| Corresponding author(s): | Harald Lund, Robert Harris | |-----------------------------------------|----------------------------| | Last updated by author(s): Oct 24, 2025 | | # Reporting Summary Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our Editorial Policies and the Editorial Policy Checklist. | | | Statistics | |--|--|------------| | | | | | | | | | | For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section. | |-----|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| | n/a | Confirmed | | | The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement | | | A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly | | | The statistical test(s) used AND whether they are one- or two-sided
Only common tests should be described solely by name; describe more complex techniques in the Methods section. | | | A description of all covariates tested | | | A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons | | | A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient)
AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) | | | For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted
Give P values as exact values whenever suitable. | | | For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings | | | For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes | | | Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated | | | Our web collection on statistics for biologists contains articles on many of the points above. | #### Software and code Policy information about availability of computer code Data collection For TEM images, sections were analyzed using a Hitachi HT7700 transmission electron microscope (Hitachi High-Technologies) operating at 80 kV. Digital images of the sections were captured with a 2kx2k Veleta CCD camera (Olympus Soft Imaging Solutions). For fluorescent imaging, data were collected with a Zeiss LSM880 confocal microscope or Zeiss Axio Imager Z1 microscope using ZEN software. Flow cytometry data were collected using either a Beckman Coulter Gallios cytometer or an Aurora Cytek 3L/5L spectral cytometer. Sequencing data were gathered with a NovaSeq 6000 platform. The NanoString nCounter system and the Bio-Rad CFX384 Touch Real-Time PCR Detection System were used for collecting transcriptional data. Gel data for Western blotting were obtained using a Bio-Rad ChemiDoc Imaging System. Spinal cord wholemount imaging data were collected with an UltraMicroscope Blaze system. Data analysis For TEM images, axon counts and axonal perimeters for each field of view were automatically measured using the 'Analyze Particles' function in ImageJ/FIJI. The analyses for g-ratio calculations and periaxonal space measurements were performed using MyelTracer software. For fluorescent imaging, images were rendered and analyzed using either or Imaris 9.8.0. or FIJI/ImageJ. Flow cytometry data were analyzed using FlowJo v10 or Kaluza. Bulk RNA-seq data were analyzed using the BGI Dr. Tom Data Visualization Solution. snRNA-seq data were analyzed using R 4.3.2 software with the following packages: Seurat, ClusterProfiler, FastCar, DoubletFinder, scvelo and scanpy. NanoString data were analyzed using NanoString nSolver Analysis Software. qRT-PCR data were analyzed using CFX Maestro Software. Western blot bands were analyzed using Bio-Rad Image Lab software. 3D images were reconstructed using MACS iQ View (3D large volume software, Version 1.0.0) and Imaris 9.8.0. Statistical analyses were conducted using GraphPad Prism 10. The codes used for snRNA-seq analyses are available on Github: https://github.com/Kenny19920122/TGFb\_snRNAseq For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information. ### Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: - Accession codes, unique identifiers, or web links for publicly available datasets - A description of any restrictions on data availability - For clinical datasets or third party data, please ensure that the statement adheres to our policy Bulk RNA-seq data of microdissected microglia and NanoString nCounter raw data were uploaded to NCBI GEO database under the accession number GSE254915 and GSE283721, respectively. snRNA-seq data was available at BioProject with the accession number PRJNA1185825. The raw sequencing results related to the genes in the TGF-β signaling pathway of the human spinal cord were uploaded to the European Genome-phenome Archive (EGA); due to European Data Protection Regulations (GDPR) and Karolinska Institutet's data governance policy, the individual-level human sequencing data will be available upon request to the corresponding author, following institutional and ethical approval procedures. ### Research involving human participants, their data, or biological material Policy information about studies with human participants or human data. See also policy information about sex, gender (identity/presentation), and sexual orientation and race, ethnicity and racism. Reporting on sex and gender Post-mortem samples were mixed in terms of biological sex, and this detail is not relevant to the respective conclusion. Reporting on race, ethnicity, or other socially relevant groupings We did not include this information in the analysis as it is out of the scope of the section in the study. Population characteristics All donor post-mortem samples were healthy in the spinal cord, between age 17 to 77. Recruitment All post-mortem human spinal cords were collected at the Montpellier Hospital from organ-donor patients (accidental death) in strict agreement with the French bioethics laws (articles L1232-1 and -6) and after approval by the French institution for organ transplantation. An informed consent from the families was obtained by the organ procurement organization for this study. Ethics oversight Ethical approval was granted by the French Agence de la biomédecine (n° SPGED19-3-4692; April 2nd, 2019) for human spinal cord samples. Informed consent was obtained from the families by the organ procurement organization for this study. Note that full information on the approval of the study protocol must also be provided in the manuscript. # Field-specific reporting | Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. | | | | | |------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------|---------------------------------------------------|--|--| | Life sciences | Behavioural & social sciences | Ecological, evolutionary & environmental sciences | | | | | For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf | | | | # Life sciences study design All studies must disclose on these points even when the disclosure is negative. Sample size Sample size was determined based on previous similar studies (McNamara et al. 2023 Nature; Qin et al. Cell 2018; Cantuti-Castelvetri et al. Science 2018; Bosch-Queralt et al. Nature Metabolism 2021; ) and our experiences (Lund et al. Nature Immunology 2018; Lund et al. Nature Communications 2018; Zhu et al. EMBO Reports 2022; Zhu et al. Glia 2020). Normally, at least 3 samples per group were used for statistical analyses, and around 10 mice per group were used for behavioral evaluation. Data exclusions For bulk RNA-seq data, spinal cord tissue from one mouse at D28 following Tgfbr2 deletion was excluded as the sample did not pass the quality control. For snRNA-seq data, one D10 sample was excluded due to failed library construction. These exclusions were pre-determined. For qRT-PCR analyses, an unbiased outlier removal process was performed using the 'Identify Outliers' function in GraphPad Prism using the ROUT method with a Q value of 1%. Replication All attempts at replicating the phenotype of mice deficient in microglial Tgfbr2 or Tgfb1 were successful; at least 3 individual experiments were performed. Subsequent analyses, including immunostaining and flow cytometry analyses, were repeated at least twice. Bulk RNA-seq, snRNA-seq, NanoString nCounter, and TEM analyses were performed once, but the conclusions were supported by validation using other experimental methods. Randomization Samples were randomly allocated into different groups. Blinding Blinding was employed for behavioral assessment, clinical scoring, snRNA-seq analysis, TEM analysis, image analysis, and gel analysis. # Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. | Materials & experimental systems | Methods | |----------------------------------|--------------------------------------------------------------------------------------------------------------------------| | n/a
Involved in the study | n/a
Involved in the study | | Antibodies | ChIP-seq | | Eukaryotic cell lines | Flow cytometry | | Palaeontology and archaeology | MRI-based neuroimaging | | Animals and other organisms | | | | | | Clinical data | | | Dual use research of concern | | | Plants | | | | | | Antibodies | | | Antibodies used | 1. rabbit-anti-IBA1 (1:500, 019-19741, FUJIFILM WAKO) | | | 2. goat-anti-IBA1 (1:400, ab5076, Abcam) | | | 3. rat-anti-MHC-II (1:200, 14-5321-82, Thermo) | | | 4. rat-anti-F4/80 (1:200, MCA497GA, Bio-Rad)
5. rabbit-anti-GPNMB (1:500, ab188222, Abcam) | | | 6. rat-anti-Galectin-3 (1:200, 14-5301-82, Thermo) | | | 7. chicken-anti-PLP1 (1:200, NB100-1608, Novus Biologicals) | | | 8. mouse-anti-Tuj1 (1:100, 801213, BioLegend) | | | 9. rat-anti-MBP (1:500, MCA409S, Bio-Rad),
10. R667-conjugated-anti-MBP (1:200, 130-131-152, Miltenyi Biotec), | | | 11. rabbit-anti-dMBP (1:1000, AB5864, Millipore Sigma), | | | 12. rabbit-anti-PLIN2 (1:200, 15294-1-AP, Proteintech), | | | 13. mouse-anti-CC-1 (1:200, OP80, Millipore Sigma), | | | 14. goat-anti-Serpina3n (1:200, AF4709, R&D Systems)
15. Fluoromyelin (1:300, F34651, Invitrogen) | | | 16. LipidSpot (1:1000, 70069, Biotium) | | | 17. anti-TGFβR2 (1:1000, SAB5700739, Sigma) | | | 18. anti-TGFB1 (1:1000, MA121595, Thermo) | | | 19. anti-SMAD2/3 (1:1000, 8685T, CST) | | | 20. anti-phospho SMAD2 (1:1000, AB3849-I, MERCK) | | | 21. CD11b-BV786 (clone M1/70, 417-0112-82, Thermo Fisher Scientific)
22. CD45-AF488 (clone 30-F11, 103122, Biolegend) | | | 23. CX3CR1-AF700 (clone SA011F11, 149036, Biolegend) | | | 24. GPNMB-eFluor660 (clone CTSREVL, 50-5708-82, Thermo Fisher Scientific) | | | 25. Galectin-3-PE/Cy7 (clone M3/38, 125418, Biolegend) | | | 26. Dectin-1-PerCP-eFluor710 (clone bg1fpj, 46-5859-82, Biolegend)
27. CD74-BUV737 (clone In-1, 741798, BD) | | | 28. LAP/TGFβ1-BV421 (clone TW7-16B4, 141407, Biolegend) | | | 29. CD140a-BV480 (clone APA5, 746581, BD) | | | 30. O4-APC (clone REA576, 130-119-897, Miltenyi) | | | 31. GLAST-PE (clone ACSA-1, 130-118-344, Miltenyi) | | | 32. CD11b-PerCP/Cy5.5 (clone M1/70, 101228, Biolegend)
33. CD45-PE/Cy7 (clone 30-F11, 103114, Biolegend) | | | 34. Ly6C-PE (clone HK1.4, 128008, BioLegend) | | | 35. F4/80-APC (clone BM8, 123116, BioLegend) | | | 36. MHC-II-AF700 (clone M5/114.15.2, 107622, BioLegend) | | | 37. Ly6C-AF700 (clone HK1.4, 128024, Biolegend) | | | 38. Ly6G-V450 (clone 1A8, 560603, BD)
39. MHC-II-PE-Dazzle-594 (clone M5/114.15.2, 107648, Biolegend) | | | 40. MSR1-AF647 (clone 1F8C33, 154719, Biolegend) | | | 41. Galectin-3-PE (clone M3/38, 125405, Biolegend) | | | 42. F4/80-BV421 (clone BM8, 123137, Biolegend) | | | | Validation 1. https://labchem-wako.fujifilm.com/us/product/detail/W01W0101-1974.html Species reactivity: human, mouse, rat Application: ICC, IHC (Frozen) Clonality: Polyclonal Reference: PMIDs-32059778, 35593064 2. https://www.abcam.com/en-us/products/primary-antibodies/iba1-antibody-ab5076 Species reactivity: human, rat, mouse (published reference) Application: IHC-P, WB Clonality: Polyclonal References: PMID 38480892 3. https://www.thermofisher.com/antibody/product/MHC-Class-II-I-A-I-E-Antibody-clone-M5-114-15-2-Monoclonal/14-5321-82 Species reactivity: human, mouse Application: Flow, ICC/IF, IHC Clonality: monoclonal antibody (M5/114.15.2) References: PMIDs-37563136, 36067217 4. https://www.bio-rad-antibodies.com/monoclonal/mouse-f4-80-antibody-cl-a3-1-mca497.html?f=purified Species reactivity: mouse Application: Flow, IF/IHC/WB Clonality: monoclonal antibody (Cl:A3-1) References:PMIDs-15883173, 15085191 5. https://www.abcam.com/en-us/products/primary-antibodies/gpnmb-antibody-epr18226-147-ab188222 Species reactivity: mouse Application: IP, WB, ICC/IF, Flow Clonality: monoclonal antibody (EPR18226-147) References: PMID-37138705 6. https://www.thermofisher.com/antibody/product/Galectin-3-Antibody-clone-eBioM3-38-M3-38-Monoclonal/14-5301-82 Species reactivity: human, mouse, rat Application: WB, IHC, ICC/IF, Flow, Elisa, IP Clonality: monoclonal antibody (eBioM3/38 (M3/38)) References: PMIDs-35977488, 33507881 7. https://www.novusbio.com/products/myelin-plp-antibody\_nb100-1608? srsltid=AfmBOoqYHe0a1OyeXUcW55K3CFBVlYq3PxWvp70pvtgIhj89f5pE4XyR Species reactivity: human, mouse, rat Applications: WB, ICC/IF, IHC Clonality: polyclonal References: PMIDs-24057669, 28282586 8. https://www.biolegend.com/de-de/products/purified-anti-tubulin-beta-3-tubb3-antibody-11580?GroupID=GROUP686 Species reactivity: human, mouse, rat Applications: IHC-P, WB, ICC, flow Clonality: monoclonal antibody (TUJ1) References: PMIDs-28281529, 17099708 9. https://www.bio-rad-antibodies.com/monoclonal/cow-bovine-mbp-antibody-12-mca409.html?f=s%2Fn Species reactivity: mouse, rabbit, rat, guinea pig, sheep, human, chicken, pig Applications: IF, ELISA, WB Clonality: monoclonal antibody (12) References: PMIDs-39023138, 37291151 10. https://www.miltenyibiotec.com/US-en/products/mbp-antibody-anti-human-mouse-reafinity-rea1154.html#conjugate=vio- r667:size=600-ul Species reactivity: mouse, human, rat Applications: 3D-IF, IF, IHC Clonality: monoclonal antibody (REA1154) References: PMIDs-8544862 11. https://www.sigmaaldrich.com/SE/en/product/mm/ab5864? utm\_source=google&utm\_medium=cpc&utm\_campaign=8890193254&utm\_content=87903050565&gclid=CjwKCAiAudG5BhAREiwA WMlSjHJx4I85p5QDv5K8QtzfRnpQ6mcRH-mTDm3YPOL-RPz\_eCaGF4lYIhoC3PIQAvD\_BwE Species reactivity: human, rat, mouse (published references) Applications: IHC Clonality: polyclonal antibody References: PMIDs-32127495, 36966295 12. https://www.ptglab.com/products/ADRP-Antibody-15294-1-AP.htm? srsltid=AfmBOoo9thP0L6ArOnG5Po5bI7U11xC149V8b05lwHYyBBYyVtmv5zql Species reactivity: human, mouse, rat Applications: WB, IHC, IF/ICC, ELISA Clonality: polyclonal antibody References: PMIDs-37923718, 35614132 13. https://www.emdmillipore.com/US/en/product/Anti-APC-Ab-7-Mouse-mAb-CC-1,EMD\_BIO-OP80 Species reactivity: human, mouse, rat Applications: IF, IHC Clonality: monoclonal antibody (CC-1) References: PMIDs-18371179, 17553990 14. https://www.rndsystems.com/products/mouse-serpin-a3n-antibody\_af4709 Species reactivity: mouse Applications: WB, IP, IF/IHC (published references) Clonality: polyclonal antibody References: PMIDs-32183784, 36517604, 36658241, 36952346 15. https://www.thermofisher.com/order/catalog/product/F34651 Species reactivity: NA Applications: IF Clonality: NA References: PMIDs-36229601, 34555490 16. https://biotium.com/product/lipidspot-488-lipid-droplet-stain-1000x/ Species reactivity: NA Applications: IF Clonality: NA References: PMIDs-38480892, 38243233 17. https://www.sigmaaldrich.com/SE/en/product/sigma/sab5700739? srsltid=AfmBOooE7MsSXEpqPT9vKVqx6h6N-2gZl8nJCHIFeR4gcmZ6WTFCKJT4 Species reactivity: rat, human, mouse Clonality: polyclonal antibody Applications: IF, IHC, WB References: NA 18. https://www.thermofisher.com/antibody/product/TGF-beta-1-Antibody-clone-TB21-Monoclonal/MA1-21595 Species reactivity: bovine, human. mouse, sheep, rabbit, rat Applications: WB, IHC, ICC/IF, Flow Clonality: monoclonal antibody (TB21) References: PMID-35197446 19. https://www.cellsignal.com/products/primary-antibodies/smad2-3-d7g7-xp-rabbit-mab/8685? srsltid=AfmBOop\_nkssE9evwvNHdgYZUb3yJCyM1czRNsIUek3G7ATT\_3CQSoKg Species reactivity: human, mouse, rat, monkey Applications: WB, IP, IF, Flow, ChIP Clonality: monoclonal antibody (D7G7) References: PMIDs-12040180, 9393997 20. https://www.emdmillipore.com/US/en/product/Anti-phospho-SMAD2-Antibody-Ser465-467,MM\_NF-AB3849-I? ReferrerURL=https%3A%2F%2Fwww.google.com%2F Species reactivity: human, mouse (published references) Applications: WB Clonality: monoclonal antibody (Ser465/467) References: PMIDs-33494271, 35285550, 33619376 21. https://www.thermofisher.com/antibody/product/CD11b-Antibody-clone-M1-70-Monoclonal/417-0112-82 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (M1/70) 22. https://www.biolegend.com/fr-ch/products/alexa-fluor-488-anti-mouse-cd45-antibody-3100?GroupID=BLG1932 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (30-F11) 23. https://www.biolegend.com/fr-ch/products/alexa-fluor-700-anti-mouse-cx3cr1-antibody-13474 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (SA011F11) 24. https://www.thermofisher.com/antibody/product/GPNMB-Antibody-clone-CTSREVL-Monoclonal/50-5708-82 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (CTSREVL) 25. https://www.biolegend.com/ja-jp/products/pe-cyanine7-anti-mousehuman-mac-2-galectin-3-antibody-15096 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (M3/38) 26. https://www.thermofisher.com/antibody/product/CD369-Clec7a-Dectin-1-Antibody-clone-bg1fpj-Monoclonal/46-5859-82 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (bg1fpj) 27. https://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies- ruo/buv737-rat-anti-mouse-cd74.741798 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (In-1) 28. https://www.biolegend.com/fr-ch/products/brilliant-violet-421-anti-mouse-lap-tgf-beta1-antibody-8180 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (TW7-16B4) 29. https://www.bdbiosciences.com/en-fr/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodiesruo/bv480-rat-anti-mouse-cd140a.746581 Species reactivity: mouse Apllication: flow Clonality: monoclonal antibody (APA5) 30. https://www.miltenyibiotec.com/US-en/products/o4-antibody-anti-human-mouse-rat-reafinity- rea576.html#conjugate=apc:size=100-tests-in-200-ul Species reactivity: mouse Application: flow Clonality: monoclonal antibody (REA576) 31. https://www.miltenyibiotec.com/US-en/products/glast-acsa-1-antibody-anti-human-mouse-rat- acsa-1.html#Conjugate=PE:size=100-tests-in-200-uL Species reactivity: human, mouse, rat Application: flow, IF, IHC Clonality: monoclonal antibody (ACSA-1) 32. https://www.biolegend.com/fr-ch/products/percp-cyanine5-5-anti-mouse-human-cd11b-antibody-4257 Species reactivity: mouse, human, cynomolgus, rhesus Application: flow Clonality: monoclonal antibody (M1/70) 33. https://www.biolegend.com/fr-ch/products/pe-cyanine7-anti-mouse-cd45-antibody-1903 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (30-F11) 34. https://www.biolegend.com/fr-ch/products/pe-anti-mouse-ly-6c-antibody-4904 Species reactivity: mouse Application: mouse Clonality: monoclonal antibody (HK1.4) 35. https://www.biolegend.com/fr-ch/products/apc-anti-mouse-f4-80-antibody-4071 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (BM8) 36. https://www.biolegend.com/fr-ch/products/alexa-fluor-700-anti-mouse-i-a-i-e-antibody-3413 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (M5/114.15.2) 37. https://www.biolegend.com/fr-ch/products/alexa-fluor-700-anti-mouse-ly-6c-antibody-6757 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (HK1.4) 38. https://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies- ruo/v450-rat-anti-mouse-ly-6g.560603 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (1A8) 39. https://www.biolegend.com/en-gb/explore-new-products/pe-dazzle-594-anti-mouse-i-a-i-e-antibody-12088?GroupID=BLG11931 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (M5/114.15.2) 40. https://www.biolegend.com/en-gb/products/alexa-fluor-647-anti-mouse-cd204-antibody-20748 Species reactivity: mouse Application: flow Clonality: monoclonal antibody (1F8C33) 41. https://www.biolegend.com/en-gb/products/pe-anti-mouse-human-mac-2-galectin-3-antibody-4937 Species reactivity: mouse, human | Application: flow | | | |-------------------|--------------------------------------------------------------------------------------------------|--| | | Clonality: monoclonal antibody (M3/38) | | | | | | | | 42. https://www.biolegend.com/en-gb/products/brilliant-violet-421-anti-mouse-f4-80-antibody-7199 | | | | Species reactivity: mouse | | | Application: flow | | | | | Clonality: monoclonal antibody (BM8) | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | ### Palaeontology and Archaeology | Specimen provenance | NA | | | |------------------------------------------------------------------------------------------------------------------------|----|--|--| | Specimen deposition | NA | | | | Dating methods | NA | | | | Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information. | | | | | Ethics oversight | NA | | | | | | | | Note that full information on the approval of the study protocol must also be provided in the manuscript. # Animals and other research organisms Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in Research Laboratory animals Cx3cr1CreER-YFP (#021160) and Tgfb1 fl/fl (#065809) mice were obtained from the Jackson Laboratory. Tgfbr2fl/fl mice were kindly offered by M. Li at Sloan Kettering Institute. C57BL/6NTac mice (Taconic) were bred at the local animal facility. Most experiments used female mice of different ages to investigate age-related effects, while both males and females were included to compare disease progression and to analyze the spinal cord transcriptome using NanoString nCounter. For TEM analyses, female mice aged 2– 3 months, 6–8 months, and 16–18 months were used to examine age-related alterations in myelin ultrastructure. For NanoString nCounter analyses, we included both female and male mice aged 2–3 months or 12–13 months to compare age-related molecular changes in the spinal cord, with the presented data obtained from female mice. For snRNA-seq, female mice were around 12 months. To investigate age-related upregulation of TGF-β signaling in Fig. 2, young mice were 2–3 months old, while older mice were approximately 12 months or older. Microglia isolated from microdissected spinal cord regions for bulk RNA-seq analysis and flow cytometry were obtained from adult mice aged 4-5 months. Immunostaining and other flow cytometry results in Figs. 3-7 were obtained from Cx3cr1CreER:Tgfbr2fl/fl or Cx3cr1CreER:Tgfb1fl/fl mice aged 8-12 months. Detailed age information is provided in the figure legends. Wild animals This study did not involve wild animals. Reporting on sex Sex was accounted for in the study, with both female and male mice included in the study. A more pronounced phenotype was observed in females upon deletion of Tgfbr2 in microglia, and therefore, females were used for most of the experiments. Field-collected samples This study did not involve samples collected from field. Ethics oversight Animal experiments were performed under the guidelines from the European Community Council Directive and the Swedish National Board for Laboratory Animals (86/609/EEC) under the ethical permits N138/14, followed by 9328-2019, and 23561-2022, approved by the local ethical review board the North Stockholm Animal Ethics Committee. Note that full information on the approval of the study protocol must also be provided in the manuscript. | | Plants | |--|--------| | Seed stocks | NA | |-----------------------|----| | Novel plant genotypes | NA | | Authentication | NA | ## Flow Cytometry #### Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC). The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers). All plots are contour plots with outliers or pseudocolor plots. A numerical value for number of cells or percentage (with statistics) is provided. #### Methodology Sample preparation Mouse spinal cord tissues were physically dissociated followed by enzymatic digestion with papain (1:100 diluted in L15 medium; LS003126, Worthington) and 0.2 mg/mL DNase I (10104159001, Roche). Myelin was removed using 38% isotonic Percoll (P1644, Sigma) by centrifugating at 800 g (4x acceleration, no brake) for 10 min. Cell pellets were suspended for flow cytometry or cell sorting. Instrument Cytek Aurora (3 or 5 lasers) or Beckman Coulter Gallios Flow Cytometer Software Flowjo V10 or Kaluza. Cell population abundance From the whole spinal cord tissue, approximately 5,000 microglial cells were obtained. In the case of micro-dissected spinal cord subregions (dorsal column (DC), ventral column (VC), and gray matter (GM)), about 800 cells were collected from the microglial population. Gating strategy FSC/SSC parameters were used to roughly separate the cell population from cell debris and myelin debris. FSC-A and FSC-H parameters were employed to gate singlets. Dead cells were excluded using LIVE/DEAD Fixable Stain Kits. Microglia were identified and gated based on their expression levels: CD11b-high, CD45-intermediate, Cx3cr1-high, Ly6C-low, and Ly6G-low. Oligodendrocyte lineage cells were CD45-negative, O4-high; astrocytes were CD45-negative, GLAST-high. The boundaries between positive and negative staining cell populations are defined by comparing with the wildtype mice. Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.