz M, Schmid-Antomarchi H, Millet MA, Breittmayer V, Rossi B, Schmid-Alliana A. Signal transduction pathways involved in soluble fractalkine-induced monocytic cell adhesion. Blood. 2001;97:2031–7.PubMedView ArticleGoogle Scholar
- Ding XM, Pan L, Wang Y, Xu QZ. Baicalin exerts protective effects against lipopolysaccharide-induced acute lung injury by regulating the crosstalk between the CX3CL1-CX3CR1 axis and NF-kappaB pathway in CX3CL1-knockout mice. Int J Mol Med. 2016. Doi 10.3892/ijmm.2016.2456
- Nakayama T, Watanabe Y, Oiso N, Higuchi T, Shigeta A, Mizuguchi N, Katou F, Hashimoto K, Kawada A, Yoshie O. Eotaxin-3/CC chemokine ligand 26 is a functional ligand for CX3CR1. J Immunol. 2010;185:6472–9. doi:10.4049/jimmunol.0904126.PubMedView ArticleGoogle Scholar
- Pope SM, Fulkerson PC, Blanchard C, Akei HS, Nikolaidis NM, Zimmermann N, Molkentin JD, Rothenberg ME. Identification of a cooperative mechanism involving interleukin-13 and eotaxin-2 in experimental allergic lung inflammation. J Biol Chem. 2005;280:13952–61. doi:10.1074/jbc.M406037200.PubMedView ArticleGoogle Scholar