Departments of Biochemistry and Neurology Alzheimers Disease Research Center Emory University School of Medicine Brain phosphoproteome network analysis discriminates Alzheimers Disease from other ID: 1043482
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1. Eric B. Dammer Research AssociateDepartments of Biochemistry and NeurologyAlzheimer’s Disease Research CenterEmory University, School of MedicineBrain phosphoproteome network analysis discriminates Alzheimer’s Disease from other tauopathies
2. 2Tau Phosphorylation in neurodegenerationDestabilizes microtubules (MT) - “Loss of function”Facilitates tau aggregation into Paired Helical Filaments (PHF) – “Gain of Function”Both thought to cause neuronal toxicity
3. AD and other tauopathiespTau Pathology Alzheimer's Disease (AD)Corticobasal Degeneration (CBD)Progressive Supranuclear Palsy (PSP)Pick’s DiseaseFrontotemporal Dementia w/ Parkinsonism (FTDP-17)Chronic Traumatic Encephalopathy (CTE)Are the phosphorylation patterns on Tau in these diseases distinct?
4. AD and other tauopathiespTau Pathology Alzheimer's Disease (AD)Corticobasal Degeneration (CBD)Progressive Supranuclear Palsy (PSP)Pick’s DiseaseFrontotemporal Dementia w/ Parkinsonism (FTDP-17)Chronic Traumatic Encephalopathy (CTE)Are the phosphorylation patterns on Tau in these diseases distinct?
5. Phosphoproteomics in ADObjectiveDevelop methods to identify, quantify site-specific changes in protein phosphorylation in brainHypothesis: Does the brain phosphoproteome network discriminate AD from other tauopathies?
6. Quantifying PTMs: PhosphorylationDammer EB et. al., Proteomics 2015 15:508-19.
7. Classifying AD cases by phosphorylation signaturesDammer EB et. al., Proteomics2015 ;15:508-19.
8. Quantifying Tau Phosphorylation2N4RDammer EB et. al., Proteomics 2015 15:508-19.
9. Quantifying Tau Phosphorylation86 phosphopeptideson tau were enriched in AD representing ~30 total pS/T/Y sitesDammer EB et. al., Proteomics2015 ;15:508-19.
10. CBDCtlADResults: Proteins Identified & QuantifiedStarting with 2 µg peptides, we identified 4,479 peptides mapping to 1,161 gene products (1% FDR) across all 21 case samples Orbitrap XL MSNanoAcquityLCHypothesis: Specific brain phosphoprotein networks are differentially enriched in AD and corticobasal degeneration (CBD).
11. MS quantification of phosphopeptides on TauDammer EB, et. al., unpublished
12. Phospho-tau Hotspots in AD or non-AD tauopathyS46S64T71T175S184S199S210S214T220S235S238S289S356S396T403S422S61T69S113T181S185S202T212S217T231S237S262S305Y394S400S404S433ControlADCBD1 2 3 4 5 6 71 2 3 4 5 6 71 2 3 4 5 6 72N4R
13. Tau Phosphorylation Differentiates AD from CBD
14. Stratifying AD and CBD cases by phosphorylation signaturesClustering based on distance in 2-D principal component space
15. Phospho-tau Hotspots in AD or non-AD tauopathyS46S64T71T175S184S199S210S214T220S235S238S289S356S396T403S422S61T69S113T181S185S202T212S217T231S237S262S305Y394S400S404S433ControlADCBD1 2 3 4 5 6 71 2 3 4 5 6 71 2 3 4 5 6 72N4R
16. Phospho-tau Hotspots in AD or non-AD tauopathypS262
17. Phospho-tau Hotspots in AD or non-AD tauopathypS262Log2 Sample Intensity/meanControlsADCBDSummed Peptide Intensity
18. Western Blot Confirmation of MS Intensities
19. Western Blot Confirmation of MS Intensities
20. Correlation of Phospho-tau Site-specific MeasurespS262ControlsADCBDLog2 Sample Intensity/meanSumpS262*pS400pS400*pT403*pT231*pT217Y394/S400*pS396*pT231 WBpS262 WBBest pTau Correlations*Individual Peptide Intensity
21. KIGSTENTau S262Cell-ECM interactionCAMK-like activity,associated with polymerizedPresynaptic Caskin1, Mint1/APPGFAP S323Astrocyte filamentsand cytoskeletonDPYSL3/CRMP-4T623CDK2, GSK3ß, Wnt activitiesaxon elongationBIN1 [S/T]PS265, S267, S272, T276Synaptic vesicleendocytosisVYKSPVVTau S396(PHF epitope)SPP1Osteopontin S219 (isoform 5 S205)CASK/Lin-2S55 (novel)P.GLSTEDpart trypticCAMK2B S395+397dendritic spines,neuronal plasticity,Ca++ regulation ESSDSANCorrelation of Tau pS262 to Other Proteins, Functions
22. Global Phosphopeptide Co-enrichment Network0.60.70.80.91.0HeightModule ColorsCTADCBDBRAAK StageNC Plaque Freq.PMIAgeDurationApoESexFC NFT Freq.Trait correlations range from -1 (blue) to +1 (red)
23. Topology Overlap Matrix (TOM) & Selected ModuleDisease Specificity
24. Conclusions and ImplicationsNetwork analysis of phosphopeptide co-enrichment identifies AD- and non-AD tauopathy hotspots in Tau phosphorylation within postmortem frontal cortex.Co-occurrence of phosphorylation at sites across the phosphoproteome is inferred from coenrichment/correlation.Interrogation of correlated phosphopeptide intensities in the network may yet uncover possible causal interactions between site-specific phosphorylation events and disparate protein functions.
25. AcknowledgementsAllan LeveyNicholas SeyfriedJim LahIan DinerDuc DuongFunding: Marla GearingRujing RenAndrew LeeChad HalesGiovanni CoppolaDan GeschwindVivek Swarup