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Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice

Nature Neuroscience volume 18, pages 1183–1189 (2015)Cite this article

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Abstract

The microtubule-associated protein tau has been implicated in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. Reducing tau levels ameliorates AD-related synaptic, network, and behavioral abnormalities in transgenic mice expressing human amyloid precursor protein (hAPP). We used mass spectrometry to characterize the post-translational modification of endogenous tau isolated from wild-type and hAPP mice. We identified seven types of tau modifications at 63 sites in wild-type mice. Wild-type and hAPP mice had similar modifications, supporting the hypothesis that neuronal dysfunction in hAPP mice is enabled by physiological forms of tau. Our findings provide clear evidence for acetylation and ubiquitination of the same lysine residues; some sites were also targeted by lysine methylation. Our findings refute the hypothesis of extensive O-linked N-acetylglucosamine (O-GlcNAc) modification of endogenous tau. The complex post-translational modification of physiological tau suggests that tau is regulated by diverse mechanisms.

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Figure 1: PTMs identified in endogenous mouse tau.
Figure 2: Differential modification of tau in the PSD of wild-type mice.

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Acknowledgements

We thank M. Finucane for help with the statistical analysis, S. Lee, W. Guo, J. Kang, X. Wang, D. Kim and G.-Q. Yu for technical assistance, S. Ordway for editorial assistance, and M. Dela Cruz and A. Cheung for administrative assistance. The study was supported by US National Institutes of Health grants to L.M. (NS041787) and to A.L.B. (GM103481).

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Author notes

  1. Jonathan C Trinidad

    Present address: Present address: Department of Chemistry, Indiana University, Bloomington, Indiana, USA.,

  2. Meaghan Morris and Giselle M Knudsen: These authors contributed equally to this work.

Authors and Affiliations

  1. Gladstone Institute of Neurological Disease, San Francisco, California, USA

    Meaghan Morris, Sumihiro Maeda & Lennart Mucke

  2. Department of Biological Chemistry, Biochemistry, Cellular and Molecular Biology Graduate Program, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Meaghan Morris

  3. Department of Pharmaceutical Chemistry, Mass Spectrometry Facility, University of California, San Francisco, California, USA

    Giselle M Knudsen, Jonathan C Trinidad, Alexandra Ioanoviciu & Alma L Burlingame

  4. Department of Neurology, University of California, San Francisco, California, USA

    Sumihiro Maeda & Lennart Mucke

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Contributions

M.M. designed and conducted the mouse experiments, the statistical analyses and wrote the manuscript. G.M.K. analyzed and curated the PTM mapping data, conducted the quantitative mass spectrometry experiments and wrote the manuscript. S.M. conducted PSD fractionation and western blots, and wrote the manuscript. J.C.T. assisted in mass spectrometry method development and wrote the manuscript. A.I. conducted PTM mapping experiments by mass spectrometry. A.L.B. and L.M. supervised the project and wrote the manuscript.

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Correspondence to Lennart Mucke.

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Competing interests

L.M. receives research support from Bristol-Myers Squibb.

Integrated supplementary information

Supplementary Figure 1 Comparison of post-translational modifications in wildtype and hAPP mice.

Tau was isolated from hAPP and wildtype mice and the levels of all quantifiable post-translational modifications were assessed by mass spectrometry. Wildtype tau modification levels in each cohort were defined as 1.0. (a) Tau was isolated from whole cortical and hippocampal lysates either by selective solubility in perchloric acid from wildtype or hyperactive hAPP mice (Cohort A, n = 3 mice per group, 6 months of age) or by immunoprecipitation in randomly selected wildtype and hAPP mice (Cohort B, n=12 mice per group, 7–10 months of age). (b) Tau was immunoprecipitated from PSD fractions of three cohorts of randomly selected mice (Cohorts B, D, and E, n = 10 or 12 mice per group, ages 7–10 months, Supplementary Table 4). After correcting Student’s t-test p-values for multiple comparisons using a Benjamini-Hochberg correction, no p-values were ≤0.05. p=0.06 for pS416 in Cohort A (a) and p=0.29 for tri-p198-210 in Cohort E (b) prior to correction for multiple comparisons. A Student's t-test with Welch's correction was used if the variance differed significantly between groups. Modified amino acids are shown that were unambiguously assigned and conserved in human tau 441. Multiple unambiguous modifications are listed with ‘+’ between residues. If the site was ambiguous, a peptide sequence containing the modified sites was indicated with ‘di-‘ or ‘tri-‘ indicating doubly and triply modified peptides, respectively. Ac, acetylation; GG, GlyGly; Me, methylation; p, phosphorylation; WT, wildtype; hAPP, human amyloid precursor protein transgenic; PCA, isolated by perchloric acid solubility; IP, immunoprecipitated. Quantitative values are means ± SEM.

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Supplementary Figure 2 Comparison of S400 O-GlcNAc modification of tau in wildtype and hAPP mice.

(a) O-GlcNAc modification of tau at S400 (gS400) was measured relative to total tau by western blotting using Ab3925 and Tau5 antibodies in cortical and hippocampal homogenates. (Cohort C, n = 4 mice per group, 7–8 months of age, Supplementary Table 4). Mice lacking endogenous tau served as negative controls. (b) Quantification of S400 O-GlcNAc modification relative to total tau (t(6) = 1.251, p = 0.257 by unpaired, two-tailed Student’s t-test). Quantitative values are means ± SEM.

Supplementary Figure 3 Comparison of PHF1 immunoreactivity in the PSD fraction of wildtype and hAPP mice.

PSD fractions were prepared from combined cortex and hippocampus homogenates. (a) Phosphorylation of tau at the PHF1 epitope (pS396, pS404) in Cohort F (n = 5 WT and 6 hAPP mice per group, 5 months of age, Supplementary Table 4). Similar results were obtained in independent groups of mice from Cohort G (n = 6 mice per group, 5-7 months of age, data not shown). Mice lacking endogenous tau served as negative controls, and actin was used as a loading control. The panels were cropped from the same image of a single membrane to facilitate the comparison of different genotypes. (b) Quantification of PHF1 relative to actin (t(9) = 0.053, p = 0.958 by unpaired two-tailed Student’s t-test). Quantitative values are means ± SEM.

Supplementary Figure 4 Full-length images of the blots presented in the main text.

Western blot corresponding to Figure 2a showing PSD95, tau and α-synuclein levels in two different mice from a replicate cohort at each step of post-synaptic density fractionation.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 618 kb)

Supplementary Methods Checklist (PDF 417 kb)

Supplementary Table 1

Summary of experiments used to identify post-translational modifications of tau (reporting data available for viewing through MASSive). (XLSX 17 kb)

Supplementary Table 2

Peptide identifications for modified and unmodified species of tau (data also provided in MS-Viewer format). (XLSX 69 kb)

Supplementary Table 3

Summary of OGlcNAc-Modified Peptides Identified by LWAC Enrichment. (XLSX 113 kb)

Supplementary Table 4

Mouse cohorts used for quantitative experiments. (XLSX 10 kb)

Supplementary Table 5

Quantified tau peptides from hippocampal and cortical whole lysate of cohort A (data also provided in MS-Viewer format). (XLSX 13 kb)

Supplementary Table 6

Quantified tau peptides from hippocampal and cortical whole lysate, and the post-synaptic density fraction of cohort B (data also provided in MS-Viewer format). (XLSX 20 kb)

Supplementary Table 7

Quantified tau peptides from post-synaptic density fraction of cohort D (data also provided in MS-Viewer format). (XLSX 16 kb)

Supplementary Table 8

Quantified tau peptides from post-synaptic density fraction of cohort E (data also provided in MS-Viewer format). (XLSX 22 kb)

Supplementary Table 9

Comparison of tau modifications identified in this study and previously reported modifications identified in mouse/rat or human brain tissue. (XLSX 28 kb)

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Morris, M., Knudsen, G., Maeda, S. et al. Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice. Nat Neurosci 18, 1183–1189 (2015). https://doi.org/10.1038/nn.4067

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