Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander Disease severity

  1. Rachel A Battaglia
  2. Adriana S Beltran
  3. Samed Delic
  4. Raluca Dumitru
  5. Jasmine A Robinson
  6. Parijat Kabiraj
  7. Laura E Herring
  8. Victoria J Madden
  9. Namritha Ravinder
  10. Erik Willems
  11. Rhonda A Newman
  12. Roy Andrew Quinlan
  13. James E Goldman
  14. Ming-Der Perng
  15. Masaki Inagaki
  16. Natasha T Snider  Is a corresponding author
  1. University of North Carolina at Chapel Hill, United States
  2. Thermo Fisher Scientific, United States
  3. Durham University, United Kingdom
  4. Columbia University, United States
  5. National Tsing Hua University, Taiwan, Republic of China
  6. Mie University Graduate School of Medicine, Japan

Abstract

Alexander Disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for mass spec results in Figure 1 and Supplemental Figure 6.

Article and author information

Author details

  1. Rachel A Battaglia

    Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    No competing interests declared.
  2. Adriana S Beltran

    Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    No competing interests declared.
  3. Samed Delic

    Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    No competing interests declared.
  4. Raluca Dumitru

    Human Pluripotent Stem Cell Core Facility, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    No competing interests declared.
  5. Jasmine A Robinson

    Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    No competing interests declared.
  6. Parijat Kabiraj

    Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    No competing interests declared.
  7. Laura E Herring

    Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    No competing interests declared.
  8. Victoria J Madden

    Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7909-7743
  9. Namritha Ravinder

    Thermo Fisher Scientific, Carlsbad, United States
    Competing interests
    Namritha Ravinder, is a paid employee of ThermoFisher Scientific, whose products were used to complete parts of the study.
  10. Erik Willems

    Thermo Fisher Scientific, Carlsbad, United States
    Competing interests
    Erik Willems, is a paid employee of ThermoFisher Scientific, whose products were used to complete parts of the study.
  11. Rhonda A Newman

    Thermo Fisher Scientific, Carlsbad, United States
    Competing interests
    Rhonda A Newman, is a paid employee of ThermoFisher Scientific, whose products were used to complete parts of the study. ThermoFisher Scientific had no role in the study design, data analysis, decision to publish, or preparation of the manuscript.
  12. Roy Andrew Quinlan

    Department of Biosciences, Durham University, Durham, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0644-4123
  13. James E Goldman

    Department of Pathology, Columbia University, New York, United States
    Competing interests
    No competing interests declared.
  14. Ming-Der Perng

    Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
    Competing interests
    No competing interests declared.
  15. Masaki Inagaki

    Department of Physiology, Mie University Graduate School of Medicine, Mie, Japan
    Competing interests
    No competing interests declared.
  16. Natasha T Snider

    Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    For correspondence
    ntsnider@med.unc.edu
    Competing interests
    Natasha T Snider, is a member of the Scientific Advisory Board for Elise's Corner Fund, which supported part of this work.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7663-4585

Funding

Elise's Corner Fund (Research Grant)

  • Natasha T Snider

United Leukodystrophy Foundation (Research Grant)

  • Natasha T Snider

National Science Foundation (Graduate Research Fellowship)

  • Rachel A Battaglia

University of North Carolina at Chapel Hill (Department of Cell Biology and Physiology)

  • Natasha T Snider

National Institutes of Health (P30 CA016086)

  • Victoria J Madden

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Kang Shen, Howard Hughes Medical Institute, Stanford University, United States

Publication history

  1. Received: April 18, 2019
  2. Accepted: November 4, 2019
  3. Accepted Manuscript published: November 4, 2019 (version 1)
  4. Version of Record published: December 23, 2019 (version 2)

Copyright

© 2019, Battaglia et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,591
    Page views
  • 370
    Downloads
  • 12
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Rachel A Battaglia
  2. Adriana S Beltran
  3. Samed Delic
  4. Raluca Dumitru
  5. Jasmine A Robinson
  6. Parijat Kabiraj
  7. Laura E Herring
  8. Victoria J Madden
  9. Namritha Ravinder
  10. Erik Willems
  11. Rhonda A Newman
  12. Roy Andrew Quinlan
  13. James E Goldman
  14. Ming-Der Perng
  15. Masaki Inagaki
  16. Natasha T Snider
(2019)
Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander Disease severity
eLife 8:e47789.
https://doi.org/10.7554/eLife.47789

Further reading

    1. Cell Biology
    2. Computational and Systems Biology
    Théo Aspert et al.
    Tools and Resources

    Automating the extraction of meaningful temporal information from sequences of microscopy images represents a major challenge to characterize dynamical biological processes. So far, strong limitations in the ability to quantitatively analyze single-cell trajectories have prevented large-scale investigations to assess the dynamics of entry into replicative senescence in yeast. Here, we have developed DetecDiv, a microfluidic-based image acquisition platform combined with deep learning-based software for high-throughput single-cell division tracking. We show that DetecDiv can automatically reconstruct cellular replicative lifespans with high accuracy and performs similarly with various imaging platforms and geometries of microfluidic traps. In addition, this methodology provides comprehensive temporal cellular metrics using time-series classification and image semantic segmentation. Last, we show that this method can be further applied to automatically quantify the dynamics of cellular adaptation and real-time cell survival upon exposure to environmental stress. Hence, this methodology provides an all-in-one toolbox for high-throughput phenotyping for cell cycle, stress response, and replicative lifespan assays.

    1. Cell Biology
    2. Microbiology and Infectious Disease
    Alice L Herneisen et al.
    Research Article

    Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.