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Oligodendrocyte death initiates synchronous remyelination to restore cortical myelin patterns in mice

Abstract

Myelin degeneration occurs in neurodegenerative diseases and aging. In these conditions, resident oligodendrocyte progenitor cells (OPCs) differentiate into oligodendrocytes that carry out myelin repair. To investigate the cellular dynamics underlying these events, we developed a noninflammatory demyelination model that combines intravital two-photon imaging with a single-cell ablation technique called two-photon apoptotic targeted ablation (2Phatal). Oligodendrocyte 2Phatal in both sexes results in a myelin degeneration cascade that triggers rapid forms of synchronous remyelination on defined axons. This remyelination is driven by oligodendrocytes differentiated from a subset of morphologically distinct, highly branched OPCs. Moreover, remyelination efficiency depends on the initial myelin patterns, as well as the age of the organism. In summary, using 2Phatal, we show a form of rapid synchronous remyelination, mediated by a distinct subset of OPCs, capable of restoring the original myelin patterning in adulthood but not aging.

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Fig. 1: On-demand focal cortical demyelination with oligodendrocyte 2Phatal.
Fig. 2: Myelin sheath degeneration occurs via distinct phases of retraction and membrane decompaction.
Fig. 3: Divergent fates of single OPCs after oligodendrocyte 2Phatal.
Fig. 4: OPC fate is linked to morphological complexity and GPR17 expression during remyelination.
Fig. 5: Myelin patterns impact the success and spatiotemporal dynamics of remyelination.
Fig. 6: Distinct forms of synchronous and rapid remyelination.
Fig. 7: Age-related myelin degeneration occurs via sheath loss and cell death with limited repair.
Fig. 8: Failed myelin repair after oligodendrocyte 2Phatal in aging.

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Data availability

All data used for quantification and statistical comparisons are provided in the source data file associated with each figure. All imaging data files are available on an unrestricted basis from the corresponding author. Source data are provided with this paper.

References

  1. Zalc, B., Goujet, D. & Colman, D. The origin of the myelination program in vertebrates. Curr. Biol. 18, R511–R512 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Stadelmann, C., Timmler, S., Barrantes-Freer, A. & Simons, M. Myelin in the central nervous system: structure, function, and pathology. Physiol. Rev. 99, 1381–1431 (2019).

    Article  CAS  PubMed  Google Scholar 

  3. Franklin, R. J. M. & Ffrench-Constant, C. Remyelination in the CNS: from biology to therapy. Nat. Rev. Neurosci. 9, 839–855 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Nave, K.-A. Myelination and support of axonal integrity by glia. Nature 468, 244–252 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Peters, A. & Kemper, T. A review of the structural alterations in the cerebral hemispheres of the aging rhesus monkey. Neurobiol. Aging 33, 2357–2372 (2012).

    Article  PubMed  Google Scholar 

  6. Almeida, R. G. & Lyons, D. A. On myelinated axon plasticity and neuronal circuit formation and function. J. Neurosci. 37, 10023–10034 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Fields, R. D. A new mechanism of nervous system plasticity: activity-dependent myelination. Nat. Rev. Neurosci. 16, 756–767 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chapman, T. W. & Hill, R. A. Myelin plasticity in adulthood and aging. Neurosci. Lett. 715, 134645 (2020).

    Article  CAS  PubMed  Google Scholar 

  9. Nishiyama, A. Polydendrocytes: NG2 cells with many roles in development and repair of the CNS. Neuroscientist 13, 62–76 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Bergles, D. E. & Richardson, W. D. Oligodendrocyte development and plasticity. Cold Spring Harb. Perspect. Biol. 8, a020453 (2015).

    Article  PubMed  Google Scholar 

  11. Kirby, B. B. et al. In vivo time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during zebrafish development. Nat. Neurosci. 9, 1506–1511 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Hughes, E. G., Kang, S. H., Fukaya, M. & Bergles, D. E. Oligodendrocyte progenitors balance growth with self-repulsion to achieve homeostasis in the adult brain. Nat. Neurosci. 16, 668–676 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Auer, F., Vagionitis, S. & Czopka, T. Evidence for myelin sheath remodeling in the CNS revealed by in vivo imaging. Curr. Biol. 28, 549–559 (2018).

    Article  CAS  PubMed  Google Scholar 

  14. Hill, R. A., Patel, K. D., Goncalves, C. M., Grutzendler, J. & Nishiyama, A. Modulation of oligodendrocyte generation during a critical temporal window after NG2 cell division. Nat. Neurosci. 17, 1518–1527 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Spitzer, S. O. et al. Oligodendrocyte progenitor cells become regionally diverse and heterogeneous with age. Neuron 101, 459–471 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Viganò, F., Möbius, W., Götz, M. & Dimou, L. Transplantation reveals regional differences in oligodendrocyte differentiation in the adult brain. Nat. Neurosci. 16, 1370–1372 (2013).

    Article  PubMed  Google Scholar 

  17. Marisca, R. et al. Functionally distinct subgroups of oligodendrocyte precursor cells integrate neural activity and execute myelin formation. Nat. Neurosci. 23, 363–374 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hill, R. A., Patel, K. D., Medved, J., Reiss, A. M. & Nishiyama, A. NG2 cells in white matter but not gray matter proliferate in response to PDGF. J. Neurosci. 33, 14558–14566 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Shen, S. et al. Age-dependent epigenetic control of differentiation inhibitors is critical for remyelination efficiency. Nat. Neurosci. 11, 1024–1034 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chacon-De-La-Rocha, I. et al. Accelerated dystrophy and decay of oligodendrocyte precursor cells in the APP/PS1 model of Alzheimer’s-like pathology. Front. Cell. Neurosci. 14, 575082 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Koudelka, S. et al. Individual neuronal subtypes exhibit diversity in CNS myelination mediated by synaptic vesicle release. Curr. Biol. 26, 1447–1455 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Goebbels, S. et al. A neuronal PI(3,4,5)P3-dependent program of oligodendrocyte precursor recruitment and myelination. Nat. Neurosci. 20, 10–15 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Seidl, A. H. Regulation of conduction time along axons. Neuroscience 276, 126–134 (2014).

    Article  CAS  PubMed  Google Scholar 

  24. Osso, L. A. & Chan, J. R. Architecting the myelin landscape. Curr. Opin. Neurobiol. 47, 1–7 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hines, J. H., Ravanelli, A. M., Schwindt, R., Scott, E. K. & Appel, B. Neuronal activity biases axon selection for myelination in vivo. Nat. Neurosci. 18, 683–689 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Mitew, S. et al. Pharmacogenetic stimulation of neuronal activity increases myelination in an axon-specific manner. Nat. Commun. 9, 306 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Mensch, S. et al. Synaptic vesicle release regulates myelin sheath number of individual oligodendrocytes in vivo. Nat. Neurosci. 18, 628–630 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Bacmeister, C. M. et al. Motor learning promotes remyelination via new and surviving oligodendrocytes. Nat. Neurosci. 23, 819–831 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Orthmann-Murphy, J. et al. Remyelination alters the pattern of myelin in the cerebral cortex. eLife 9, e56621 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Snaidero, N. et al. Myelin replacement triggered by single-cell demyelination in mouse cortex. Nat. Commun. 11, 4901 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hill, R. A., Damisah, E. C., Chen, F., Kwan, A. C. & Grutzendler, J. Targeted two-photon chemical apoptotic ablation of defined cell types in vivo. Nat. Commun. 8, 15837 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Damisah, E. C. et al. Astrocytes and microglia play orchestrated roles and respect phagocytic territories during neuronal corpse removal in vivo. Sci. Adv. 6, eaba3239 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Deng, Y. et al. Direct visualization of membrane architecture of myelinating cells in transgenic mice expressing membrane-anchored EGFP. Genesis 52, 341–349 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hill, R. A., Li, A. M. & Grutzendler, J. Lifelong cortical myelin plasticity and age-related degeneration in the live mammalian brain. Nat. Neurosci. 21, 683–695 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Davalos, D. et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8, 752–758 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Schain, A. J., Hill, R. A. & Grutzendler, J. Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy. Nat. Med. 20, 443–449 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hill, R. A. & Grutzendler, J. In vivo imaging of oligodendrocytes with sulforhodamine 101. Nat. Methods 11, 1081–1082 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Fard, M. K. et al. BCAS1 expression defines a population of early myelinating oligodendrocytes in multiple sclerosis lesions. Sci. Transl. Med. 9, eaam7816 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Guo, F. & Wang, Y. TCF7l2, a nuclear marker that labels premyelinating oligodendrocytes and promotes oligodendroglial lineage progression. Glia 71, 143–154 (2023).

    Article  CAS  PubMed  Google Scholar 

  40. Lecca, D., Raffaele, S., Abbracchio, M. P. & Fumagalli, M. Regulation and signaling of the GPR17 receptor in oligodendroglial cells. Glia 68, 1957–1967 (2020).

    Article  PubMed  Google Scholar 

  41. Peters, A. The effects of normal aging on myelin and nerve fibers: a review. J. Neurocytol. 31, 581–593 (2002).

    Article  PubMed  Google Scholar 

  42. Marshall-Phelps, K. L. H. et al. Neuronal activity disrupts myelinated axon integrity in the absence of NKCC1b. J. Cell Biol. 219, e201909022 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Traka, M. et al. A genetic mouse model of adult-onset, pervasive central nervous system demyelination with robust remyelination. Brain 133, 3017–3029 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Gardner, C. et al. Cortical grey matter demyelination can be induced by elevated pro-inflammatory cytokines in the subarachnoid space of MOG-immunized rats. Brain 136, 3596–3608 (2013).

    Article  PubMed  Google Scholar 

  45. Sachs, H. H., Bercury, K. K., Popescu, D. C., Narayanan, S. P. & Macklin, W. B. A new model of cuprizone-mediated demyelination/remyelination. ASN Neuro 6, 1759091414551955 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Matsushima, G. K. & Morell, P. The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system. Brain Pathol. 11, 107–116 (2001).

    Article  CAS  PubMed  Google Scholar 

  47. Czopka, T., Ffrench-Constant, C. & Lyons, D. A. Individual oligodendrocytes have only a few hours in which to generate new myelin sheaths in vivo. Dev. Cell 25, 599–609 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Snaidero, N. et al. Myelin membrane wrapping of CNS axons by PI(3,4,5)P3-dependent polarized growth at the inner tongue. Cell 156, 277–290 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Snaidero, N. et al. Antagonistic functions of MBP and CNP establish cytosolic channels in CNS myelin. Cell Rep. 18, 314–323 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Yeung, M. S. Y. et al. Dynamics of oligodendrocyte generation in multiple sclerosis. Nature 566, 538–542 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Jäkel, S. et al. Altered human oligodendrocyte heterogeneity in multiple sclerosis. Nature 566, 543–547 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Neely, S. A. et al. New oligodendrocytes exhibit more abundant and accurate myelin regeneration than those that survive demyelination. Nat. Neurosci. 25, 415–420 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Marques, S. et al. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science 352, 1326–1329 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Chen, Y. et al. The oligodendrocyte-specific G protein-coupled receptor GPR17 is a cell-intrinsic timer of myelination. Nat. Neurosci. 12, 1398–1406 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Ou, Z. et al. Olig2-targeted G-protein-coupled receptor Gpr17 regulates oligodendrocyte survival in response to lysolecithin-induced demyelination. J. Neurosci. 36, 10560–10573 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Wang, J. et al. Robust myelination of regenerated axons induced by combined manipulations of GPR17 and microglia. Neuron 108, 876–886 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Boda, E. et al. The GPR17 receptor in NG2 expressing cells: focus on in vivo cell maturation and participation in acute trauma and chronic damage. Glia 59, 1958–1973 (2011).

    Article  PubMed  Google Scholar 

  58. Haroutunian, V. et al. Myelination, oligodendrocytes, and serious mental illness. Glia 62, 1856–1877 (2014).

    Article  CAS  PubMed  Google Scholar 

  59. Sim, F. J., Zhao, C., Penderis, J. & Franklin, R. J. M. The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation. J. Neurosci. 22, 2451–2459 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kotter, M. R., Li, W.-W., Zhao, C. & Franklin, R. J. M. Myelin impairs CNS remyelination by inhibiting oligodendrocyte precursor cell differentiation. J. Neurosci. 26, 328–332 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Tse, K.-H. & Herrup, K. DNA damage in the oligodendrocyte lineage and its role in brain aging. Mech. Ageing Dev. 161, 37–50 (2017).

    Article  CAS  PubMed  Google Scholar 

  62. Zhu, X. et al. Age-dependent fate and lineage restriction of single NG2 cells. Development 138, 745–753 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Yona, S. et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38, 79–91 (2013).

    Article  CAS  PubMed  Google Scholar 

  64. Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    Article  CAS  PubMed  Google Scholar 

  65. Lecca, D. et al. The recently identified P2Y-like receptor GPR17 is a sensor of brain damage and a new target for brain repair. PLoS ONE 3, e3579 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Ciana, P. et al. The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor. EMBO J. 25, 4615–4627 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Ferreira, T. A. et al. Neuronal morphometry directly from bitmap images. Nat. Methods 11, 982–984 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the following grant nos. R00NS099469, P20GM113132, R01NS122800 (National Institutes of Health) and by a New Vision Award through the Donors Cure Foundation, a Fay/Frank Seed Grant from the Brain Research Foundation and funding from the Esther A. & Joseph Klingenstein Fund and Simons Foundation to R.A.H. G.E.O. was supported by a Graduate Assistance in Areas of National Need Department of Education grant no. P200A210064. We thank D. Lecca and M. Abbracchio (Università degli Studi di Milano) for graciously sharing the antibody to GPR17 and Dartmouth colleagues L. Petrov and S. Gregorace for contributions to data analysis. We thank members of the Hoppa laboratory at Dartmouth for critical feedback throughout the project.

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Authors and Affiliations

Authors

Contributions

T.W.C. and R.A.H. conceived, designed and performed all the experiments and most of the data analysis and quantification. G.E.O. performed the OPC morphological and migration analyses and contributed to quantification associated with sheath and node remyelination. Xh.B. contributed to data analysis and quantification. E.P. contributed to OPC fate analysis and imaging data collation. T.W.C. and R.A.H. wrote the paper and R.A.H. supervised the study.

Corresponding author

Correspondence to Robert A. Hill.

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The authors declare no competing interests.

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Nature Neuroscience thanks Jason Plemel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Microglia do not acutely respond to oligodendrocyte 2Phatal and cuprizone-induced myelin pathology and dynamics of degeneration.

(a) In vivo images from dual-reporter, triple transgenic mice with oligodendrocytes (green) and microglia (magenta) labeling. Experiments were repeated three times. Microglia exhibited no immediate chemotactic response towards oligodendrocytes targeted with 2Patal (orange arrowheads) demonstrating no disruption of the targeted oligodendrocyte cell membrane during 2Phatal photobleaching. (b) Additional examples of control (red arrowheads) or 2Phatal (yellow arrowheads) oligodendrocyte soma again showing no chemotactic response by the microglia up to 1 day later. These examples were selected to show that while occasionally there were microglial cell processes and thus fluorescent signals detected in the quantification adjacent to the soma 1 day later, there is not a phagocytic response. Experiments were repeated three times for controls and three times for 2Phatal. (c) Paradigm used for cuprizone induced demyelination experiments. Mice were fed 0.2% w/w cuprizone mixed in ground chow. In-vivo imaging was done weekly, starting at week 0, through week 10. (d) Cuprizone induced widespread demyelination and oligodendrocyte cell loss (orange arrowheads) after 41 dwc (days with cuprizone). Remyelination was largely complete by 31 dpc (days post cuprizone). Green arrowheads denote points of reference for position orientation. Experiments were repeated three times. (e) Representative images of common myelin pathology observed during cuprizone intoxication, including partial loss of compaction (top, between arrows), visualized by a lack of SCoRe signal, complete loss of compaction with myelin debris (middle), and balloon formation (bottom, arrowheads). Experiments were repeated three times. (f) Temporal dynamics of sheath degeneration during cuprizone intoxication. Each trace represents sheaths produced from a single oligodendrocyte (n = 8 cells, 116 sheaths, 3 mice).

Extended Data Fig. 2 The Cnp-mEGFP:Cspg4-creER:tdTomato mouse line identifies newly generated oligodendrocytes.

(a) Example in-vivo image showing a previously generated, tdTomato-, oligodendrocyte (orange arrow) and tdTomato+ OPCs (white arrows) at day 0. (b) Time series showing the differentiation of new dual-labeled oligodendrocytes (yellow arrows) over the course of the experiment, while a previously established single labeled oligodendrocyte (orange arrow) is maintained. (c) Representative image of a Cnp-mEGFP:Cspg4-creER:tdTomato mouse at day 60 showing all surviving oligodendrocytes (arrows) are dual labeled with mEGFP+ and tdTomato + . For this example all cells at day 0 that were mEGFP+ were targeted with 2Phatal (d) Time series showing the presence of a new mEGFP + /tdTomato+ oligodendrocyte (orange arrow, top images) at day 60. During all experiments we encountered only a single tdTomato- oligodendrocyte, that was generated between day 0 and day 60 (white arrow, bottom images). (e) Of the 107 remyelinating sheaths analyzed, a single sheath was tdTomato-. As it was in the vicinity of the tdTomato- cell shown in d, it is likely to have originated from that cell.

Extended Data Fig. 3 Increased oligodendrocyte generation compensates for cell degeneration.

(a) Cell maps of imaging positions in control animals, denoting the locations of oligodendrocytes present at the start of imaging (non-targeted oligodendrocytes, black dots) and oligodendrocytes produced over 60 days (new oligodendrocytes, cyan dots) (scale bars are 50 µm). (b) Cell maps of imaging positions in animals targeted with 2Phatal on day 0, denoting the locations of oligodendrocytes targeted with 2Phatal (red dots), non-targeted oligodendrocytes present at day 0 (black dots), and new oligodendrocytes produced over 60 days post 2Phatal (cyan dots) (scale bars are 50 µm). (c) Average number of new oligodendrocytes generated per day, over 60 days, in mice not targeted with 2Phatal (control, n = 4 mice) and mice with oligodendrocytes targeted with 2Phatal (blue, n = 4 mice). There were significantly more oligodendrocytes produced per day in mice targeted with 2Phatal than in control animals (two-tailed, unpaired t test, error bars are SEM). (d) Average fold-change in total oligodendrocytes in each position, between day 0 and day 60, in mice without oligodendrocyte 2Phatal (control, n = 4 mice) and mice with oligodendrocyte 2Phatal (2Phatal, n = 4 mice). (two-tailed, Unpaired t test, error bars are SEM).

Source data

Extended Data Fig. 4 Local OPC migration is unaffected by oligodendrocyte 2Phatal and OPC morphological complexity during remyelination.

(a) Representative MAX projection of a single time point, showing OPCs (magenta), used to determine the migration of OPCs over time. Experiments were repeated eight times. (b) Example OPC migration tracks between day 28 and day 60 from a single imaging location. Each line represents the migration track of a single cell. (c) The total distance traveled (left) and net displacement (right) of OPCs in control mice compared to mice targeted with 2Phatal. There was no significant difference in overall migration behavior between the two groups (control n = 48 cells, 2Phatal n = 45 cells, two-tailed, unpaired t test, dots indicate single cells, error bars are SEM). (d) Sholl analyses of two representative OPCs with the total number of cell process intersections plotted relative to the cell center. (e) Images showing the morphology of the five most complex OPCs (top row) and five least complex OPCs (bottom row) paired with the fate of each of those cells. (f) Linear regression analyses revealed no correlation between total intersections and the time from Sholl analysis to final fate outcome in control (left), days post analysis refers to the time of fate determination relative to the day of Sholl analysis (day 28 after 2Phatal) (n = 12 differentiating cells, blue, and n = 14 dying cells, green) and 2Phatal (right) conditions (n = 34 differentiating, blue, and n = 21 dying, green). (g) Single OPCs imaged over 23 days showing relatively stable morphological complexity. Experiments were repeated four times. (h) The total of intersections captured from multiple Sholl analysis and the same cell over time, again showing relatively stable morphological complexity over weeks.

Source data

Extended Data Fig. 5 Localization maps of OPC morphological complexity.

(a) Single position captured at day 28 after oligodendrocyte 2Phatal showing OPCs, oligodendrocytes and a corresponding cell map of the targeted oligodendrocytes (red dots) and OPCs (blue dots). Numbers indicate the cell ID and total intersections for that cell. Experiments were repeated four times. (b) Additional cell maps of all positions used in the 2Phatal conditions showing OPC location and morphological complexity (cell ID + total intersections for that cell) and locations of targeted oligodendrocytes (red dots) (c) The total intersections of all cells that remained OPCs, differentiated, or died either adjacent to or away from targeted oligodendrocytes. Remained OPC n = 19 adjacent, 17 away, oligodendrocyte differentiation n = 28 adjacent, 7 away; OPC death n = 15 adjacent, 6 away. One way ANOVA with Sidak’s multiple comparison’s test, error bars are SEM.

Source data

Extended Data Fig. 6 Multifocal 2Phatal enables molecular interrogation of OPCs during oligodendrocyte death.

(a) Pipeline demonstrating our approach to multifocal 2Phatal. Oligodendrocytes (orange arrowheads) throughout the cranial window were targeted with 2Phatal. 28 days post 2Phatal, targeted cells (green arrow) can be easily identified by their bright, condensed somas compared to non-targeted cells (orange arrows). Animals used in multifocal experiments were perfused at day 28 which is prior to the majority demyelination (n = 19 cells, 183 sheaths, 4 mice, error is SEM). Experiments were repeated four times. (b) Brain sections can then be immunolabeled. Analysis was localized to layer I cortex (box), as this was where 2Phatal was performed. Experiments were repeated four times. (c) 2Phatal-targeted oligodendrocytes (green arrows) can be easily identified in fixed tissue by their condensed soma compared to non-targeted oligodendrocytes (orange arrows). Nuclear condensation was also a reliable method for identification. Experiments were repeated four times. (d) Schematic identifying genes of interest for our pipeline. GPR17 is known to label subpopulations of OPCs. TCF7L2 and BCAS1 were used as markers to identify active differentiation. CNP was used as a marker to exclude oligodendrocytes from our analysis. (e) Representative images of immunostaining for BCAS1 (left) and GPR17 (right). Experiments were repeated four times.

Source data

Extended Data Fig. 7 Identity and density of OPCs following multifocal 2Phatal.

(a) Representative images of BCAS1 staining following multifocal 2Phatal and quantification of BCAS1 positive CNP negative cell density in layer I cortex (n = 4 animals, two-tailed, unpaired t-test, error bars are SEM). Sholl was performed on tdTomato labeled BCAS1 positive CNP negative cells (left), BCAS1 positive cells with CNP co-labeling and/or attached sheaths (right) were excluded. (b) Representative image of TCF7L2 and OLIG2 staining following multifocal 2Phatal. Density of TCF7L2 cells is also reported (n = 4 animals, two-tailed, unpaired t-test, error bars are SEM). (c) Representative image showing GPR17 staining following multifocal 2Phatal. GPR17 subcellular localization to the membrane (left arrow) and cytosol (middle arrow) can be visualized, as well as no expression (right arrow). Density of combined GPR17 negative, CNP negative cells is reported (n = 4 mice, two-tailed, unpaired t-test, error bars are SEM). Grouped data showing the total intersections of GPR17 + cells with membrane (cyan) or cytosolic (orange) localization in control and 2Phatal conditions (Membrane n = 21 cells, cytosolic n = 32 cells, 4 animals, one-way ANOVA with Tukey correction for multiple comparisons, error bars are SEM).

Source data

Extended Data Fig. 8 Initial node of Ranvier locations are reestablished after remyelination.

(a) In vivo images of a node of Ranvier present at day 0 between mEGFP only labeled sheaths, which reformed at its original location between two mEGFP and tdTomato double labeled sheaths, as seen at day 60. (b) Nodes from 2Phatal mice were randomly selected on day 0, blind to the cell of origin, and evaluated on day 60, to determine if they were stable (mEGFP+ only) were reformed through remyelination (mEGFP+tdTomato+) or degenerated (80 nodes from n = 4 mice) (c) Proportion of nodes that were displaced between days 0 and 60 for both nodes that remained stable (n = 31, from 4 mice) and nodes that were regenerated following remyelination (n = 41, from 4 mice). All error bars are SEM.

Source data

Extended Data Fig. 9 Synchronous remyelination facilitates rapid remyelination in vivo and spatiotemporal maps of asynchronous and synchronous remyelination.

(a) Original images used to generate the traced time series shown in Fig. 6. The initiation of remyelination is evident by emergence of tdTomato signal (arrowheads) on day 31. (b) Additional example of synchronous remyelination without score loss throughout the repair process. (c) Representative imaging positions in CNP-mEGFP mice with oligodendrocytes targeted with 2Phatal (left), highlighting sheaths produced by targeted oligodendrocytes (middle, cyan). The same sheaths are then presented based on eventual fate post-2Phatal (right), including sheaths that were never repaired (lost, black), fully degenerated and then repaired (SCoRe and mEGFP lost, grey), underwent synchronous remyelination with loss of score (SCoRe lost mEGFP maintained, magenta), and underwent synchronous remyelination without losing SCoRe (SCoRe and mEGFP maintained, red). Targeted oligodendrocytes are shown as black dots (right).

Extended Data Fig. 10 Myelin pathology and dynamics of degeneration in aged mice.

(a) In-vivo image of myelin, in layer I of the somatosensory cortex, in an aged mouse, acquired using SCoRe and fluorescence microscopy. Experiments were repeated three times. (b) Aged mice displayed widespread myelin pathology (arrowheads) including myelin swellings (left), debris accumulation (middle), and balloons (right). Experiments were repeated three times. (c) Additional examples of myelin swellings (arrows) in aged animals. Experiments were repeated three times. (d) Oligodendrocyte death (yellow arrows) and myelin degeneration (yellow arrowheads) followed by the emergence of a newly generated oligodendrocyte (green arrows) and remyelinating sheaths (green arrowheads). This was the only example of remyelination due to the differentiation of a new oligodendrocyte we observed in aged animals. Experiments were repeated three times. (e) Representative time series showing failed remyelination following myelin degeneration (orange arrowheads) and successful remyelination after sheath loss (green arrowheads). Experiments were repeated three times. (f) Sheath repair was observed to occur following balloon formation (green arrowheads). Experiments were repeated three times. (g) Severe myelin pathology, seen in aged mice, often resulted in full sheath degeneration (bottom, orange arrowheads). Experiments were repeated three times.

Supplementary information

Reporting Summary

Oligodendrocyte degeneration after 2Phatal. In vivo time series showing the degeneration of an mEGFP-labeled oligodendrocyte after 2Phatal. The dashed line represents the soma diameter before 2Phatal. Soma condensation occurs within the first 14 d, while final cell death does not occur until 49 d after photobleaching. Images were acquired in layer I of the somatosensory cortex (frame rate: 2 frames per second).

OPC division. In vivo time series in a Cnp-mEGFP:Cspg4-creER:tdTomato mouse showing OPC (magenta) division events. The arrows denote OPC that undergo division. Images were acquired in layer I of the somatosensory cortex (frame rate: 2 frames per second).

OPC apoptosis. In vivo time series in a Cnp-mEGFP:Cspg4-creER:tdTomato mouse showing OPC (magenta) apoptosis. Note the appearance of apoptotic bodies on day 62, which are cleared by day 64. Images were acquired in layer I of the somatosensory cortex (frame rate: 2 frames per second).

Generation of oligodendrocytes from OPCs. In vivo time series in a Cnp-mEGFP:Cspg4-creER:tdTomato mouse showing the differentiation of an OPC (magenta) into a myelinating oligodendrocyte. After differentiation, the soma contracts and becomes stationary. Newly formed myelin is evident when dual-labeled with mEGFP (green) and tdTomato (magenta) (frame rate: 2 frames per second).

Long-term in vivo imaging of OPCs and oligodendrocytes. In vivo time series in a Cnp-mEGFP:Cspg4-creER:tdTomato control mouse, not targeted with 2Phatal, demonstrating the behavior of oligodendrocytes (green) and OPCs in layer I of the somatosensory cortex. As new oligodendrocytes are generated, myelin density increases. The dates represent days postnatal (postnatal 62–102) (frame rate: 2 frames per second).

Dynamics of oligodendrocyte degeneration, OPC fate and rapid remyelination after oligodendrocyte 2Phatal. In vivo time series showing the degeneration of an oligodendrocyte (green) targeted with 2Phatal and local OPC (magenta) behavior. Oligodendrocyte degeneration (orange arrowhead) occurs over 40 d and is characterized by rapid soma condensation. Throughout and after the degeneration, neighboring OPCs respond to the death via differentiation and remyelination.

Remyelination after myelin sheath degeneration. Expanded time series of the sheath degeneration and repair sequence used in Fig. 5, including SCoRe microscopy (yellow). Remyelination is clearly visible due to the presence of tdTomato fluorescence (magenta) in the newly forming sheath. Note that cytoplasmic tdTomato fluorescence intensity is highest when the remyelinating sheath is first formed (day 44) and not yet compact, as evident by the lack of SCoRe signal (frame rate: 2 frames per second).

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Chapman, T.W., Olveda, G.E., Bame, X. et al. Oligodendrocyte death initiates synchronous remyelination to restore cortical myelin patterns in mice. Nat Neurosci 26, 555–569 (2023). https://doi.org/10.1038/s41593-023-01271-1

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