Abstract
Microglia—resident immune cells in the central nervous system—undergo morphological and functional changes in response to signals from the local environment and mature into various homeostatic states. However, niche signals underlying microglial differentiation and maturation remain unknown. Here, we show that neuronal micronuclei (MN) transfer to microglia, which is followed by changing microglial characteristics during the postnatal period. Neurons passing through a dense region of the developing neocortex give rise to MN and release them into the extracellular space, before being incorporated into microglia and inducing morphological changes. Two-photon imaging analyses have revealed that microglia incorporating MN tend to slowly retract their processes. Loss of the cGAS gene alleviates effects on micronucleus-dependent morphological changes. Neuronal MN-harboring microglia also exhibit unique transcriptome signatures. These results demonstrate that neuronal MN serve as niche signals that transform microglia, and provide a potential mechanism for regulation of microglial characteristics in the early postnatal neocortex.
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Data availability
The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE110 partner repository with the dataset identifier PXD056089. The dataset of mouse genome (mm10, GRCm38) is available at http://nov2020.archive.ensembl.org/Mus_musculus/Info/Index. Sequence data have been deposited in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive under the accession code DRA015927. The data in Extended Data Figs. 9e–r and 10l–v can be obtained from the Brain RNA-seq database (https://www.brainrnaseq.org/)58,59. Source data are provided with this paper.
Code availability
The MATLAB CAMDi software is available in Supplementary Software 1.
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Acknowledgements
We thank laboratory members for helpful discussions and technical support. We thank M Kengaku (iCeMS, Kyoto University) for technical advice of the neuronal migration experiments, M. Urushitani (Shiga University of Medical Science) for distributing BV2 cells and S. Hattori (NIMS) for technical support of mass spectrometry analysis. We thank L.J. Irving (University of Tsukuba) for editing the paper. The mass spectrometry analysis was supported by ‘Nanotechnology Platform Project’ operated by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (grant no. JPMXP09S19NM0031). We wish to acknowledge the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine, for analysis with IMARIS. This study was supported by the Cooperative Study Program (24NIPS330) of National Institute for Physiological Sciences. This work was supported by Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan JSPS KAKENHI [16KK0158 (F.T.), 20K05951 (F.T.), 23H04214 (Y.K.), 24K02020 (Y.K.), JP20H05688 (K.N.), JP22K19365 (K.N.), JSPS Research Fellowship for Young Scientists (19J20619 (S.Y.), 23KJ0285 (T.T.)], AMED PRIME [24028934 (F.T.)], Kao foundation for health science (F.T.), Gout and uric acid foundation (F.T.), Asahi Glass Foundation (Y.K.), SECOM Science and Technology Foundation (Y.K.) and was partly supported by Center for Quantum and Information Life Sciences (F.T.).
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S.Y. and F.T. designed the research. S.Y., N.A., Y.K., I.T., H.K., Y.H., A.K., K.H., K.-i.K., C.M., C.H., R.i.T., T.T., K.A., T.O-T., B.S. and F.T. performed research. S.Y., N.A., Y.K., I.T., H.K., M.S., C.H., C.M., R.R., T.T., K.A., T.N. and F.T. analyzed the data. T.N developed the MATLAB program. Y.K., Y.H., H.W., Y.G. and T.I. provided materials. S.Y. and F.T. wrote the paper. H.W., Y.G., K.N., T.C. and F.T. supervised the study.
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Extended data
Extended Data Fig. 1 Physical stress promotes micronuclear formation.
(a) Immunostaining of MAP2+ neurons. Primary cultured neurons (5 DIV) were stimulated with the recombinant 100 ng/ml Reelin for 6 hours. (b) The graph shows the percentage of MN+ neurons. 10 fields, n=3 independent samples, The numbers represent the total counted DAPI+ cells. mean ± SEM, p value was calculated by two-tailed Student's t-test. (c) Schematic illustration for injecting recombinant Reelin. (d) The recombinant Reelin (100 ng) was stimulated into the mice brain (P7) for 6 hours, and the brain sections were stained using DAPI. (e) The graph shows the percentage of MN. 15 images obtained from three independent brain, mean ± SEM, p value was calculated by two-tailed Student's t-test. (f) Schematic illustration for an in vitro migration assay. (g) Immunostaining of migrated neurons at the bottom sides of the trans-well. Nuclear shapes were detected by Lamin B1 staining. Asterisks indicate the marks of the hole. Arrow indicates the micronucleus. Illustration indicates the condition of migration, herniation, and micronuclear formation. (h) Immunostaining of migrated neurons at the bottom side of the trans-well. Yellow arrows indicate MN. (i)(j) The graph shows the population of MN+ neurons.(i) n=6, (j) n=3 trans-wells. More than 200 neurons were analyzed from one trans-well. (i) pore size; 3 μm, (j) pore size 12 μm, mean ± SEM, p value was calculated by two-tailed Student's t-test. (k) Schematic illustration for an in vitro mechanical stress assay. Neuro2A cells received mechanical stresses by pumping. (l) Neuro2A cells were stained with Hoechst. MN were generated by mechanical stress. (m) The graph shows the percentage of population of MN+ cells. Low; 5.0 x 104 cells/300μl, High; 5.0 x 104 cells/150μl, n=9 images, mean ± SEM, p values were calculated by one-way ANOVA Dunnett's multiple comparison test.
Extended Data Fig. 2 The autophagy pathway is involved in micronuclear secretion.
(a) Immunostaining of MAP2+ neurons in the cerebral cortex of either WT or NexCre:Atg7f/f at P21. Yellow arrows indicate MN. (b) The graph shows the population of MN in the cerebral cortex. n=20 images, 10 images obtained from two independent brain; mean ± SEM, p value was calculated by two-tailed Student's t-test. (c) Immunostaining of Iba1+ microglia in the cerebral cortex of either WT or NexCre:Atg7f/f at P21. Yellow arrows indicate MN. (d) The graph shows the percentage of MNs+ microglia in the cerebral cortex. Data were combined from 3 independent brains (8 images per brain, total 24 images), mean ± SEM, p value was calculated by two-tailed Student's t-test. (e) Immunoblotting of the extracellular MN (P15) obtained from primary cortical neurons in the presence or absence of 100 nM BafA treatment. Equal amounts of conditioned medium and total cell lysates were loaded onto separate gels and subjected to SDS-PAGE, followed by western blot analysis using anti-tubulin and anti-Lamin B1 antibodies, respectively. (f) Primary cortical neurons were stimulated with either 300 nM BafA for 3 hours or 50 μM etoposide (Eto, positive control for inducing apoptosis) for 24 hours. Immunostaining of cleaved-caspase 3 and MAP2+ cortical neurons (5 DIV). (g) The graph shows the fluorescence intensity of cleaved-caspase 3 with MAP2. n=4 images (DMSO) and 5 images (BafA and Eto), mean ± SEM, p values were calculated by one-way ANOVA Dunnett's multiple comparison test. (h) Schematic illustration of the collection of extracellular micronuclear for immunocytochemistry. (i) Immunostaining of Tubulin and Rab35 resided in the extracellular MN. (j) Immunostaining of transfected TCP subunits in the migrated neurons at the bottom side of the trans-well (3 DIV). Yellow arrows indicate MN. (k) Immunostaining of GFP+-MN in the cerebral cortex. A small amount of Tubulin resided in GFP+-MN. P14. Yellow allow indicates Tubulin+ signal. (l) Immunostaining of GFP+ MN in the cerebral cortex. MAP2 and LC3 resided in GFP+-MN. P14. (m) Working hypothesis of micronuclear secretion dependent on autophagy-lysosome machinery. TCP subunit is a potential receptor linking the micronucleus to LC3.
Extended Data Fig. 3 Micronuclei are incorporated into microglia.
(a) Schematic illustration for an in vitro micronuclear transfer assay. (b) TEM image of BV2 cells after treatment with conditioned medium. NE: nuclear envelope, MN: micronucleus, Cyto: cytoplasm. (c) Immunostaining of BV2 cells with GFP−-and GFP+-MN. (d) Immunostaining of GFP−- and GFP+-MN in BV2. Yellow arrow indicates micronucleus. (e) The graph shows the percentage of neuronal MN (GFP+ MN) containing BV2 cells after treatment with a conditioned medium. Each spot indicates the combined value of 10 images from one experiment. n=3 independent experiments. NB; fresh neurobasal medium, mean ± SEM, p values were analyzed by one-way ANOVA Dunnett's multiple comparisons test. (f) Schematic illustration for eliminating MN. (g) The graph shows MN+ BV2 cells 6 hours after treatment with a conditioned medium. mean ± SEM, p value was calculated by one-way ANOVA Tukey's multiple comparisons tests. Each spot indicates the combined value of more than 9 images from one experiment. Each column indicates a set of 3 independent experiments. (h) Live imaging of Cx3cr1-EGFP;H2B-mCherry cortical slice culture (P3). The interval of taking images was every 5 min. Yellow arrowheads indicate MN in the microglia. Cyan asterisks indicate MN out of the microglia. No.1-6, 89 z-stack images/field, No. 7-24, 92 z-stack images/field, 1.5 µm pitch.
Extended Data Fig. 4 Microglial morphology differs based on the cortical layer.
(a) Tailing immunostained images of Iba1+ microglia in the cerebral cortex. WT, P14. (b) Representative images of microglia in Layer 1 (L1) and Layers 2-6 (L2-6) from the insets of Extended Data Fig. 4a. (c)(d) The graphs show the analyses of microglial characters. 3 brains. (c) Process length, (d) the number of the process. mean ± SEM, p value was calculated by two-tailed Student's t-test.
Extended Data Fig. 5 Involvement of micronuclei on microglial gene expression.
(a) Immunostaining of Iba1+ and CD68+ microglia with or without MN (MN−; micronuclei-negative, MN+; micronuclei-positive). NexCre:LSL-Sun1GFP mice, P14. (b) The graph shows the expression level of CD68. The numbers counted are shown in the graph. 5 brains. mean ± SEM, p values were analyzed by one-way ANOVA Dunnett's multiple comparisons test. (c) Immunostaining of Iba1+ and Tmem119+ microglia with or without MN. NexCre:LSL-Sun1GFP mice, P14. (d) The graph shows the expression level of Tmem119. The numbers counted are shown in the graph. 3 brains. mean ± SEM, p values were analyzed by one-way ANOVA Dunnett's multiple comparisons test. (e) Immunostaining of Iba1+ and P2ry12+ microglia with or without MNs. The numbers counted are shown in the graph. NexCre:LSL-Sun1GFP mice, P14. (f) The graph shows the expression level of P2ry12. 5 brains, mean ± SEM, p values were analyzed by one-way ANOVA Dunnett's multiple comparisons test.
Extended Data Fig. 6 Two-photon imaging of microglial dynamics obtained from two distinct mice.
(a) to (j'') Quantification of individual microglial morphology. The line graph shows the change in the length of individual processes. Specific processes were measured every minute for one hour. The red outlines present MN+ processes. The violin plot shows the distribution of the lengths of individual processes in the line graph. The red outlines present MN+ processes. The blue and red line indicates the median and quartiles. The stack bars graph indicates the type and the number of individual processes (primary, secondary, tertiary, and quaternary processes) at 0, 30, and 60 minutes. The individual dot plots of stack bar graphs are shown in Supplementary Fig. 1. mean±SEM. Blue title: MN− microglia, n=10 cells, Red title: MN+ microglia, 10 each microglia obtained from 2 individual mice (mice identification number; Mo1 and Mo2).
Extended Data Fig. 7 Two-photon imaging of microglial dynamics obtained from three distinct mice.
(a) to (j'') Quantification of individual microglial morphology. The line graph shows the change in the length of individual processes. Specific processes were measured every minute for one hour. The red outlines present MN+ processes. The violin plot shows the distribution of the lengths of individual processes in the line graph. The red outlines present MN+ processes. The blue and red line indicates the median and quartiles. The stack bars graph indicates the type and the number of individual processes (primary, secondary, tertiary, and quaternary processes) at 0, 30, and 60 minutes. The individual dot plots of stack bar graphs are shown in Supplementary Fig. 2. mean±SEM. Blue title: MN− microglia, n=10 cells, Red title: MN+ microglia, 10 each microglia obtained from 3 individual mice (mice identification number; Mo2, Mo3, and Mo4).
Extended Data Fig. 8 cGAS is involved in morphological changes of microglia.
(a) Immunostaining of Iba1+ microglia in the cerebral cortex of either WT or cGAS−/− mice at P6. (b)-(d) The graphs show the analyses of microglial state. 2 brains. The numbers in the graph represent the numbers of cells counted. (b) The number of processes, (c) process length, (d) expression level of CD68. mean ± SEM, p values were analyzed by two-tailed Student's t-test. (e) Immunostaining of CD68 in Iba1+ microglia of either WT or cGAS−/− mice at P6. (f) The graph show the expression level of CD68. The numbers in the graph represent the numbers of cells counted. mean ± SEM, 2 brains. p values were analyzed by one-way ANOVA Tukey's multiple comparisons test. (g) Immunostaining of CD68 in Iba1+ microglia of either WT or cGAS−/− mice, MN−; micronuclei-negative, MN+; micronuclei-positive. P14. (h) The graph show the expression level of CD68. The numbers in the graph represent the numbers of cells counted. mean ± SEM, 2 brains. p values were analyzed by one-way ANOVA Tukey's multiple comparisons test.
Extended Data Fig. 9 Effect of micronuclei on gene expression in microglia.
(a) Representative gating strategy for microglia in NexCre:LSL-Sun1GFP mice. (b) The GO enrichment analysis of DEG in GFPHigh MN+ microglia (1,663 genes). Data was analyzed by Enrichr analysis tool. (c) The scatter plot indicates the ratio and differences of FPKM between GFPNeg MN− microglia and GFPHigh MN+ microglia. FPKM: GFPHigh/GFPNeg>5, GFPHigh-GFPNeg>200, (d) The Heat map indicates the comparison of FPKM among GFPNeg MN− microglia, GFPLow MN+ microglia, and GFPHigh MN+ microglia. FPKM: GFPHigh/GFPNeg>5, GFPHIgh-GFPNeg>200 (e)-(r) The graph shows the expression of each gene obtained from bulk-RNAseq analysis in GFPHigh MN+ microglia [ASC; astrocyte, NEUR; neuron (red), OPC; oligodendrocyte precursor cell, iOLG; immature oligodendrocyte, mOLG; myelinating oligodendrocyte, MG/MAC; microglia/macrophage (blue), EC; Endothelial cell, All others except NEUR and MG/MAC are shown in the black bar. mean, n=2]. This data was obtained from the Brain RNA-Seq database (https://www.brainrnaseq.org/).
Extended Data Fig. 10 Changing microglia-related genes by micronuclei transfer.
(a)-(k) The bar graphs show the value of FPKM obtained from bulk-RNAseq analysis in GFPLow MN+ microglia (P8, 3 mice; P9, 3 mice; P10, 3 mice). Top 11 genes out of 591 genes (Fig. 5c center and Fig. 5f) are represented, FPKM: GFPLow/ GFPNeg>2, GFPLow-GFPNeg>50. n=9, mean ± SEM, p values were analyzed by one-way ANOVA Tukey's multiple comparisons test. (l)-(v) The graph shows the expression of each gene obtained from bulk-RNAseq analysis in GFPHigh MN+ microglia. (ASC; astrocyte, NEUR; neuron (red), OPC; oligodendrocyte precursor cell, iOLG; immature oligodendrocyte, mOLG; myelinating oligodendrocyte, MG/MAC; microglia/macrophage (blue), EC; Endothelial cell, All others except NEUR and MG/MAC are shown in the black bar. mean, n=2). This data was obtained from the Brain RNA-Seq database (https://www.brainrnaseq.org/).
Supplementary information
Supplementary Information (download PDF )
Supplementary Figs. 1–3.
Supplementary Table 1 (download XLSX )
Identification of components of the extracellular micronucleus using LC/MS.
Supplementary Table 2 (download XLSX )
Genes with expression variations in microglia induced by neuronal MN.
Supplementary Table 3 (download XLSX )
Primer sequence for quantitative RT–PCR.
Supplementary Video 1 (download AVI )
Cortical slice culture imaging using Cx3cr1-GFP/H2B-mCherry mice brain.
Supplementary Video 2 (download AVI )
In vivo two-photon imaging using Cx3cr1-GFP/H2B-mCherry mice brain.
Supplementary Video 3 (download AVI )
In vivo two-photon imaging using Cx3cr1-GFP/H2B-mCherry mice brain.
Supplementary Video 4 (download AVI )
In vivo two-photon imaging using Cx3cr1-GFP/H2B-mCherry mice brain.
Supplementary Video 5 (download AVI )
In vivo two-photon imaging using Cx3cr1-GFP/H2B-mCherry mice brain.
Supplementary Video 6 (download AVI )
In vivo two-photon imaging using Cx3cr1-GFP/H2B-mCherry mice brain.
Supplementary Video 7 (download AVI )
In vivo two-photon imaging using Cx3cr1-GFP/H2B-mCherry mice brain.
Supplementary Video 8 (download AVI )
In vivo two-photon imaging using Cx3cr1-GFP/H2B-mCherry mice brain.
Supplementary Video 9 (download AVI )
in vivo two-photon imaging using Cx3cr1-GFP/H2B-mCherry mice brain.
Supplementary Video 10 (download AVI )
In vivo two-photon imaging using Cx3cr1-GFP/H2B-mCherry mice brain.
Supplementary Software (download ZIP )
The package containing the code of CAMDi software.
Source data
Source Data Figs. 1–5 (download XLSX )
Statistical Source Data for main Figs. 1–5.
Source Data Extended Data Figs. 1–10 (download XLSX )
Statistical Source Data for Extended Data Figs. 1–10.
Source Data Fig.2 and Extended Data Fig.2 (download PDF )
Uncropped blot data for Fig. 2 and Extended Data Fig. 2.
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Yano, S., Asami, N., Kishi, Y. et al. Propagation of neuronal micronuclei regulates microglial characteristics. Nat Neurosci 28, 487–498 (2025). https://doi.org/10.1038/s41593-024-01863-5
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DOI: https://doi.org/10.1038/s41593-024-01863-5
