Central nervous system regeneration is driven by microglia necroptosis and repopulation

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Abstract

Failed regeneration of CNS myelin contributes to clinical decline in neuroinflammatory and neurodegenerative diseases, for which there is an unmet therapeutic need. Here we reveal that efficient remyelination requires death of proinflammatory microglia followed by repopulation to a pro-regenerative state. We propose that impaired microglia death and/or repopulation may underpin dysregulated microglia activation in neurological diseases, and we reveal therapeutic targets to promote white matter regeneration.

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Fig. 1: Microglia death occurs during transition in activation following in vivo demyelination.
Fig. 2: Proinflammatory microglia undergo necroptosis before onset of remyelination.
Fig. 3: Microglia repopulation is associated with remyelination in mouse and human white matter and depends on type-1 IFN signaling.

Data availability

The data that support the findings of this study are available from the corresponding author on request. The RNA sequencing data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through the series GSE118450; accession numbers are GSM3330371 (3-dpl microglia sample 1), GSM3330372 (3-dpl microglia sample 2), GSM3330373 (3-dpl microglia sample 3), GSM3330374 (10-dpl microglia sample 4), GSM3330375 (10-dpl microglia sample 5) and GSM3330376 (10-dpl microglia sample 6). Raw data (FPKM) are shown in Fig. 1d,f and Supplementary Figs. 1b,d,e,g,h,k,m and 10a,b. Processed data are shown in Fig. 1, Supplementary Figs. 1 and 2 and Supplementary Tables 13.

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Acknowledgements

This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC)-Collaborative Award in Science and Engineering studentship in collaboration with GlaxoSmithKline (V.E.M., J.C.R.; grant no. BB/M502777/1), a Medical Research Council and United Kingdom Multiple Sclerosis Society Career Development Award (V.E.M.; grant no. MR/M020827/1), funds from the Medical Research Council Centre for Reproductive Health (grant no. MR/N02256/1) and the Wellcome Trust (J.W.P.; grant no. 101067/Z/13/Z), and a Momentum Award from the United Kingdom Dementia Research Institute (J.P.). The cuprizone studies were supported by the German Research Foundation (T.K.; grant no. SFB-TR128-B7). The Cx3cr1-CreER experiments were supported by the German Research Foundation (J.P.; grant no. SFB-TR167). The LNC studies were supported by grants from F.R.S.-FNRS (A.d.R. and Y.L.), the Fondation Charcot Stichting and the International Foundation for Research in Paraplegia (A.d.R.). We thank the United Kingdom Multiple Sclerosis Society Tissue Bank for providing tissue, F. Roncaroli for neuropathological diagnosis of multiple sclerosis lesions and R. Nicholas for providing clinical history of patients with multiple sclerosis. We also thank I. Molina-Gonzalez, M. Tzioras, N. Fullerton, C. Watkins, C. Böttcher and J. Jamal El-Din for technical support, and O. Dando for helpful discussions.

Author information

A.F.L. co-designed the study, carried out the experiments, analyzed and interpreted the data, and wrote the manuscript. C.L.D. carried out lesioning experiments, optimized lesion isolation protocols, assisted with flow cytometry, and performed experiments and analysis for RNA sequencing. R.K.H. assisted in lesioning experiments and optimized human tissue staining and analysis protocols. Y.L., D.C. and A.d.R. developed and tested LNCs for microglia targeting. G.I. assisted with genotyping. A.D. and D.S. developed remyelination index quantification protocols. E.B. and A.W. provided corpus callosum lesion tissue. J.C.R. provided guidance for experimental design. A.W. and J.W.P. co-supervised the project and assisted with experimental design and interpretation and manuscript editing. T.K. provided cuprizone tissue and edited the manuscript. A.W. provided human tissue neuropathological mapping. J.P. assisted in experimental design, data interpretation and manuscript editing. V.E.M. co-designed the study, supervised the project and guided experimental design, data interpretation and manuscript preparation.

Correspondence to Veronique E. Miron.

Ethics declarations

Competing interests

A.F.L.’s salary and experiments for this study were co-funded by GlaxoSmithKline. J.C.R. was a full-time employee at GlaxoSmithKline at the time of the study.

Additional information

Journal peer review information: Nature Neuroscience thanks Oleg Butovsky and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Integrated supplementary information

Supplementary Figure 1 RNA sequencing of microglia during in vivo remyelination.

a. FACS-based isolation of microglia from corpus callosum demyelinated lesions at 3 and 10 dpl, by gating on live cells, CD11b+ cells (PeCy7), excluding Ly6G+ (PerCP) and CD3+ cells (APC), and selecting CD45lo cells (BV605). b. Mean expression (FPKM) ± s.e.m. at 3 and 10 dpl of microglia signature genes. No significance detected (2-tailed paired t-test, P = 0.8942, t = 0.1353, df = 15), all Log2 Fold Changes <2 and/or non-significant. N = 3 mice per time point. c. Mean gene expression of Csf1r and P2ry12 in microglia from non-lesioned white matter and lesions at 3 and 10 days post LPC (dpl) as indicated by 2-ΔCt ± s.e.m. **P = 0.0036 non-lesion vs 10 dpl (2-tailed paired Student’s t-test, t = 16.73, df=2, N = 3 mice for 3 dpl, 3 mice for 10 dpl, and 2 mice for non-lesion). d. CD45lo cells lack expression (mean FPKM ± s.e.m.) of genes for which border-associated macrophages (BAMs) or bone marrow derived cells (BMdCs) are positive (ND = not detected or average FPKM <0.05), and express genes (Siglech, Sall1) for which these cells are negative. e & f. Expression of genes associated with microglia driving developmental myelination, as shown by mean FPKM ± s.e.m. (e; *P = 0.0168, 2-tailed paired Student’s t-test, t = 2.635, df=18) and significantly upregulated with a Log2 Fold Change ≥2 (f; Gpnmb, P = 0.04502; Fabp5, P = 0.002945; Atp6v0d2, P = 0.03932). N = 3 mice per time point. g. Mean expression (FPKM) ± s.e.m. of genes associated with damage-associated microglia (DAM) signature. ***P = 0.0006 (2-tailed paired Student’s t-test, t = 4.237, df = 17). No significant Log2 Fold Changes >2. N = 3 mice per time point. h & i. Expression of genes associated with neurodegenerative microglia (MGnD) signature, as shown by mean FPKM ± s.e.m. (h; *P = 0.0117, 2-tailed paired Student’s t-test, t = 3.076, df = 10) and significant Log2 Fold Change >2 (i; Gpnmb, P = 0.04502; Fabp5, P = 0.002945). N = 3 mice per time point. j. Volcano plot of mean Log2 Fold Change (3 dpl over 10 dpl) versus –Log10 (p value) with significantly upregulated genes of interest indicated in magenta for 3 dpl, and green for 10 dpl. N = 3 mice/time point. Significance was determined by empirical Bayesian analysis. k. Transcriptional regulators with anti-inflammatory function, represented as mean FPKM ± s.e.m. *P = 0.0186 (2-tailed paired Student’s t-test, t = 4.661, df = 3). Log2 Fold Changes indicated in Supplementary Table 1. N = 3 mice per time point. l. Significantly upregulated genes in 10 dpl microglia associated with a white matter-supportive signature with a Log2 Fold Change (3 dpl over 10 dpl) ≥2. P values for the following genes are 0.04618 (Matn2), 0.003924 (Osm), 0.02387 (Fgf1), 0.01856 (Cd300lf), 0.006616 (Bmp1), 0.003652 (Cd69), 0.002945 (Fabp5), 0.03804 (Cp), 0.02563 (Nit1). N = 3 mice per time point. Significance was determined by empirical Bayesian analysis. m. Transcriptional regulators with cell death-associated function, represented as mean FPKM ± s.e.m. ****P < 0.0001 (2-tailed paired Student’s t-test, t = 8.858, df = 15). Log2 Fold Changes indicated in Supplementary Table 1. N = 3 mice per time point.

Supplementary Figure 2 Gene Ontology term enrichment in microglia during remyelination in vivo.

Genes that mapped significantly at p < 0.01 were assessed for Gene Ontology (GO) term enrichment. GO terms are on the Y axis and enrichment score on the X axis represented as –Log10(p) enrichment (p < 0.05), with top 100 terms displayed for each time point. Individual GO terms were assessed for significant enrichment by assessing whether the number of significantly differentially expressed genes within a term is more than expected by chance given the total number of genes. The P value was determined using a hypergeometric test and this was corrected for tests over multiple terms using the Benjamini and Hochberg method to yield an adjusted P value. a. GO terms enriched at 3 dpl, including those relating to chronic inflammation (black arrow) and phagocytosis/ breakdown of myelin debris (magenta arrows). b. GO terms enriched at 10 dpl, including those relating to anti-inflammatory function (black arrow) and phagocytosis/ breakdown of myelin debris (green arrows).

Supplementary Figure 3 Controls for microglia death following demyelination in vivo.

a. Gating strategy for all living and dying cells excluding debris (labelled ‘cells’). Myeloid cells were detected by CD11b expression (PerCP). Detection of microglia was based on low expression of CD45 (BV605). Dying cells were detected by 7-AAD and Annexin-V (FITC). b. Flow cytometry plot of sham-lesioned mouse (taken at 7 days post-injection) showing microglia populations detected in injection site that are negative for cell death markers Annexin-V and 7-AAD. This experiment was performed with 3 mice. c. Mean number of Annexin-V+ 7-AAD+ CD11b+ CD45lo cells isolated from in vivo lesions at 3, 7, and 10 dpl ± s.e.m. *P = 0.0219, 3 vs 7 dpl (Kruskal-Wallis test). N = 3 mice per time point.

Supplementary Figure 4 Microglia death occurs during transition in activation following demyelination of ex vivo brain explants.

a. Explants were cultured for 21 days in vitro (DIV) then demyelinated with lysophosphatidyl choline (LPC) and fixed at various days post-LPC (dpl) (arrows) during remyelination. b. Demyelinated explants immunostained for iNOS (green), Arg-1 (magenta), and CD68 (blue) and counterstained with Hoechst (turquoise), at 0.5, 1 and 7 days dpl. Scale bar, 10 µM. This experiment was performed with 3 litters. c. Mean numbers of microglia (CD68+, iNOS+CD68+, Arg-1+ CD68+) from 0 – 14 dpl ± s.e.m. N = 3 litters. Quantification is from images were taken at 40X magnification. d. Demyelinated explants immunostained for PU.1 (green) and counterstained with Hoechst (blue) at 6 and 24 hours post-LPC (hpl). Scale bar, 50 µM. This experiment was performed with 3 litters. e. Mean numbers of PU.1+ cells in explants from 0 – 24 hpl ± s.e.m. N = 3 litters. P = 0.0115, 24 hpl v 6 hpl (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test). f. Mean numbers of IBA-1+ cells in explants from 0 – 18 hpl ± s.e.m. N = 3 litters. *P = 0.0279, 18 hpl v 6 hpl (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test). g. Explants treated with PBS control show intact myelin (MBP) and axons (NF-H) at 22 and 35 DIV. Scale bar, 20 µm. This experiment was performed with 3 explants. h. Explants treated with PBS control show Arg-1+ (red) CD68+ (white) cells that are iNOS negative (green) at 22 and 35 DIV. Scale bar, 10 µm. This experiment was performed with 3 explants. i. Fully myelinated explant at 21 DIV stained for MBP (red) and NF-H (green). Scale bar, 10 µM. This experiment was performed with 3 explants. j. Explants untreated (0 hours post-LPC (hpl)) and demyelinated (12 hpl) live-labelled for propidium iodide (PI) (red) then immunostained for CD68 (green), double positive cells indicated (arrows). Scale bar, 10µM. k. Mean percentage of PI+ activated microglia (CD68+) from 0 – 24 hpl ± s.e.m. N = 3 litters. *P = 0.0160 24 hpl vs 0 hpl (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test). l. Untreated (0 hpl) and demyelinated (18 hpl) explants live-labelled for PI (red) then immunostained for PU.1 (green), double positive cells indicated (arrows). Scale bar, 10µM. This experiment was performed with 3 litters. m. Mean percentage of PI+ total microglia (PU.1+) from 0 – 24 hpl ± s.e.m. N = 3 litters. *P = 0.0140, 24 hpl vs 0 hpl (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test).

Supplementary Figure 5 Microglia do not undergo death via LPC, apoptosis, or pyroptosis.

a. Images of primary rat microglia treated for 18 hours with LPC, immunostained for CD68 (white), iNOS (green) and Arg-1 (magenta) and counterstained with Hoechst (turquoise). Scale bar, 20µM. This experiment was performed with 1 litter. b. Explants treated with ethanol (EtOH) as a positive control to induce apoptosis (left), or with LPC for 12 hours post LPC (hpl; right), immunostained for Cleaved Caspase-3 (green), TUNEL assay (red) and PU.1 (white). Scale bar, 10µM. This experiment was performed with 3 explants. c. Explants untreated (0 hpl) (left) or LPC-treated at 12 hpl (right) immunostained for CD68 (green) and Cleaved Caspase-1 (red). Arrows indicate Cleaved Caspase-1+ microglia (CD68+) in both untreated and demyelinated explants. Scale bar, 10µM. This experiment was performed with 3 explants. d. Mean number of CD68+ RIPK3+ cells per field at 3 and 7 days post-demyelination in vivo ± s.e.m. N = 3 mice per time point. **P = 0.0011 (2-tailed unpaired Student’s t-test, t = 8.443, df=4).

Supplementary Figure 6 The majority of RIPK3+ cells after in vivo demyelination are microglia and not monocyte-derived macrophages.

a. Schematic of Ccr2RFP/+ peripheral monocytes migrating to the CNS where they differentiate into RFP+ monocyte-derived macrophages, distinguishable from CNS-resident RFP-negative microglia. b. Images of lesioned corpus callosum in vivo at 3 and 10 days post-LPC (dpl) from Ccr2RFP/+ reporter mice showing RIPK3+ (green) cells and monocyte-derived macrophages (Ccr2-RFP+; red). Filled arrows show examples of RFP+ RIPK3+ cells, open arrows show examples of RFP-negative RIPK3+ cells. Scale bar, 20 μM. This experiment was performed with 3 mice. c. Mean proportion of RIPK3+ cells which are positive or negative for Ccr2-RFP at 3 and 10 dpl ± s.e.m. N = 3 mice per time point. d. Mean percentage of CD68 that co-localizes with Tmem119 within lesions at 3, 7, and 10 dpl ± s.e.m. N = 3 mice per time point. e. Representative image of co-localization between Tmem119 (red) and CD68 (green) within lesions. Scale bar, 50 µm. This experiment was performed with 3 mice per time point.

Supplementary Figure 7 Microglia necroptosis is associated with remyelination in two additional in vivo models of demyelination.

a. Schematic of cuprizone model of demyelination. b. Mean number of RIPK3+ CD68+ microglia (±s.e.m.) in mice treated with cuprizone at various phases of demyelination and remyelination: demyelination-only, demyelination and remyelination, remyelination only phases (1.5 wks post-return to normal diet, 4 wks post-return to normal diet) ± s.e.m. N = 3 mice per time point and condition. *P = 0.0365 for demyelination & remyelination vs demyelination only (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test). c. Images of tissue from cuprizone-treated mice at ‘demyelination-only’ and concomitant ‘demyelination with remyelination’ phases, immunostained for CD68 (red) and RIPK3 (green). Scale bar, 50µM. This experiment was performed with 3 mice from each time point. d. Representative image of tissue from cuprizone-treated mice at demyelination and remyelination phase immunostained for MLKL (red) and IBA-1 (green). Scale bar; 50µM. This experiment was performed with 3 mice from each time point. e. Data-mining of mean gene expression (represented as Fragments Per Kilobase of transcript per Million mapped reads (FKPM)) of Ripk3 and Mlkl in microglia in EAE at no disease and late disease phases, as assessed by clinical score indicating partial hindlimb paralysis. Dataset was generated from 6 mice (late disease) in original study in Lewis et al., 2014, J Neuroimmunol9.

Supplementary Figure 8 Controls for necroptosis assessment and necrostatin treatment.

a. Explant immunostained for oligodendrocyte lineage cell nuclei (Olig2+; green) and RIPK3 (red) at 6 hpl. Arrow indicates RIPK3+ Olig2+ cell. Scale bar, 50µM. This experiment was performed with 3 litters. b. Mean numbers of total Olig2+ cells and Olig2+ RIPK3+ cells in demyelinated explants at 6 hpl ± s.e.m. N = 3 litters. c. Explant immunostained for neuronal nuclei (NeuN+; green) and RIPK3 (red) at 6 hpl. Arrows indicate RIPK3+ NeuN+ cells. Scale bar, 50 µM. This experiment was performed with 1 litter. d. Number of total NeuN+ cells and NeuN+ RIPK3+ cells in demyelinated explant at 6 hpl. N = 1 litter. e. Treatment of fully myelinated explants with necrostatin-1 (NEC-1) for 1 day (22 DIV) or 14 days (35 DIV), equivalent to treatment times in demyelinated explants, showing intact myelin (MBP; green) around axons (NF-H; green). Scale bar, 10 µm. This experiment was performed with 3 explants. f. Treatment of fully myelinated explants with NEC-1 for 1 day (22 DIV) or 14 days (35 DIV), showing activated microglia (CD68+; white) positive for Arg-1 (red) but negative for iNOS (green). Scale bar, 5 µm. This experiment was performed with 3 explants. g. Image demonstrating accumulation of DiD LNCs (white) in IBA-1+ cells (red) within demyelinated lesions; CC1+ oligodendrocytes shown in green. Scale bar, 25 µm. This experiment was performed with 3 mice. h. 3D rendering of internalization of DiD-LNCs (white) within IBA-1+ cells (green); Oligodendrocyte lineage cells shown in red (CC1+ in left column, Olig1+ in right panel). Scale bar, 5 µm. This experiment was performed with 3 mice. i. Mean percentage of CD68+ cells which are RIPK3+ (± s.e.m.) in Veh LNC and NEC-1 LNC-treated lesions at 3 dpl. N = 3 mice per condition. j. Mean number of Ki67+PU.1+ cells per field (± s.e.m.) in Veh LNC and NEC-1 LNC-treated lesions at 3 and 10 dpl. **P = 0.0045, 2-tailed Student’s t-test (t = 6.502, df = 2). N = 3 mice per condition. k. In vivo lesions treated with vehicle-loaded LNCs or NEC-1 LNCs showing no accumulation of myelin debris (MBP+; red); IBA-1 in green. Scale bar, 50 µm. This experiment was performed with 3 mice per condition.

Supplementary Figure 9 Lineage tracing of microglia source during repopulation following demyelination.

a. Flow cytometry gating on CD11b+CD45lo microglia, isolated from in vivo demyelinated lesions in Nes-CreERT2;RCL-tdT mice. This experiment was performed on 3 mice per time point. b. Mean number of tdT(Nestin)+ CD11b+CD45lo cells ± s.e.m. at 3, 7, and 10 days post lysolecithin (dpl) detected in in vivo lesions. N = 3 mice per time point. c. Schematic of 4-hydroxytamoxifen (4-OHT) treatment in Nes-CreERT2;RCL-tdT explants ex vivo. d. Representative explant from Nes-CreERT2; RCL-tdT mice at 21 days in vitro (DIV) with 4-OHT treatment immunostained with Nestin (green) shows high recombination efficiency. Scale bar, 20 μM. This experiment was performed with 3 mice. e. Explants from Nes-CreERT2;RCL-tdT mice at 0 dpl, 2 dpl, and 7 dpl immunostained for IBA-1 (green). Scale bar, 20 µm. e’: magnified view of tdT+ cells in 0 dpl and 2dpl, showing an area of high tdT+ IBA-1+ cell density at 2 dpl. Scale bar, 25 µm. Arrows demonstrate tdT+ IBA-1+ cells. This experiment was performed with 4 mice per time point. f. Mean proportion of IBA-1+ cells positive and negative for tdTomato(Nestin) at 0-2 dpl ± s.e.m in explants. N = 4 mice per time point. **P = 0.00154 0 dpl vs 2 dpl (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test). g. Explants at 21 DIV show that tdT(Nestin)+ cells are positive for Musashi-1 (Msh-1; green) and Sox-2 (green), but negative for GFAP (green). Scale bar, 20 µm. This experiment was performed with 3 explants. h. Schematic of 4-OHT treatment in Cx3cr1-CreER;RCL-tdT explants. i. Representative explant from Cx3cr1-CreER; RCL-tdT mice at 21 days in vitro (DIV) with 4-OHT treatment immunostained with IBA-1 (green) shows partial recombination. Scale bar, 20 μm. j. Representative explant from Cx3cr1-CreER; RCL-tdT stained for oligodendrocyte lineage cells (Olig2+; green) shows no co-localization with tdTomato (red). Scale bar, 50 μM. This experiment was performed with 3 explants. k. Explants from Cx3cr1-CreER;RCL-tdT mice at 0 dpl and 2 dpl immunostained for IBA-1 (green) and Nestin (magenta). Scale bar, 20 µm. This experiment was performed with 4 mice per time point. l. Mean proportion of IBA-1+ cells positive and negative for tdTomato(Cx3cr1) at 0-2 dpl ± s.e.m in explants. N = 4 mice per time point. m. Mean number of IBA-1+ cells positive for tdTomato(Cx3cr1) at 0-3 dpl ± s.e.m. in explants. N = 4 mice per time point. n. Mean proportion of tdTomato(Cx3cr1)+ cells positive and negative for Nestin at 0-2 dpl ± s.e.m in explants. *P = 0.0106, 0 dpl vs 2 dpl (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test). N = 4 mice per time point. o. Mean number of tdTomato(Cx3cr1)+ cells positive for Nestin at 0-3 dpl ± s.e.m in explants. N = 4 mice per time point.

Supplementary Figure 10 Type-1 interferon signaling regulates microglia repopulation during remyelination.

a. Mean expression (FPKM) of genes (± s.e.m.) encoding Type-1 interferon receptor subunits Ifnar1 and Ifnar2 by microglia at 3 and 10 days post LPC (dpl). N = 3 mice per time point. b. Mean expression (FPKM) of Type-1 interferon-associated genes by microglia at 3 and 10 dpl (±s.e.m.). *P = 0.0178 (2-tailed paired Student’s t-test, t = 3.884, df=4). N = 3 mice per time point. c. Mean number of phospho-STAT1+ PU.1+ cells per field (± s.e.m.) at 7 dpl in demyelinated brain explants treated with goat IgG or anti-IFNAR2 IgG. *P = 0.0411 (2-tailed Student’s t-test, t = 2.972, df=4). N = 3 mice per condition.

Supplementary information

Supplementary Figures 1–10 and Supplementary Tables 2–4.

Reporting Summary

Supplementary Table 1

RNA sequencing log2 (fold changes) and significantly upregulated genes in microglia during remyelination.

Supplementary Video 1

Microglia do not undergo cell death in untreated explants: live imaging of untreated Csf1r-eGFP (MacGreen) explants shows healthy microglia (green) over 12 h.

Supplementary Video 2

Microglia undergo cell death following demyelination of explants: live imaging of demyelinating Csf1r-eGFP (MacGreen) explants shows microglia retracting processes, rounding up and rupturing cell membranes over 12 h. The video was started 20 min after addition of LPC (due to technical setup).

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Lloyd, A.F., Davies, C.L., Holloway, R.K. et al. Central nervous system regeneration is driven by microglia necroptosis and repopulation. Nat Neurosci 22, 1046–1052 (2019) doi:10.1038/s41593-019-0418-z

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