title: Phosphoproteomic analysis reveals site-specific changes in GFAP and NDRG2 phosphorylation in frontotemporal lobar degeneration.
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 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.
authors.affiliation: Department of Neurology, the Center for Neurodegenerative Diseases, Laboratory Medicine, and Proteomics Service Center, Emory University School of Medicine, Atlanta, Georgia 30322, United States.
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Herskowitz
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Seyfried
authors.forename: Duc M
authors.initials: DM
authors.lastname: Duong
authors.forename: Qiangwei
authors.initials: Q
authors.lastname: Xia
authors.forename: Howard D
authors.initials: HD
authors.lastname: Rees
authors.forename: Marla
authors.initials: M
authors.lastname: Gearing
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
authors.forename: James J
authors.initials: JJ
authors.lastname: Lah
authors.forename: Allan I
authors.initials: AI
authors.lastname: Levey
mesh_terms: D000368:Aged; D000595:Amino Acid Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism / Q000473:pathology; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D005260:Female; D057174:Frontotemporal Lobar Degeneration / Q000378:metabolism* / Q000473:pathology; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D011180:Postmortem Changes; D040901:Proteomics / Q000379:methods*; D012694:Serine / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D025521:Tumor Suppressor Proteins / Q000032:analysis* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D005904:Glial Fibrillary Acidic Protein; C466228:NDRG2 protein, human; D010748:Phosphopeptides; D010750:Phosphoproteins; D025521:Tumor Suppressor Proteins; D012694:Serine