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

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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.

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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.

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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.
Adriana S Beltran

Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, United States

Competing interests
No competing interests declared.
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.
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.
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.
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.
Laura E Herring

Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, United States

Competing interests
No competing interests declared.
Victoria J Madden

Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-7909-7743
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.
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.
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.
Roy Andrew Quinlan

Department of Biosciences, Durham University, Durham, United Kingdom

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-0644-4123
James E Goldman

Department of Pathology, Columbia University, New York, United States

Competing interests
No competing interests declared.
Ming-Der Perng

Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, Republic of China

Competing interests
No competing interests declared.
Masaki Inagaki

Department of Physiology, Mie University Graduate School of Medicine, Mie, Japan

Competing interests
No competing interests declared.
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.

"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.

Copyright

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.