In sporadic Alzheimer's disease (AD), neurofibrillary lesion formation is preceded by extensive post-translational modification of the microtubule associated protein tau. To identify the modification signature associated with tau lesion formation at single amino acid resolution, immunopurified paired helical filaments were isolated from AD brain and subjected to nanoflow liquid chromatography-tandem mass spectrometry analysis. The resulting spectra identified monomethylation of lysine residues as a new tau modification. The methyl-lysine was distributed among seven residues located in the projection and microtubule binding repeat regions of tau protein, with one site, K254, being a substrate for a competing lysine modification, ubiquitylation. To characterize methyl lysine content in intact tissue, hippocampal sections prepared from post mortem late-stage AD cases were subjected to double-label confocal fluorescence microscopy using anti-tau and anti-methyl lysine antibodies. Anti-methyl lysine immunoreactivity colocalized with 78 ± 13% of neurofibrillary tangles in these specimens. Together these data provide the first evidence that tau in neurofibrillary lesions is post-translationally modified by lysine methylation.
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Acknowledgments This work was supported by National Institutes of Health Grants MH59786 and AG25323 to A.Y. J.K is supported by AG14452. Y.W. and S.T. were supported by the Maryland Cigarette Restitution Fund. The access to the confocal microscope at Ohio State University was supported by Core Center grant P30-NS045758. The authors thank Diane Cripps for excellent technical assistance in the preparation of PHF-tau for LC–MS/MS analysis and also for the optimization of the LC–MS/MS platform used for the studies
- Alzheimer's disease
- Mass spectrometry
- Neurofibrillary tangles
- Paired helical filaments