Two studies in this issue of Cell, by Bintu et al. and Brann et al., overturn the long-standing zonal model of olfactory receptor organization. Using image-based spatial transcriptomics, they reveal that receptors occupy reproducible positions along continuous gradients, coupling receptor choice to axonal targeting through a shared molecular code.

Calcium (Ca2+) signaling is a key regulator of brain function and development. Here, we comprehensively analyze the Ca2+ signaling transcriptome in the adult mouse brain and the developing human brain to reveal the basis of signaling specificity. We show that neurons organize into non-stochastic Ca2+ states that reflect cell-type identity and capture subtle functional differences. These states arise from lineage-specific developmental Ca2+ programs that are detectable already in progenitor stages, and may precede differentiation into mature neuronal cell types. During neocortical development, many Ca2+ signaling genes, such as ADGRV1, NCALD, and CREB5, peak at distinct developmental stages, are evolutionarily conserved, and reflect transcriptional heterogeneity within progenitors associated with cell-fate decisions. Together, our findings provide an in-depth understanding of how a tightly regulated Ca2+ signaling transcriptome encodes cell-state-specific signaling programs and demonstrate that Ca2+ signaling is precisely tailored to distinct cell states.

Epstein-Barr virus (EBV) infection constitutes a prerequisite for multiple sclerosis (MS) development, and cross-reactivity between EBV nuclear antigen 1 (EBNA1) and anoctamin-2 (ANO2) antibodies was previously demonstrated in persons with MS (pwMS). Here, we show that ANO2-specific CD4+ T cells are more frequent in pwMS. Immunization of SJL/J mice with ANO2 or EBNA1 led to cross-reactive CD4+ T cell and antibody responses. ANO2 pre-immunization led to exacerbated experimental autoimmune encephalomyelitis (EAE), an effect mediated by CD4+ T cells, as confirmed by adoptive transfer experiments. T cell clones with cross-reactivity to EBNA1 and ANO2 could be isolated from natalizumab-treated pwMS, and sequencing of EBNA1- and ANO2-specific T cell receptors (TCRs) revealed a significant repertoire overlap. We thus report the first mechanistic evidence that EBNA1 CD4+ T cells can target the MS autoantigen ANO2, thereby establishing a link between EBV infection and neuroinflammation.

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.

Microglia survey and regulate central nervous system myelination during embryonic development and adult homeostasis. However, whether microglia–myelin interactions are spatiotemporally regulated remains unexplored. Here, by examining spinal cord white matter tracts in mice, we determined that myelin degeneration was particularly prominent in the dorsal column (DC) during normal aging. This was accompanied by molecular and functional changes in DC microglia as well as an upregulation of transforming growth factor beta (TGF)β signaling. Disrupting TGFβ signaling in microglia led to unrestrained microglial responses and myelin loss in the DC, accompanied by neurological deficits exacerbated with aging. Single-nucleus RNA-sequencing analyses revealed the emergence of a TGFβ signaling-sensitive microglial subset and a disease-associated oligodendrocyte subset, both of which were spatially restricted to the DC. We further discovered that microglia rely on a TGFβ autocrine mechanism to prevent damage of myelin in the DC. These findings demonstrate that TGFβ signaling is crucial for maintaining microglial resilience to myelin degeneration in the DC during aging. This highlights a previously unresolved checkpoint mechanism of TGFβ signaling with regional specificity and spatially restricted microglia–oligodendrocyte interactions. Zhu et al. find that aging causes region-specific myelin damage in the spinal cord, which is counteracted by enhanced TGFβ signaling in microglia, revealing a protective mechanism for healthy aging.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a small vessel disease caused by cysteine-altering NOTCH3 gene variants, leading to vascular smooth muscle cell degeneration, compromised cerebral blood flow, subcortical ischaemic infarcts, cognitive decline and often ultimately vascular dementia. Little is known about the cellular and molecular effects downstream of the cerebral ischaemia in CADASIL, or whether brain regions known to be involved in dementia, such as the hippocampus, are particularly susceptible to such pathological downstream changes. In this study, we used a humanized CADASIL mouse model harbouring the p.(Arg182Cys) variant (R182C-TgN3), post-mortem human CADASIL brain sections with four different NOTCH3 gene variants and primary human cerebral vascular smooth muscle cells (VSMCs) harbouring the p.R133C NOTCH3 variant as primary cellular models to characterize the properties and contribution of mutant VSMCs to cognitive impairment. To specifically evaluate neuronal, mitochondrial and neurovascular function, we performed ex vivo electrophysiology, immunohistochemistry [confocal and immunolabelling-enabled 3D imaging of solvent-cleared organs (iDISCO+) methods], western blotting, Seahorse assay, quantitative PCR and single-cell RNA sequencing. In the CADASIL mice, hippocampal gamma oscillation patterns were impaired along with significant decreases in neuronal fibre length and aberrant neuronal morphology. The latter two phenotypes were also observed in post-mortem brain tissue from CADASIL patients. Consistent with these findings, we noted significantly lower levels of mitochondrial respiratory complexes in the CADASIL mouse hippocampus, isolated mouse brain vessels and primary human cerebral VSMCs. The human cerebral VSMCs exhibited reduced oxygen consumption rates leading to reduced ATP production as well as decreased glycolytic capacity in conjunction with increased pro-inflammatory gene expression, suggesting a broader impact on cellular energy metabolism and a neuroinflammatory process. In the CADASIL mice, we also observed extensive accumulation of the NOTCH3 extracellular domain in hippocampal vessels. Light sheet imaging with iDISCO+ clearing demonstrated substantial VSMC loss and reduced vessel density in the hippocampus at 9 months of age. Additionally, 3D imaging showed increased microglial attachment to vessels and enlargement of the size of the vessel-associated microglia in CADASIL mice. Single-cell RNA sequencing revealed a microglial subcluster expressing genes involved in mitochondrial respiration and inflammation. Collectively, our results reveal how small vessel pathology in CADASIL leads to significant neuronal pathology in the hippocampus involving metabolic and neuroinflammatory changes and highlight the critical role of the neurovascular unit. Our findings pave the way for future research and potential therapeutic strategies.

The emerging field of 3D histology centers on the molecular profiling of intact organs for a comprehensive understanding of biological systems. While current methods focus largely on protein visualization, techniques for whole-organ RNA transcript imaging remain underdeveloped. Here we present a detailed protocol for Tris buffer-mediated retention of in situ hybridization chain reaction signal in cleared organs (TRISCO), a method for single-cell RNA three-dimensional mapping across tissue volumes. By ensuring homogeneous and well-preserved labeling throughout the entire tissue, TRISCO has been successfully applied to several mouse organs, including the brain, lung, heart, kidney and spinal cord, as well as to rat and guinea pig brains. The protocol is straightforward, is adaptable to diverse laboratory setups and research questions and avoids complex instrumentation, specialized expertise or harsh chemical treatments that might limit its applicability. The workflow, which can be completed in 10-15 days, includes flexible stopping points for convenient scheduling and is suitable for users with expertise in light-sheet microscopy and animal handling.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of midbrain dopaminergic (mDA) neurons in the substantia nigra, leading to motor symptoms. Current pharmacological treatments are insufficient in halting disease progression, making cell therapy a promising alternative for replacing lost neurons. Advances in stem cell research have enabled the generation of mDA progenitors from human pluripotent stem cells (hPSCs), demonstrating success in preclinical models and clinical trials. However, challenges such as dyskinesias from non-mDA contaminations, graft rejection, and the need for more specific cell types persist. This study focuses on utilizing single-cell multiomic and transcriptomic data to design mDA progenitor differentiation protocols, aiming to generate more specific subtypes of mDA neurons, particularly those vulnerable to degeneration in PD. By refining the protocol with enhanced ventralization and prolonged Wnt activation, we achieved improved midbrain patterning, cell viability, and the accelerated generation of SOX6-expressing mDA neurons in vitro with proven therapeutical properties in grafted hemiparkinsonian mice models. This approach aims to present a protocol designed using single-cell analysis to enrich the most PD-affected mDA neuron populations, offering potential for better cell modelization, and more targeted cell replacement therapies. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, PreciseCellPD 884608/EA European Union’s Horizon 2020 research and innovation programme, NSC-Reconstruct 874758/EA, 899687 - HS-SEQ, MSCA-ITN ASCTN/EA Knut and Alice Wallenberg Foundation, 2018.0232/EA Vetenskaprådet, VR2020-01426/EA Chan Zuckerberg Initiative (United States), 2018-191929/EA Aligning Science Across Parkinson’s, ASAP-20505/EA Hjärnfonden, FO2024-0195/EA Deutsche Forschungsgemeinschaft, 516641042 Wenner-Gren Foundations, UPD2022-0159

Cells continuously experience fluctuating intracellular calcium (Ca²⁺) signals that orchestrate diverse processes such as transcription, proliferation, and apoptosis. Temporal features of Ca²⁺ dynamics, including oscillation frequency, are hypothesized to encode information, allowing cells to discriminate between relevant and stochastic signals. However, the mechanisms of frequency decoding and their transcriptional consequences remain incompletely understood. To address this, we investigated how defined Ca²⁺ oscillation frequencies are translated into signaling cascades and gene expression programs in human non-excitable cells. Using optogenetic control of melanopsin-mediated Ca²⁺ influx, we induced slow (8 mHz) or fast (15 mHz) oscillations with identical single-pulse kinetics to isolate the effect of frequency. We found that TNF and IL8 transcription via NF-κB displayed sigmoidal frequency dependence, strictly requiring regular periodic stimulation, while random or low-frequency inputs with equal cumulative Ca²⁺ exposure were ineffective. Bulk RNA sequencing revealed a MYC -centered transcriptional response, with 116 of 215 differentially expressed genes predicted as MYC targets, despite unchanged MYC mRNA levels. Label-free phosphoproteomics identified PRKDC, CHEK2 and ATM as the top upstream kinases, forming a network linking Ca²⁺ oscillations to cell cycle and stress signaling. These findings demonstrate that cells can decode Ca²⁺ oscillation frequency through a multi-kinase network that tunes transcription via NF-κB and MYC, providing mechanistic insight into how temporal dynamics of second messengers shape cellular decision-making. ### Competing Interest Statement The authors have declared no competing interest. Swedish Research Council, https://ror.org/03zttf063, 2022-02185, 2009-3364, 2010-451 4392, 2013-3189

Background Only twice have variants in the ITPR1 gene been described among patients with ataxia and miosis. Functional characterization of these variants is lacking. Objective To characterize a family affected by congenital ataxia and miosis associated with a novel ITPR1 variant and to provide a functional assessment for it and two previously reported variants. Methods Clinical characterization, genetic investigations, and segregation were performed. A novel variant c.7697T>C in ITPR1 was identified, HEK cells were transfected with vectors carrying our variant and two other previously published variants associated with ataxia and miosis. Results Ataxia was non-progressive in the reported family, the c.7697T>c ITPR1 variant segregated with disease. Functional validation showed that all the three ITPR1 variants were associated with reduced intracellular calcium release. Conclusions Here, we present for the first time evidence of pathogenicity for 3 heterozygous ITPR1 variants in association with ataxia and miosis. Despite being localized in different ITPR1 protein domains, these variants converged on common functional defects. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement M.P. research was supported by the Promobilia Foundation, Region Stockholm and NeuroSweden. P.S. has obtained funding from Region Stockholm and is a Wallenberg Clinical Scholar. I.E. was supported by a SSMF Postdoctoral Grant 2023 (PG-22-0462). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by Swedish Ethical Review Authority (EPN dnr 2016/2538-32). Informed written and oral consent was obtained from the patients for participation in this study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

The ability to spatially map multiple layers of omics information across developmental timepoints enables exploration of the mechanisms driving brain development1, differentiation, arealization and disease-related alterations. Here we used spatial tri-omic sequencing, including spatial ATAC-RNA-protein sequencing and spatial CUT&Tag-RNA-protein sequencing, alongside multiplexed immunofluorescence imaging (co-detection by indexinng (CODEX)) to map dynamic spatial remodelling during brain development and neuroinflammation. We generated a spatiotemporal tri-omic atlas of the mouse brain from postnatal day 0 (P0) to P21 and compared corresponding regions with the human developing brain. In the cortex, we identified temporal persistence and spatial spreading of chromatin accessibility for a subset of layer-defining transcription factors. In the corpus callosum, we observed dynamic chromatin priming of myelin genes across subregions and identified a role for layer-specific projection neurons in coordinating axonogenesis and myelination. In a lysolecithin neuroinflammation mouse model, we detected molecular programs shared with developmental processes. Microglia exhibited both conserved and distinct programs for inflammation and resolution, with transient activation observed not only at the lesion core but also at distal locations. Overall, this study reveals common and differential mechanisms underlying brain development and neuroinflammation, providing a rich resource for investigating brain development, function and disease.

Background: Personalized medicine in breast cancer requires a detailed understanding of the tumor and its microenvironment at the molecular level. Current diagnostic strategies, primarily based on 2D imaging methods, are limited by the lack of spatial molecular 3D mapping. Our study aims to bridge this gap by developing a 3D imaging methodology for simultaneously profiling RNA and protein within breast cancer tissues, providing a holistic view of tumor biology. Methods: We developed a novel 3D imaging method to achieve high-resolution 3D mapping of RNA and protein expressions in breast cancer tissue samples. This method integrates 3D in situ hybridization chain reaction, 3D immunostaining, and a non-toxic tissue clearing technique. We applied this method to intact triple-negative breast cancer (TNBC) samples, performing multiplex labeling for ErbB-2 (mRNA), HER2 (protein), and CD34 for co-mapping tumor cells with the vascular network. Machine-learning-based topological analysis was utilized to characterize spatial blood vessel organization across samples. Various visualization techniques, including scatter plots, histograms and cumulative plots, were employed to investigate the interactions between tumor cells and their nearest vessels. Results: Our approach successfully generated 3D maps of RNA and protein distribution within breast cancer sample blocks. Topological analysis of the vascular network identified metrics such as vessel length density (VLD), vessel volume density (VVD), connection density (CD), mean angle (MA), mean diameter (MD), and straightness. The results indicated that VLD and VVD positively correlated with prognosis, while CD and MA showed a tendency towards negative correlation. Notably, a subset of cells with ErbB2-mediate expression and HER2-negative expression (ErbB2++/HER2-) was prevalent in many TNBC cases. By comparing these findings with simulated random cell distribution experiments, we observed that the attraction distance between ErbB2++/HER2- cells and blood vessels in TNBC with lymph node metastasis was 58.77μm, significantly smaller than the 401.20μm observed in TNBC without lymph node metastasis. This implies a closer association of ErbB2++/HER2- cells with blood vessels in metastatic TNBC. The analysis revealed a statistically significant increase in the proportion of perivascular ErbB2++/HER2- cells in TNBC samples associated with lymph node metastasis compared to those without lymph node involvement (P<0.05). Conclusion: The simultaneous 3D mapping of RNA and protein provides unprecedented insights into the molecular architecture of breast cancer. Our findings underscore the critical importance of spatial context in molecular profiling. This newly developed method offers a powerful tool for identifying spatially therapeutic targets, redefining our understanding of tumor biology, and guiding clinical decisions. Citation Format: Yue Li, Shigeaki Kanatani, Per Uhlén. Simultaneous 3D Mapping of RNA and Protein to Advance Personalized Medicine in Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-09-05.

Objective Pathogenic variants in B-cell receptor-associated protein (BCAP31) are associated with X-linked, deafness, dystonia and cerebral hypomyelination (DDCH) syndrome. DDCH is congenital and non-progressive, featuring severe intellectual disability (ID), variable dysmorphism, and sometimes associated with shortened survival. BCAP31 encodes one of the most abundant chaperones, with several functions including acting as a negative regulator of endoplasmic reticulum (ER) calcium ion (Ca2+) concentration. Here, we characterize an X-linked syndrome, its underlying genotype, and a functional evaluation of the identified candidate genetic variant. Methods Evaluation of motor features, neuroimaging studies, neurophysiological, and cognitive tests. Whole exome sequencing (WES) was applied, a plasmid encoding BCAP31 with and without a candidate variant was transfected into SH-SY5Y cells to assess subcellular location and to measure Ca2+ levels in the cytoplasm. Results Adult-onset ataxia, cognitive impairment, and hearing loss leading to deafness are the predominant features. Reduced penetrance, slow progression with preserved ability to walk in advance age, and universal cerebellar atrophy are other features for this syndrome. This condition is associated with the new variant c.22G>A (V8I) in BCAP31 at Xq28. The subcellular location of the V8I BCAP31 protein was not altered but caused significant elevation of cytosolic Ca2+. Conclusions Our findings expand the spectrum of variants in BCAP31 from neurodevelopmental syndromes to include a progressive neurodegenerative disease with variable expressivity. This is the first time ataxia is described in association with a BCAP31 variant and functional evidence of pathogenicity is provided. Additional BCAP31 cases featuring ataxia are needed to establish an association. (c) 2025 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Colorectal cancer (CRC) is a leading cause of cancer mortality and is characterized by a high tumor mutational burden, making it responsive to immunotherapy. We developed a bioinformatic and manufacturing pipeline for personalized cancer vaccines and evaluated it in the MC38 colon adenocarcinoma mouse model. Thirty-six high-scoring neoantigens (NAGs) were identified by exome and transcriptome analysis, produced as six purified polypeptides, and coupled to paramagnetic beads. Intralymphatic vaccination of C57BL/6 mice with NAG beads induced robust NAG-specific T cell and antibody responses, resulting in significant inhibition of MC38 tumor growth. Treated tumors displayed increased necrosis and CD8+ T cell infiltration. Compared with soluble peptides, bead-coupled antigens elicited superior protection. Studies in T cell-deficient and antibody-depleted mice confirmed that both CD4+ and CD8+ T cells mediated the antitumor effect. These findings highlight the potential of NAG bead vaccination as an effective immunotherapy for CRC. ### Competing Interest Statement The authors have declared no competing interest. Cancer- och allergifonden European Innovation Council Eurostars Swedish Research Council, https://ror.org/03zttf063 Swedish Cancer Society, https://ror.org/0527jb766 Swedish Brain Foundation European Union’s Horizon 2020 research and innovation program

Mechanical signals sensed by human stem cells are transduced via discrete signaling pathways to modulate developmental phenotype and function. Proximal tubules isolated from nephron lineage-derived kidney organoids undergo a developmental increase in abundance and/or activity of the basolateral mechanosensor PIEZO1 and Ca2+ signal transduction pathways (Carrisoza-Gaytan R, Kroll KT, Hiratsuka K, Gupta NR, Morizane R, Lewis JA, Satlin LM. Am J Physiol Cell Physiol 324: C757-C768, 2023). Here, we investigate whether human induced pluripotent stem cell (iPSC)-derived ureteric bud (UB) and collecting duct (CD) organoid cells exhibit a similar developmental increase in PIEZO1 function. Comparison of cells in tubules microdissected from UB and CD organoids cultured for 34-35 days or 62-65 days showed 1) increased intracellular Ca2+ concentration ([Ca2+]i) response to basolateral application of the selective PIEZO1 agonist Yoda1 and 2) decreased time to peak [Ca2+]i with advancing days in culture. Single-cell analyses of the Yoda1-induced [Ca2+]i response revealed 7- to 15-mHz [Ca2+]i oscillations that were more prevalent with advancing days in culture and differentiation (CD vs. UB). Concurrent exposure to inhibitors of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) or the plasma membrane Ca2+-ATPase (PMCA) dampened the amplitude of the Yoda1-induced [Ca2+]i oscillations. Bulk RNA analysis and pathway enrichment analysis revealed broad changes in genes associated with Ca2+ signaling, but not PIEZO1, with advancing days in culture and differentiation. These findings are consistent with a developmental increase in activity of PIEZO1 channels and/or maturation of associated pathways shaping Ca2+ signaling dynamics in maturing UB and CD organoids. Decoding of [Ca2+]i oscillations may identify molecular mechanisms important in morphological and functional differentiation of organoid tubules.NEW & NOTEWORTHY This investigation, focused on analyzing the role of PIEZO1 mechanotransduction in maturing human induced pluripotent stem cell (iPSC)-derived ureteric bud (UB) and collecting duct (CD) kidney organoids, unexpectedly reveals developmentally regulated Ca2+ oscillations and begins to dissect their mechanistic underpinnings. Specifically, transcriptomic analysis reveals that with time in culture and differentiation from UB to CD, organoids progressively acquire the molecular machinery necessary for complex Ca2+ signaling dynamics. These results lead us to speculate that information encoded in oscillatory signals drives renal epithelial differentiation.

Induced pluripotent stem cells (iPSCs) have significant potential for disease modeling and cell therapies. However, their wide-spread application has faced challenges, including batch-to-batch variabilities, and notable distinctions when compared to embryonic stem cells (ESCs). Some of these disparities can stem from using undefined culture conditions and the reprogramming procedure, however, the precise mechanisms remain understudied. Here, we compared gene expression data from over 100 iPSC and ESC lines cultivated under undefined and defined conditions. Defined conditions significantly reduced inter-PSC line variability, irrespective of PSC cell type, highlighting the importance of standardization to minimize PSC biases. This variability is concurrent with decreased somatic cell marker and germ layer differentiation gene expression and increased Ca2+-binding protein expression. Moreover, SERCA pump inhibition highlighted an important role for intracellular Ca2+ activity in maintaining pluripotency gene expression under defined conditions. Further understanding of these processes can help standardize and improve defined hPSC culture conditions.

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.