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J Biol Chem. 2014 Dec 5;289(49):34389-407. doi: 10.1074/jbc.M114.611368. Epub 2014 Oct 22.

Oligomer formation of tau protein hyperphosphorylated in cells.

Author information

1
From the DZNE, German Center for Neurodegenerative Diseases, 53175 Bonn, Germany, the CAESAR Research Center, 53175 Bonn, Germany.
2
the Department of Biosystems Science and Engineering, ETHZ, 4058 Basel, Switzerland.
3
the Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, 35390 Giessen, Germany, and.
4
the Joint Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg and University of Lübeck, ℅DESY, 22603 Hamburg, Germany.
5
From the DZNE, German Center for Neurodegenerative Diseases, 53175 Bonn, Germany, the CAESAR Research Center, 53175 Bonn, Germany, Eckhard.Mandelkow@dzne.de.

Abstract

Abnormal phosphorylation ("hyperphosphorylation") and aggregation of Tau protein are hallmarks of Alzheimer disease and other tauopathies, but their causative connection is still a matter of debate. Tau with Alzheimer-like phosphorylation is also present in hibernating animals, mitosis, or during embryonic development, without leading to pathophysiology or neurodegeneration. Thus, the role of phosphorylation and the distinction between physiological and pathological phosphorylation needs to be further refined. So far, the systematic investigation of highly phosphorylated Tau was difficult because a reliable method of preparing reproducible quantities was not available. Here, we generated full-length Tau (2N4R) in Sf9 cells in a well defined phosphorylation state containing up to ∼20 phosphates as judged by mass spectrometry and Western blotting with phospho-specific antibodies. Despite the high concentration in living Sf9 cells (estimated ∼230 μm) and high phosphorylation, the protein was not aggregated. However, after purification, the highly phosphorylated protein readily formed oligomers, whereas fibrils were observed only rarely. Exposure of mature primary neuronal cultures to oligomeric phospho-Tau caused reduction of spine density on dendrites but did not change the overall cell viability.

KEYWORDS:

Fluorescence Anisotropy; Neuron; Oligomers; Phosphorylation; Synapse; Tau Protein (Tau); Time-correlated Single Photon Counting; Toxicity

PMID:
25339173
PMCID:
PMC4256367
DOI:
10.1074/jbc.M114.611368
[Indexed for MEDLINE]
Free PMC Article

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