Phosphoproteomic analysis reveals site-specific changes in GFAP and NDRG2 phosphorylation in frontotemporal lobar degeneration

• Authors: Jeremy Hartford Herskowitz, Emory University; Nicholas Seyfried, Emory University; Duc Duong, Emory University; Qiangwei Xia, Emory University; Howard D Rees III, Emory University; Marla Gearing, Emory University; Junmin Peng, Emory University; James J Lah, Emory University; Allan I Levey, Emory University
• Journal Title: Journal of Proteome Research
• Volume: Volume 9, Number 12
• Publisher: American Chemical Society | 2010-12-03, Pages 6368-6379
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1021/pr100666c
• Subjects: Engineering, Chemical; Chemistry, Biochemistry
• Keywords: Frontotemporal dementia; phosphorylation; immobilized metal-affinity chromatography (IMAC); proteomics; neurodegeneration

Abstract

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 postmortem 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 90kDa 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 postmortem human brain tissue.