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Stimulation of functional neuronal regeneration from Müller glia in adult mice

Nature volume 548, pages 103107 (03 August 2017) | Download Citation

Abstract

Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Müller glia (MG) are the source of regenerated neurons1,2,3,4,5,6. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage1,7 in zebrafish and is necessary for regeneration8. Although Ascl1 is not expressed in mammalian MG after injury9, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro10 and in vivo after NMDA (N-methyl-d-aspartate) damage in young mice11. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression11. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC–seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases.

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Acknowledgements

The authors acknowledge the following funding sources for support of this work. NIH NEI 1R01EY021482 to T.A.R., EY14358 to R.O.W., Howard Hughes Medical Institute to F.R., Allen Distinguished Investigator award (Paul G. Allen Family Foundation) to T.A.R., F.R. and R.O.W., an NSF Fellowship to M.W. (DGE-0718124), a Cellular and Molecular Biology Training Grant (T32GM007270) to L.S.V., and the Vision Core Grant P30EY01730 for use of the imaging facilities. We thank members of the Reh and Bermingham-McDonogh laboratories for valuable discussion and technical advice. We thank O. Bermingham-McDonogh for constructive comments on the manuscript. We thank A. Wills and A. Chitsazan for their ATAC–seq protocol and data analytics suggestions. We thank J. Delrow, C. Bennett and A. Berger at the Fred Hutch Genomics Shared Resource and Flow Cytometry facilities for their contributions to our sequencing datasets. We thank the laboratory of R. D. Hawkins for the cChIP reagents and protocol (H3K27ac ChIP). We thank the laboratory of C. Trapnell, specifically D. Jackson and R. Chawla, for their help generating the 10× Genomics single-cell RNA-seq datasets. Lastly, the authors thank E. Levine (Vanderbilt University) for the Rlbp1-creER mouse line and M. Nakafuku (Cincinnati Children's) for the tetO-Ascl1-GFP mice.

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Affiliations

  1. Department of Biological Structure, University of Washington, Seattle, Washington 98195, USA

    • Nikolas L. Jorstad
    • , Matthew S. Wilken
    • , Stefanie G. Wohl
    • , Leah S. VandenBosch
    • , Takeshi Yoshimatsu
    • , Rachel O. Wong
    •  & Thomas A. Reh
  2. Department of Pathology, Molecular Medicine and Mechanisms of Disease Program, University of Washington, Seattle, Washington 98195, USA

    • Nikolas L. Jorstad
  3. Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195, USA

    • William N. Grimes
    •  & Fred Rieke
  4. Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA

    • Fred Rieke

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Contributions

N.L.J. and T.A.R. designed and conceived the experiments. N.L.J. performed all injections, immunohistochemistry, imaging, western blots and cell reconstructions. T.Y. and R.O.W. prepared samples for SBFSEM and provided guidance for cell reconstructions and synaptic staining. W.N.G. and F.R. performed all electrophysiological recordings. S.G.W. performed FACS on retinas for ChIP and single-cell mRNA-seq. M.S.W. designed and conceived the experiments and performed Otx2 ChIP–seq and analysis. ATAC–seq and H3K27ac ChIP data was generated by L.S.V. and analysed by L.S.V. and T.A.R. All other figures and text were prepared by N.L.J. and T.A.R. with feedback and contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Thomas A. Reh.

Reviewer Information Nature thanks Z. He, H. Song and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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https://doi.org/10.1038/nature23283

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