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Ptbp1 deletion does not induce astrocyte-to-neuron conversion

Nature volume 618, pages E1–E7 (2023)Cite this article

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Matters Arising to this article was published on 07 June 2023

The Original Article was published on 24 June 2020

arising from: H. Qian et al. Nature https://doi.org/10.1038/s41586-020-2388-4 (2020)

Although in vivo reprogramming of astrocytes into neurons holds promise for treating neurodegenerative disorders, protocols to achieve it are complex and inefficient. A recent study reported that knockdown of Ptbp1 in the cortex, striatum and substantia nigra of the midbrain efficiently reprogrammed astrocytes into functional neurons that rescued motor defects in a mouse model of Parkinson’s disease1, but it did not convincingly demonstrate that astrocyte-to-neuron conversion had actually occurred. Using genetic disruption of Ptbp1 in combination with cell-lineage analysis, we observe no astrocyte-to-neuron conversion or substantial changes in gene expression in Ptbp1-deficient astrocytes. We conclude that the results of astrocyte-to-neuron conversion in the previous study1 most likely reflect leaky neuronal expression of the Gfapcre mouse lines used to label astrocytes, and more-rigorous experimental methods must be used to support their observations of glial reprogramming in vivo.

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Fig. 1: Genetic loss of function of Ptbp1 did not result in astrocyte-to-neuron conversion in the adult mouse brain.
Fig. 2: No changes in the electrophysiological properties of cortical astrocytes, and only subtle changes in astrocyte gene expression, were observed after Ptbp1 deletion.

Data availability

All scRNA-seq data have been deposited at the Gene Expression Omnibus (GSE184933).

Code availability

Codes for processing scRNA-seq files are available at GitHub (https://github.com/dkim195/Ptbp1_Nature_MA).

References

  1. Qian, H. et al. Reversing a model of Parkinson’s disease with in situ converted nigral neurons. Nature 582, 550–556 (2020).

  2. Su, M. et al. Expression specificity of GFAP transgenes. Neurochem. Res. 29, 2075–2093 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Fujita, T. et al. Neuronal transgene expression in dominant-negative SNARE mice. J. Neurosci. 34, 16594–16604 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Wang, L.-L. et al. Revisiting astrocyte to neuron conversion with lineage tracing in vivo. Cell 184, 5465–5481 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Le, N., Appel, H., Pannullo, N., Hoang, T. & Blackshaw, S. Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina. Front. Cell Dev. Biol. 10, 914386 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Srinivasan, R. et al. New transgenic mouse lines for selectively targeting astrocytes and studying calcium signals in astrocyte processes in situ and in vivo. Neuron 92, 1181–1195 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Shibayama, M. et al. Polypyrimidine tract-binding protein is essential for early mouse development and embryonic stem cell proliferation. FEBS J. 276, 6658–6668 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Mo, A. et al. Epigenomic signatures of neuronal diversity in the mammalian brain. Neuron 86, 1369–1384 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. McNeill, J., Rudyk, C., Hildebrand, M. E. & Salmaso, N. Ion channels and electrophysiological properties of astrocytes: implications for emergent stimulation technologies. Front. Cell. Neurosci. 15, 644126 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chen, W., Zheng, Q., Huang, Q., Ma, S. & Li, M. Repressing PTBP1 fails to convert reactive astrocytes to dopaminergic neurons in a 6-hydroxydopamine mouse model of Parkinson’s disease. eLife 11, e75636 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wang, L.-L. & Zhang, C.-L. Therapeutic potential of PTBP1 inhibition, if any, is not attributed to glia-to-neuron conversion. Annu. Rev. Neurosci. https://doi.org/10.1146/annurev-neuro-092822-083410 (2022).

  12. Yang, G. et al. Ptbp1 knockdown failed to induce astrocytes to neurons in vivo. Gene Ther. https://doi.org/10.1038/s41434-023-00382-5 (2023).

  13. Zhou, H. et al. Glia-to-neuron conversion by CRISPR-CasRx alleviates symptoms of neurological disease in mice. Cell 181, 590–603 (2020).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank F. Zhou, A. Fischer, J. Ling, C. Qian and W. Yap for their comments on the manuscript; the staff at the Single Cell & Transcriptomics Core (Johns Hopkins) for sequencing of scRNA-seq libraries. Schematics in Figs. 1 and 2, Extended Data Fig. 3 and Supplementary Figs. 1–6 were created in part using BioRender. This work was supported by the NIH National Eye Institute grants R01EY020560 and U01EY027267 to S.B.; R24EY027283 to S.B.; and the Maryland Stem Cell Research Fund (2019-MSCRFF-5124) to D.W.K. S.B. is supported by a Stein Innovation Award from Research to Prevent Blindness.

Author information

Author notes

  1. Thanh Hoang

    Present address: Department of Ophthalmology, University of Michigan, Ann Arbor, MI, USA

  2. Thanh Hoang

    Present address: Department of Cell & Developmental Biology, University of Michigan, Ann Arbor, MI, USA

  3. Dong Won Kim

    Present address: Danish Research Institute of Translational Neuroscience (DANDRITE), Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus, Denmark

  4. Dong Won Kim

    Present address: Department of Biomedicine, Aarhus University, Aarhus, Denmark

  5. Juhyun Kim

    Present address: Korea Brain Research Institute, Daegu, South Korea

  6. These authors contributed equally: Thanh Hoang, Dong Won Kim

Authors and Affiliations

  1. Solomon H. Snyder Department of Neuroscience, John Hopkins University School of Medicine, Baltimore, MD, USA

    Thanh Hoang, Dong Won Kim, Haley Appel & Seth Blackshaw

  2. Center for Experimental Medicine and Systems Biology, University of Tokyo, Tokyo, Japan

    Manabu Ozawa

  3. Division of Biomedical Sciences, University of California, Riverside, Riverside, CA, USA

    Sika Zheng

  4. Center for RNA Biology and Medicine, University of California, Riverside, Riverside, CA, USA

    Sika Zheng

  5. Department of Psychiatry and Behavioral Science, John Hopkins University School of Medicine, Baltimore, MD, USA

    Juhyun Kim

  6. Department of Ophthalmology, John Hopkins University School of Medicine, Baltimore, MD, USA

    Seth Blackshaw

  7. Department of Neurology, John Hopkins University School of Medicine, Baltimore, MD, USA

    Seth Blackshaw

  8. Institute for Cell Engineering, John Hopkins University School of Medicine, Baltimore, MD, USA

    Seth Blackshaw

  9. Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Seth Blackshaw

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Contributions

T.H., D.W.K. and S.B. conceived and supervised the study. T.H. and D.W.K. generated and analysed immunohistochemistry and scRNA-seq data from the brain, assisted by H.A. M.O. and S.Z. provided reagents. J.K. performed slice electrophysiology of cortical astrocytes. T.H., D.W.K. and S.B. drafted the manuscript. All of the authors contributed to writing the manuscript.

Corresponding authors

Correspondence to Thanh Hoang, Dong Won Kim or Seth Blackshaw.

Ethics declarations

Competing interests

S.B. is a shareholder in CDI Labs, receives research support from Genentech and is/was a consultant to Third Rock Ventures and Tenpoint Therapeutics.

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Extended data figures and tables

Extended Data Fig. 1 Astrocyte-specific deletion of Ptbp1 does not lead to replenishing lost dopaminergic neurons.

a, Schematic of the experimental timeline for 6-OHDA-induced lesion of dopaminergic neurons, followed by tamoxifen-induced specific deletion of Ptbp1 and lineage tracing of astrocytes. b, Unilateral loss of Th-positive cells in the striatum and midbrain following 6-OHDA lesion. c, Representative immunostaining images of TH and GFP in the striatum and substantia nigra in an intact and lesioned area following 6-OHDA-induced lesion across genotypes. No TH/GFP-double-positive cells were observed in any of the brain regions across genotypes. Note that TH immunostaining signals are only present in non-cell bodies in the striatum (n > = 3 mice/genotype). Scale bars = 50 μm.

Extended Data Fig. 2 RNAScope showing non-astrocytic cells in the lateral ventricular/choroid plexus regions that express GFP and Otx2 mRNA.

a, Low magnification images of GFP mRNA and DAPI in Ptbp1 KO brain sections (n > = 3 mice/genotype). b, Low magnification images of Otx2 mRNA and DAPI in Ptbp1 KO brain section. c,d, High magnification images of GFP and Otx2 mRNA across genotypes. Scale bars = 50 μm.

Extended Data Fig. 3 ScRNA-Seq analysis of doublet-cells from FACS-purified GFP+ cells in mouse brain after astrocyte-specific Ptbp1 deletion.

Violin plots showing expression of neurogenic and cluster-enriched genes in all collected cells in the cortex (a), striatum (b) and substantia nigra (c) across genotypes. Note that Neurog2 and Neurod1 were not detected in the striatum and substantia nigra scRNA-Seq dataset. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).

Supplementary information

Supplementary Information

The legends for Supplementary Figs. 1–6 and Supplementary Tables 1–17, the Supplementary Methods and references.

Reporting Summary

Supplementary Fig. 1

Ptbp1 immunohistochemistry analysis in the adult mouse brain after astrocyte-specific deletion of Ptbp1. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).

Supplementary Fig. 2

Immunohistochemistry analysis of the adult mouse brain using neuronal markers after astrocyte-specific deletion of Ptbp1. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).

Supplementary Fig. 3

Immunohistochemistry analysis of the adult mouse brain using dopamine markers after astrocyte-specific deletion of Ptbp1. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).

Supplementary Figure 4

scRNA-seq analysis of subsets of astrocytes in the mouse brain after astrocyte-specific Ptbp1 deletion. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).

Supplementary Fig. 5

scRNA-seq analysis of the mouse brain after astrocyte-specific Ptbp1 deletion. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).

Supplementary Fig. 6

scRNA-seq analysis of FACS-purified GFP+ cells in the mouse brain after astrocyte-specific Ptbp1 deletion. The drawings of coronal sections were created using BioRender (https://www.biorender.com/).

Supplementary Tables 1–17

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Hoang, T., Kim, D.W., Appel, H. et al. Ptbp1 deletion does not induce astrocyte-to-neuron conversion. Nature 618, E1–E7 (2023). https://doi.org/10.1038/s41586-023-06066-9

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