Glioblastoma, IDH1 wildtype, aggressive primary brain tumors with a dismal prognosis, promote the recruitment of microglia, brain resident innate immune cells, and ultimately their activation toward a tumor-supportive phenotype that increases gliomal proliferation and invasion capability. Here, we report that upon stimulation by glioma cells, microglia transit via a reactive state holding anti-tumoral properties coupled to reduced DNA methyltransferase 3 A (DNMT3A) chromatin occupancy and DNA demethylation that promote the expression of gene sets related to the transforming growth factor beta (TGF-β)-dependent microglial homeostasis and the microglial sensome. We find that upon repression of Dnmt3a expression in microglia, those cells maintain anti-tumoral attributes in vitro and in vivo. In a syngeneic immunocompetent glioblastoma mouse model, brain delivery of antisense oligonucleotide targeting Dnmt3a expression led to microglial activation and reduced tumor growth. Taken together, our results reveal the involvement of DNA demethylation in the control of glioma cells-induced microglia activation and indicate that microglial DNMT3A is a potentially therapeutic target to treat brain neoplasms such as glioblastoma that include a microglial component.

The aging process is marked by a time-dependent deterioration in cellular functions, particularly the immune and neural systems. Understanding the phenotype acquisition of microglia, the sentinel immune cells of the brain, is crucial for understanding the nature of age-related neurological diseases. However, the specific phenotype adopted by microglia during aging remains a subject of debate and is contingent on the chosen experimental model. To address these unresolved questions, we employed a novel and highly controlled approach utilizing long-term cultivated BV-2 microglia, exempted from additional external stimuli. Our findings revealed that aged microglial cells, in comparison to their younger counterparts, acquire a distinct gene expression profile, primarily characterized by alterations in microglial immune response. Indeed, pro-inflammatory stimulated aged and young BV-2 microglia exhibited similar transcriptomic profiles, yet the response intensity to the stimulus was markedly muted in the aged microglia. Functional neurotoxic assays confirmed diminished neuronal death in coculture with aged, activated microglia, underscoring a compromised immune response. Furthermore, a subsequent comparative analysis of aged BV-2 microglia with established transcriptomic microglial datasets from aged mice and humans identified 13 overlapping genes, laying the foundation for identifying core microglial aging signature. Particularly noteworthy were SLC16A3 and P2RY13, which consistently exhibited upregulation and downregulation, respectively, across all datasets. Additionally, four other genes—CAPG, LGALS3BP, NRIP1, and P2RY12—were found to share regulatory patterns in response to both aging and extrinsic activation. An in-depth investigation focused on SLC16A3, encoding the high-affinity lactate transporter MCT4, revealed disruptions in extracellular acidification rate and lactate concentration with age. Microglial purine sensing and motility capacities, regulated by P2RY12/P2RY13, displayed age-related alterations. Remarkably, protein analysis in human brain tissue validated the observed upregulation of MCT4 and downregulation of P2RY12 in aged microglia. In conclusion, our study unveils a distinct phenotype in aged microglia characterized by compromised immune responsiveness. Through the integration of in vitro cultured BV-2 microglia with primary microglia datasets, we identify critical molecular determinants of microglial cellular aging confirmed in human-aged brain tissue. This comprehensive approach offers potential insights for understanding and potentially reprogramming aged microglia, with implications for combating age-related neurological disorders.

Glioblastoma, aggressive primary brain tumors with a dismal prognosis, promote the recruitment of microglia, brain resident innate immune cells, and ultimately their activation toward a tumor-supportive phenotype that increases gliomal proliferation and invasion capability. Here, we report that upon stimulation by glioma cells, microglia transit via a reactive state holding anti-tumoral properties coupled to reduced DNMT3A chromatin occupancy and DNA demethylation that promote microglial pro-inflammatory gene expressions. We find that upon repression of Dnmt3a expression in microglia, those cells maintain anti-tumoral attributes in vitro and in vivo. In a syngeneic immunocompetent glioblastoma mouse model, brain delivery of antisense oligonucleotide targeting Dnmt3a expression led to reduced tumor growth. Taken together, our results reveal the involvement of DNA demethylation in the control of glioma cells-induced microglia activation and indicate that microglial DNMT3A is a potentially therapeutic target to treat brain neoplasms such as glioblastoma that include a microglial component. ### Competing Interest Statement MC, and BJ are co-founder of CERVO Therapeutics AB. CH, and FK are employees of and stockholders in Ionis Pharmaceuticals, Inc. The other authors declare no competing financial interests.

Glioblastoma is a highly aggressive brain tumour that creates an immunosuppressive microenvironment. Microglia, the brain's resident immune cells, play a crucial role in this environment. Glioblastoma cells can reprogramme microglia to create a supportive niche that promotes tumour growth. However, the mechanisms controlling the acquisition of a transcriptome associated with a tumour-supportive microglial reactive state are not fully understood. In this study, we investigated changes in the transcriptional profile of BV2 microglia exposed to C6 glioma cells. RNA-sequencing analysis revealed a significant upregulation of microglial inhibitor of DNA binding 1 (Id1) and Id2, helix-loop-helix negative transcription regulatory factors. The concomitant regulation of microglial ETS proto-oncogene 2, transcription factor (ETS2)-target genes, i.e., Dusp6, Fli1, Jun, Hmox1, and Stab1, led us to hypothesize that ETS2 could be regulated by ID proteins. In fact, ID2-ETS2 protein interactions increased in microglia exposed to glioma cells. In addition, perturbation of the ID2-ETS2 transcriptional axis influenced the acquisition of a microglial tumour-supportive phenotype. ID2 and ETS2 genes were found to be expressed by the tumour-associated microglia isolated from human glioblastoma tumour biopsies. Furthermore, ID2 and ETS2 gene expressions exhibited inverse prognostic values in patients with glioma in cohorts from The Cancer Genome Atlas. Collectively, our findings indicate that the regulation of ETS2 by ID2 plays a role in the transcriptional regulation of microglia in response to stimuli originating from glioblastoma cells, information that could lead to developing therapeutic strategies to manipulate microglial tumour-trophic functions.

Molecular diversity of microglia, the resident immune cells in the CNS, is reported. Whether microglial subsets characterized by the expression of specific proteins constitute subtypes with distinct functions has not been fully elucidated. Here we describe a microglial subtype expressing the enzyme arginase-1 (ARG1; that is, ARG1+ microglia) that is found predominantly in the basal forebrain and ventral striatum during early postnatal mouse development. ARG1+ microglia are enriched in phagocytic inclusions and exhibit a distinct molecular signature, including upregulation of genes such as Apoe, Clec7a, Igf1, Lgals3 and Mgl2, compared to ARG1- microglia. Microglial-specific knockdown of Arg1 results in deficient cholinergic innervation and impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, which in turn results in impaired long-term potentiation and cognitive behavioral deficiencies in female mice. Our results expand on microglia diversity and provide insights into microglia subtype-specific functions.

Caspases are a family of proteins mostly known for their role in the activation of the apoptotic pathway leading to cell death. In the last decade, caspases have been found to fulfill other tasks regulating the cell phenotype independently to cell death. Microglia are the immune cells of the brain responsible for the maintenance of physiological brain functions but can also be involved in disease progression when overactivated. We have previously described non-apoptotic roles of caspase-3 (CASP3) in the regulation of the inflammatory phenotype of microglial cells or pro-tumoral activation in the context of brain tumors. CASP3 can regulate protein functions by cleavage of their target and therefore could have multiple substrates. So far, identification of CASP3 substrates has been performed mostly in apoptotic conditions where CASP3 activity is highly upregulated and these approaches do not have the capacity to uncover CASP3 substrates at the physiological level. In our study, we aim at discovering novel substrates of CASP3 involved in the normal regulation of the cell. We used an unconventional approach by chemically reducing the basal level CASP3-like activity (by DEVD-fmk treatment) coupled to a Mass Spectrometry screen (PISA) to identify proteins with different soluble amounts, and consequently, non-cleaved proteins in microglia cells. PISA assay identified several proteins with significant change in their solubility after DEVD-fmk treatment, including a few already known CASP3 substrates which validated our approach. Among them, we focused on the Collectin-12 (COLEC12 or CL-P1) transmembrane receptor and uncovered a potential role for CASP3 cleavage of COLEC12 in the regulation of the phagocytic capacity of microglial cells. Taken together, these findings suggest a new way to uncover non-apoptotic substrates of CASP3 important for the modulation of microglia cell physiology.

Chlorhexidine did not inhibit ATPase in intact cells of Escherichia coli K12 W1317i-, even at bactericidal concentrations, and ATP hydrolysis was greatest at the highest concentration (40 mg/l), even though no net uptake of substrate occurred. Like dinitrophenol and tribrominated salicylanilide, polymyxin and chlorhexidine collapsed the membrane potential at inhibitory concentrations. Membrane disruption, and not ATPase inactivation, is considered the lethal event in chlorhexidine action.

Additional file 5. Metascape GO term clusters for overlap with C7: immunologic signature genes.

Diversity within microglia, the resident brain immune cells, is reported. Whether microglial subsets constitute different subtypes with intrinsic properties and unique functions has not been fully elucidated. Here, we describe a microglial subtype characterized by the expression of the enzyme Arginase-1, i.e. Arg1 + microglia, which is found predominantly in the cholinergic neuron-rich forebrain region during early postnatal development. Arg1 + microglia are frequently observed in close apposition to neurons and exhibit a distinctive molecular signature reflecting a reactive profile. Arg1 deficiency in microglia results in impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, and cognitive behavioural deficiencies in female mice. Our results expand on microglia diversity and provide insights into distinctive spatiotemporal functions exerted by microglial subtypes.

Background Diffuse intrinsic pontine gliomas (DIPG), within diffuse midline gliomas are aggressive pediatric brain tumors characterized by histone H3-K27M mutation. Small-molecule inhibitors for the EZH2-H3K27 histone methyltransferase have shown promise in preclinical animal models of DIPG, despite having little effect on DIPG cells in vitro. Therefore, we hypothesized that the effect of EZH2 inhibition could be mediated through targeting of this histone modifying enzyme in tumor-associated microglia. Methods Primary DIPG tissues, and cocultures between microglia and patient-derived DIPG or -pediatric high-grade glioma (pHGG) cell lines, were used to establish the H3-K27M status of each cell type. Antisense RNA strategies were used to target EZH2 gene expression in both microglia and glioma cells. Microglia anti-tumoral properties were assessed by gene expression profile, tumor cell invasion capacity, microglial phagocytic activity, and associated tumor cell death. Results In primary DIPG tissues, microglia do not carry the H3-K27M mutation, otherwise characteristic of the cancer cells. Activation of a microglial tumor-supportive phenotype by pHGG, independently of their H3-K27M status, is associated with a transient H3K27me3 downregulation. Repression of EZH2 in DIPG cells has no impact on tumor cell survival or their ability to activate microglia. However, repression of EZH2 in microglia induces an anti-tumor phenotype resulting in decreased cancer cell invasion capability, increased microglial phagocytosis, and tumor-related cell death. Conclusions These results indicate that microglia, beyond the tumor cells, contribute to the observed response of DIPG to EZH2 inhibition. Results highlight the potential importance of microglia as a new therapeutic avenue in DIPG.

Microglia are the resident innate immune cells of the immune-privileged CNS and, as such, represent the first line of defence against tissue injury and infection. Given their location, microglia are undoubtedly the first immune cells to encounter a developing primary brain tumour. Our knowledge of these cells is therefore important to consider in the context of such neoplasms. As the heterogeneous nature of the most aggressive primary brain tumours is thought to underlie their poor prognosis, this Review places a special emphasis on the heterogeneity of the tumour-associated microglia and macrophage populations present in primary brain tumours. Where available, specific information on microglial heterogeneity in various types and subtypes of brain tumour is included. Emerging evidence that highlights the importance of considering the heterogeneity of both the tumour and of microglial populations in providing improved treatment outcomes for patients is also discussed.

Microglia, resident immunocompetent cells of the central nervous system, can display a range of reaction states and thereby exhibit distinct biological functions across development, adulthood and under disease conditions. Distinct gene expression profiles are reported to define each of these microglial reaction states. Hence, the identification of modulators of selective microglial transcriptomic signature, which have the potential to regulate unique microglial function has gained interest. Here, we report the identification of ATG7 (Autophagy-related 7) as a selective modulator of an NF-κB-dependent transcriptional program controlling the pro-inflammatory response of microglia. We also uncover that microglial Atg7 -deficiency was associated with reduced microglia-mediated neurotoxicity, and thus a loss of biological function associated with the pro-inflammatory microglial reactive state. Further, we show that Atg7 -deficiency in microglia did not impact on their ability to respond to alternative stimulus, such as one driving them towards an anti-inflammatory/tumor supportive phenotype. The identification of distinct regulators, such as Atg7 , controlling specific microglial transcriptional programs could lead to developing novel therapeutic strategies aiming to manipulate selected microglial phenotypes, instead of the whole microglial population with is associated with several pitfalls.

BACKGROUND:Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM).METHODS:RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3'UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints.RESULTS:Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients.CONCLUSION:Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively.

Until recently, drug development in oncology was focused on treating most patients for a specific cancer type without taking in account the heterogeneity between these patients in term of response to treatment. Therefore, this type of broad treatment approach excludes the treatment of patient not responding to disease-specific common drugs. In this review, we focus on the different types of epigenetic drugs currently used as DNA methylation inhibitor agents and their limits in patient care due to their lack of specificity. We also highlight the emergence of a new type of epidrug with higher target specificity due to their original mechanism of action: the disruption of protein complexes involved in the epigenetic modifications.

Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled in vitro to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by de novo DNA methyltransferase DNMT3A of MAP1LC3 loci upon autophagy stimulation leads to the observed long-term decrease of MAP1LC3 isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in vivo in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.Abbreviations: ACTB: actin beta; ATG: autophagy-related; 5-Aza: 5-aza-2'-deoxycytidine; BafA1: bafilomycin A1; CBZ: carbamazepine; CDKN2A: cyclin dependent kinase inhibitor 2A; ChIP: chromatin immunoprecipitation; Clon.: clonidine; CpG: cytosine-guanine dinucleotide: DMSO: dimethyl sulfoxide; DNA: deoxyribonucleic acid; DNMT: DNA methyltransferase; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase alpha; DNMT3B: DNA methyltransferase beta; dpf: days post-fertilization; EBSS: Earle's balanced salt solution; EM: Zebrafish embryo medium; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GRO-Seq: Global Run-On sequencing; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP1LC3B2: microtubule-associated protein 1 light chain 3 beta 2; MEM: minimum essential medium; MEF: mouse embryonic fibroblasts; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RT-qPCR: quantitative reverse transcription polymerase chain reaction; SQSTM1/p62: sequestosome 1; Starv.: starvation; Treh.: trehalose; ULK1: unc-51 like autophagy activating kinase 1.

Epigenomic mechanisms regulate distinct aspects of the inflammatory response in immune cells. Despite the central role for microglia in neuroinflammation and neurodegeneration, little is known about their epigenomic regulation of the inflammatory response. Here, we show that Ten-eleven translocation 2 (TET2) methylcytosine dioxygenase expression is increased in microglia upon stimulation with various inflammogens through a NF-κB-dependent pathway. We found that TET2 regulates early gene transcriptional changes, leading to early metabolic alterations, as well as a later inflammatory response independently of its enzymatic activity. We further show that TET2 regulates the proinflammatory response in microglia of mice intraperitoneally injected with LPS. We observed that microglia associated with amyloid β plaques expressed TET2 in brain tissue from individuals with Alzheimer's disease (AD) and in 5xFAD mice. Collectively, our findings show that TET2 plays an important role in the microglial inflammatory response and suggest TET2 as a potential target to combat neurodegenerative brain disorders.

Microglia, the immune cells of the brain, play a major role in the maintenance of brain homeostasis and constantly screen the brain environment to detect any infection or damage. Once activated by a stimulus, microglial cells initiate an immune response followed by the resolution of brain inflammation. A failure or deviation in the housekeeping function of these guardian cells can lead to multiple diseases, including brain cancer and neurodegenerative diseases such as Alzheimer’s disease (AD). A small number of studies have investigated the causal relation of both diseases, thereby revealing an inverse relationship where cancer patients have a reduced risk to develop AD and vice versa. In this review, we aim to shed light on the role of microglia in the fate to develop specifically glioma as one type of cancer or AD. We will examine the common and/or opposing genetic predisposition as well as associated pathways of these diseases to unravel a possible involvement of microglia in the occurrence of either disease. Lastly, a set of guidelines will be proposed for future research and diagnostics to clarify and improve the knowledge on the role of microglia in the decision toward one pathology or another.

Summary Epigenetic mechanisms regulate distinct aspects of the inflammatory response in various immune cell types. Despite the central role for microglia, the resident macrophages of the brain, in neuroinflammation and neurodegeneration little is known about their epigenetic regulation of the inflammatory response. Here, we show that Ten-eleven translocation 2 (TET2) methylcytosine dioxygenase expression is increased in microglia upon stimulation with various inflammogens through a NF-κB-dependent pathway. We found that TET2 regulates early gene transcriptional changes that lead to early metabolic alterations, as well as a later inflammatory response independently of its 5mC oxidation activity at the affected genes. We further show that TET2 regulates the proinflammatory response in microglia induced by intraperitoneal injection of LPS in vivo . We observed that microglia associated to amyloid β plaques, recently defined as disease-associated microglia, expressed TET2 in brain tissue from individuals with Alzheimer’s disease (AD) and in 5×FAD mice. Collectively, our findings show that TET2 plays an important role in the microglial inflammatory response, and suggest TET2 as a potential target to combat neurodegenerative brain disorders.

Microglia, resident immune cells of the central nervous system, fulfill multiple functions in the brain throughout life. These microglial functions range from participation in innate and adaptive immune responses, involvement in the development of the brain and its homeostasis maintenance, to contribution to degenerative, traumatic, and proliferative diseases; and take place in the developing, the aging, the healthy, or the diseased brain. Thus, an impressive level of cellular plasticity, appears as a requirement for the pleiotropic biological functions of microglia. Epigenetic changes, including histone modifications or DNA methylation as well as microRNA expression, are important modifiers of gene expression, and have been involved in cell phenotype regulation and reprogramming and are therefore part of the mechanisms regulating cellular plasticity. Here, we review and discuss the epigenetic mechanisms, which are emerging as contributors to this microglial cellular plasticity and thereby can constitute interesting targets to modulate microglia associated brain diseases, including developmental diseases, neurodegenerative diseases as well as cancer.