A GLOBAL (PHOSPHO)PROTEOMIC WORKFLOW SUITABLE FOR ANALYSIS OF FRESH/FROZEN BRAIN TISSUE IN TRANSGENIC MODELS OF NEURODEGENERATION

Abstract

Many neurodegenerative diseases are characterised by abnormal posttranslational modification (PTM) of one or more proteins leading to their aggregation and, by mechanisms not yet fully understood, ultimately to neuronal cell death. One of the most common forms of pathological PTM is phosphorylation, with phosphorylated forms of tau (AD), TDP-43 (ALS, FTLD) and alpha-synuclein (PD) all associated with disease, whereas decreased phosphorylation on huntingtin (HD) is thought to be protective. The role of cell signalling pathways, mostly regulated by kinase/phosphatase cascades is also increasingly being recognised as modulating key aspects of neurodegeneration such as re-activation of cell cycle in neuronal cells and activation of immune cells such as microglia and astrocytes. Accordingly, there is a strong rationale to develop and apply global phosphorylation profiling to neuronal cell cultures and brain tissues used in neurodegenerative disease research. We have recently reported SysQuant, a global phosphoproteomic workflow that employs isobaric Tandem Mass Tags (TMT) and differential chromatography to measure fractions of both enriched phosphopeptides and the un-enriched nonphosphorylated peptides derived from tryptic digestion of tumour cell lysates of up to 10 samples in a single mass spectrometry experiment(1). We have now applied SysQuant to frozen brain tissue from a mouse model of human tauopathy (TMHT Mouse, QPS Austria) treated with vehicle or vehicle + kinase inhibitors, and we are able to demonstrate significant, potentially protective effects on the phosphorylation of aggregating proteins, as well as regulation of key pathways such as glycolysis/gluconeogenesis, calcium signalling, oxidative phosphorylation, citrate cycle along with all major neurodegenerative KEGG pathways. By using a TMT 10plex labelling strategy we were able to generate a detailed, quantitative phosphoproteomic map for 3 x 3 x 3 animals treated with vehicle or one of two kinase inhibitors from hippocampus, cortex and rest of brain regions in approximately 8 weeks. Data from the hippocampus is presented here.