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

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.

SUBMITTER: Battaglia RA

PROVIDER: S-EPMC6927689 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Battaglia Rachel A RA Beltran Adriana S AS Delic Samed S Dumitru Raluca R Robinson Jasmine A JA Kabiraj Parijat P Herring Laura E LE Madden Victoria J VJ Ravinder Namritha N Willems Erik E Newman Rhonda A RA Quinlan Roy A RA Goldman James E JE Perng Ming-Der MD Inagaki Masaki M Snider Natasha T NT

eLife 20191104

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 mo ...[more]

PMID: 31682229

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Project description:Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disease characterized by behavioral abnormalities, personality changes, language dysfunction, and can co-occur with the development of motor neuron disease. One major pathological form of FTLD is characterized by intracellular deposition of ubiquitinated and phosphorylated TAR DNA binding protein-43 (TDP-43), suggesting that dysregulation in phosphorylation events may contribute to disease progression. However, to date systematic analysis of the phosphoproteome in FTLD brains has not been reported. In this study, we employed immobilized metal affinity chromatography (IMAC) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify phosphopeptides from FTLD and age-matched control post-mortem human brain tissue. Using this approach, we identified 786 phosphopeptides in frontal cortex (control and FTLD), in which the population of phosphopeptides represented approximately 50% of the total peptides analyzed. Label-free quantification using spectral counts revealed six proteins with significant changes in the FTLD phosphoproteome. N-myc-Downstream regulated gene 2 (NDRG2) and glial fibrillary acidic protein (GFAP) had an increased number of phosphospectra in FTLD, whereas microtubule associated protein 1A (MAP1A), reticulon 4 (RTN4; also referred to as neurite outgrowth inhibitor (Nogo)), protein kinase C gamma (PRKCG), and heat shock protein 90 kDa alpha, class A member 1(HSP90AA1) had significantly fewer phosphospectra compared to control brain. To validate these differences, we examined NDRG2 phosphorylation in FTLD brain by immunoblot analyses, and using a phosphoserine-13 (pSer13) GFAP monoclonal antibody we show an increase in pSer13 GFAP levels by immunoblot concomitant with increased overall GFAP levels in FTLD cases. These data highlight the utility of combining proteomic and phosphoproteomic strategies to characterize post-mortem human brain tissue.

Dataset Information

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

Publications

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

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