Cx3cr1- deficient microglia exhibit a premature aging transcriptome

“DESeq2 (Love et al, 2014) was used to identify differential chromatin binding between Cx3cr1 -KO and -WT mice.”

“The raw Rbp2 or H3K27Ac ChIP sequences were mapped to mm10 genome by Bowtie 2 (Langmead & Salzberg, 2013).”

“Raw count fi les were aligned to mouse genome build mm10 using STAR and transcripts quanti fi ed with RSEM; in general, the uniquemappingratewas>80%.”

“ChIP-seq library preparation, sequencing, and data analysis ChIP-seq libraries were prepared from recovered DNA by blunting, A-tailing, and adapter ligation using barcoded adapters (NextFlex; Bioo Scienti fi c) as previously described (Heinz et al, 2010; Gosselin et al, 2014).”

“Heatmap visualizations of the DEGs were made by using custom R scripts based on ComplexHeatmap (Gu et al, 2016).”

“In multiple conditions, mouse microglia demonstrate a core conserved reactive-state transcriptome, which is extensively modi fi ed to yield disease-speci fi c transcriptomes (Chiu et al, 2013; Keren-Shaul et al, 2017; Friedman et al, 2018).”

“In WT mice, the microglial expression pro fi le obtained here is consistent with the published microglial transcriptomes (Table S1) (Hickman et al, 2013; Butovsky et al, 2014; Zhang et al, 2014).”

“…Cx3cr1 expression show altered brain development during embryogenesis, dysregulated neuronal fi ring in the fi rst few weeks after birth, and abnormal results in tests designed to mimic sociopsychological behaviors (Paolicelli et al, 2011; Rogers et al, 2011; Zhan et al, 2014; Bolós et al, 2017).”

“Indeed, Kl (Klotho) is down-regulated in Cx3cr1 -de fi cient microglia, and Klotho protein has been linked to aging and homeostatic support of brain function (Kuro-o et al, 1997).”

“CX3CR1 is involved in intestinal macrophage sensing of the microbiota (Niess et al, 2005), and gross disruption of the microbiota can affect microglial transcriptomes (Erny et al, 2015; Thion et al, 2018a).”

“Cx3cr1 -de fi cient mice, in which both copies of Cx3cr1 are disrupted by the gene for eGFP (Jung et al, 2000), were initially obtained from The Jackson Laboratory and then bred to maintain them on the C57Bl/6J background.”

“Finally, because ofits high andconsistent expression, Cx3cr1 is also often used as a driver in reporter constructs, such as Cx3cr1-eGFP mice, or for Cre-mediated gene deletion (Jung et al, 2000; Goldmann et al, 2013; Parkhurst et al, 2013).”

“They support brain development and adult CNS homeostasis, functions which are often dysregulated in acute or chronic neurological conditions (Xavier et al, 2014; Casano & Peri, 2015; Ransohoff, 2016; Hickman et al, 2018; Thion et al, 2018b).”

“They support brain development and adult CNS homeostasis, functions which are often dysregulated in acute or chronic neurological conditions (Xavier et al, 2014; Casano & Peri, 2015; Ransohoff, 2016; Hickman et al, 2018; Thion et al, 2018b).”

“CX3CR1 is involved in intestinal macrophage sensing of the microbiota (Niess et al, 2005), and gross disruption of the microbiota can affect microglial transcriptomes (Erny et al, 2015; Thion et al, 2018a).”

“…in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson ’ s disease and in tauopathies such as frontotemporal dementia, but promotes amyloid clearance in models of amyloid β deposition (Cardona et al, 2006; Bhaskar et al, 2010; Lee et al, 2010; Liu et al, 2010).”

“In WT mice, the microglial expression pro fi le obtained here is consistent with the published microglial transcriptomes (Table S1) (Hickman et al, 2013; Butovsky et al, 2014; Zhang et al, 2014).”

“…and neurodegenerative conditions and the emerging importance of gender differences in the role of microglia in response to multiple perturbations (Sorge et al, 2015; Hanamsagar et al, 2017; Thion et al, 2018a), an in-depth examination of gender differences is warranted, but beyond the scope of…”

“ChIP-seq library preparation, sequencing, and data analysis ChIP-seq libraries were prepared from recovered DNA by blunting, A-tailing, and adapter ligation using barcoded adapters (NextFlex; Bioo Scienti fi c) as previously described (Heinz et al, 2010; Gosselin et al, 2014).”

“…Cx3cr1 expression show altered brain development during embryogenesis, dysregulated neuronal fi ring in the fi rst few weeks after birth, and abnormal results in tests designed to mimic sociopsychological behaviors (Paolicelli et al, 2011; Rogers et al, 2011; Zhan et al, 2014; Bolós et al, 2017).”

“The transcriptome of young Cx3cr1 -de fi cient mice resembles that of aged WT mice Age is the dominant risk factor for neurodegenerative diseases and also alters the microglial transcriptome both in mice and humans (Grabert et al, 2016; Olah et al, 2018).”

“Moreover, Tnf , a classical in fl ammatory gene, had higher expression in Cx3cr1 - KO microglia by RNA-seq and a trend for differential Rbp2 binding by ChIP-seq (irreproducible discovery rate [IDR] = 0.0553; Fig 2D).”

“This LPS injection paradigm differs from the one we used and generates a different activation status in microglia (Wendeln et al, 2018).”

“…conditions and the emerging importance of gender differences in the role of microglia in response to multiple perturbations (Sorge et al, 2015; Hanamsagar et al, 2017; Thion et al, 2018a), an in-depth examination of gender differences is warranted, but beyond the scope of this study.”

“Finally, because ofits high andconsistent expression, Cx3cr1 is also often used as a driver in reporter constructs, such as Cx3cr1-eGFP mice, or for Cre-mediated gene deletion (Jung et al, 2000; Goldmann et al, 2013; Parkhurst et al, 2013).”

“In multiple conditions, mouse microglia demonstrate a core conserved reactive-state transcriptome, which is extensively modi fi ed to yield disease-speci fi c transcriptomes (Chiu et al, 2013; Keren-Shaul et al, 2017; Friedman et al, 2018).”

“In multiple conditions, mouse microglia demonstrate a core conserved reactive-state transcriptome, which is extensively modi fi ed to yield disease-speci fi c transcriptomes (Chiu et al, 2013; Keren-Shaul et al, 2017; Friedman et al, 2018).”

“…in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson ’ s disease and in tauopathies such as frontotemporal dementia, but promotes amyloid clearance in models of amyloid β deposition (Cardona et al, 2006; Bhaskar et al, 2010; Lee et al, 2010; Liu et al, 2010).”

“In addition, lack of Cx3cr1 expression reduces adult neurogenesis in the HC and olfactory bulb, and impairs the integration of newborn neurons (Bachstetter et al, 2011; Rogers et al, 2011; Xiao et al, 2015; Sellner et al, 2016; Reshef et al, 2017).”

“The transcription factor Sall1 is essential for the expression of microglia signature genes, which differentiate microgliafromothertissuemacrophagesandmyeloidcells(Buttgereit et al, 2016).”

“Microgliaundergosubstantialchangesin gene expression and morphology with age (Poliani et al, 2015; Grabert et al, 2016; Olah et al, 2018), but it is not known whether the changes in microglia occur as a consequence of aging or help drive the process.”

“Microglial identity is conferred by their yolk-sac origin and the brain environment (Bennett et al, 2018).”

“…in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson ’ s disease and in tauopathies such as frontotemporal dementia, but promotes amyloid clearance in models of amyloid β deposition (Cardona et al, 2006; Bhaskar et al, 2010; Lee et al, 2010; Liu et al, 2010).”

“The transcriptome of young Cx3cr1 -de fi cient mice resembles that of aged WT mice Age is the dominant risk factor for neurodegenerative diseases and also alters the microglial transcriptome both in mice and humans (Grabert et al, 2016; Olah et al, 2018).”

“…conditions and the emerging importance of gender differences in the role of microglia in response to multiple perturbations (Sorge et al, 2015; Hanamsagar et al, 2017; Thion et al, 2018a), an in-depth examination of gender differences is warranted, but beyond the scope of this study.”

“Microglial morphologyaccessedbythe totalIba1area andcell body – associated Iba1 dendritic process area and perimeter in the cerebellum was much lower thaninthe cortex, consistentwithless rami fi ed microglia in this brain region (Ayata et al, 2018).”

“…conditions and the emerging importance of gender differences in the role of microglia in response to multiple perturbations (Sorge et al, 2015; Hanamsagar et al, 2017; Thion et al, 2018a), an in-depth examination of gender differences is warranted, but beyond the scope of this study.”

“In addition, lack of Cx3cr1 expression reduces adult neurogenesis in the HC and olfactory bulb, and impairs the integration of newborn neurons (Bachstetter et al, 2011; Rogers et al, 2011; Xiao et al, 2015; Sellner et al, 2016; Reshef et al, 2017).”

“Only genes with FDR < 0.05, fold change > 30%, and average expression > 4 TPM were included in the enrichment analysis. augmented up-regulation of Klf2 , among other genes, could mediate durable functional responses by microglia (Das et al, 2006; de Bruin et al, 2016; Roberts et al, 2017).”

“…in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson ’ s disease and in tauopathies such as frontotemporal dementia, but promotes amyloid clearance in models of amyloid β deposition (Cardona et al, 2006; Bhaskar et al, 2010; Lee et al, 2010; Liu et al, 2010).”

“In addition, Cx3cr1 -KO microglia failed to up-regulate the adhesion molecule Ninj1 after LPS, which could play a role in the neuronal cell death after recurrent systemic LPS treatment (Araki & Milbrandt, 1996; Cardona et al, 2006).”

“Only genes with FDR < 0.05, fold change > 30%, and average expression > 4 TPM were included in the enrichment analysis. augmented up-regulation of Klf2 , among other genes, could mediate durable functional responses by microglia (Das et al, 2006; de Bruin et al, 2016; Roberts et al, 2017).”

“Thus, microglia establish and then alter their gene expression and function, based on contextual cues that modulate the activity of an enhancer landscape, primed through the action of lineage-determining transcription factors (Holtman et al, 2017).”

“They support brain development and adult CNS homeostasis, functions which are often dysregulated in acute or chronic neurological conditions (Xavier et al, 2014; Casano & Peri, 2015; Ransohoff, 2016; Hickman et al, 2018; Thion et al, 2018b).”

“It has been reported that CX3CL1 mRNA and protein expression decrease in the aged rodent brain (Lyons et al, 2009; Bachstetter et al, 2011), suggesting that reduced CX3CL1-CX3CR1 signaling may contribute to the transition of microglia to an aged phenotype.”

“In CNS disease models, mice de fi cient for only one copy of Cx3cr1 willunpredictablyeithershowphenotypessimilartoWTmice(Fumagalli et al, 2013) or intermediate between WT and KO (Lee et al, 2010; Rogers etal, 2011).”

“Furthermore, peripheral CX3CR1 can affect brain processes, such as cognition and memory (Garr´e et al, 2017).”

“These fi ndings were surprising because deletion of Cx3cr1 consistently enhanced in-fl ammatory responses in CNS disease models (Cardona et al, 2006; Bhaskar et al, 2010; Lee et al, 2014).”

“In addition, lack of Cx3cr1 expression reduces adult neurogenesis in the HC and olfactory bulb, and impairs the integration of newborn neurons (Bachstetter et al, 2011; Rogers et al, 2011; Xiao et al, 2015; Sellner et al, 2016; Reshef et al, 2017).”

“Both protective and detrimental effects have also been reported for Cx3cr1 deletion in varied models of traumatic brain injury, including at different time points in the same model (Febinger et al, 2015; Erturk etal, 2016; Zanier etal, 2016).”

“In addition, lack of Cx3cr1 expression reduces adult neurogenesis in the HC and olfactory bulb, and impairs the integration of newborn neurons (Bachstetter et al, 2011; Rogers et al, 2011; Xiao et al, 2015; Sellner et al, 2016; Reshef et al, 2017).”

“DESeq2 (Love et al, 2014) was used to identify differential chromatin binding between Cx3cr1 -KO and -WT mice.”

“Reassuringly, our results with isolatedmicrogliawereconsistentwithanalysisofgene expressionin hippocampal lysates from WT or Cx3cr1 -de fi cient mice (Rimmerman et al, 2017).”

“Only genes with FDR < 0.05, fold change > 30%, and average expression > 4 TPM were included in the enrichment analysis. augmented up-regulation of Klf2 , among other genes, could mediate durable functional responses by microglia (Das et al, 2006; de Bruin et al, 2016; Roberts et al, 2017).”

“In addition, lack of Cx3cr1 expression reduces adult neurogenesis in the HC and olfactory bulb, and impairs the integration of newborn neurons (Bachstetter et al, 2011; Rogers et al, 2011; Xiao et al, 2015; Sellner et al, 2016; Reshef et al, 2017).”

“This expression profile concurs with other studies of DAM [10,37,78,124,194].”

“CX3CR1/CX3CL1 signaling contributes to changes in microglia morphology and expression of immune proteins such as chemokines and chemokine receptors (Gyoneva et al., 2019).”

“The first one determined that Cx3cr1−/− microglia present a lower phagocytic and migratory capacity per se [242], while the second revealed that Cx3cr1−/− microglia of young animals are very similar to the wild type (WT) microglia of older animals [243].”