Bichmann et al. Molecular Neurodegeneration (2021) 16:46 https://doi.org/10.1186/s13024-021-00468-x RESEARCH ARTICLE Open Access SETD7-mediated monomethylation is enriched on soluble Tau in Alzheimer’s disease Maria Bichmann1, Nuria Prat Oriol1,2, Ebru Ercan-Herbst1, David C. Schöndorf1,3, Borja Gomez Ramos1,4,5, Vera Schwärzler1, Marie Neu3, Annabelle Schlüter3, Xue Wang6, Liang Jin6, Chenqi Hu6, Yu Tian6, Janina S. Ried7, Per Haberkant8, Laura Gasparini3 and Dagmar E. Ehrnhoefer1,3* Abstract Background: Human tauopathies including Alzheimer’s disease (AD) are characterized by alterations in the post- translational modification (PTM) pattern of Tau, which parallel the formation of insoluble Tau aggregates, neuronal dysfunction and degeneration. While PTMs on aggregated Tau have been studied in detail, much less is known about the modification patterns of soluble Tau. Furthermore, PTMs other than phosphorylation have only come into focus recently and are still understudied. Soluble Tau species are likely responsible for the spreading of pathology during disease progression and are currently being investigated as targets for immunotherapies. A better understanding of their biochemical properties is thus of high importance. Methods: We used a mass spectrometry approach to characterize Tau PTMs on a detergent-soluble fraction of human AD and control brain tissue, which led to the discovery of novel lysine methylation events. We developed specific antibodies against Tau methylated at these sites and biochemically characterized methylated Tau species in extracts from human brain, the rTg4510 mouse model and in hiPSC-derived neurons. Results: Our study demonstrates that methylated Tau levels increase with Tau pathology stage in human AD samples as well as in a mouse model of Tauopathy. Methylated Tau is enriched in soluble brain extracts and is not associated with hyperphosphorylated, high molecular weight Tau species. We also show that in hiPSC-derived neurons and mouse brain, methylated Tau preferentially localizes to the cell soma and nuclear fractions and is absent from neurites. Knock down and inhibitor studies supported by proteomics data led to the identification of SETD7 as a novel lysine methyltransferase for Tau. SETD7 specifically methylates Tau at K132, an event that facilitates subsequent methylation at K130. Conclusions: Our findings indicate that methylated Tau has a specific somatic and nuclear localization, suggesting that the methylation of soluble Tau species may provide a signal for their translocation to different subcellular compartments. Since the mislocalization and depletion of Tau from axons is associated with tauopathies, our findings may shed light onto this disease-associated phenomenon. Keywords: Lysine methylation , Protein methyl transferase , Nuclear tau , Posttranslational modification * Correspondence: [email protected] 1BioMed X Institute, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany 3AbbVie Deutschland GmbH & Co. KG, Neuroscience Discovery, Knollstr. 50, 67061 Ludwigshafen am Rhein, Germany Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bichmann et al. Molecular Neurodegeneration (2021) 16:46 Page 2 of 18 Background Bank and the Southwest Dementia Brain Bank, members Tau is a predominantly cytoplasmic protein that local- of the Brains for Dementia Research Network, as well as izes to axons in mature neurons, where it stabilizes the the Netherlands Brain Bank (NBB), Netherlands Institute microtubule cytoskeleton. Mislocalization of Tau to the for Neuroscience, Amsterdam. Donor characteristics are soma and dendrites occurs early during the development described in Supplementary Tables S1 and S3. All Ma- of AD and other tauopathies [1], and molecular studies terial has been collected from donors for whom a writ- have shown that acetylation at specific lysine residues ten informed consent for a brain autopsy and the use of can modulate this process [2]. Additionally, nuclear or the material and clinical information for research pur- perinuclear localization of Tau has been described in poses had been obtained by the respective brain bank. cultured cells as well as in human brain [3–5]. The mo- lecular mechanism for this nuclear translocation remains Transgenic mouse brain samples unclear, since Tau does not contain a nuclear import or Heterozygous rTg4510 mice and the corresponding localization signal, and no PTM associated with nuclear wild-type (WT) line (licensed from the Mayo Clinic, Tau has been identified to date. Jacksonville Florida, USA) [21] were used in the study. Tau is subject to a large variety of PTMs, with signifi- All mice were bred for AbbVie by Charles River Labora- cantly altered modification patterns in AD and other neu- tories (Sulzfeld, Germany). The mice were in a rodegenerative diseases [6–10]. In AD, Tau accumulates temperature- and humidity-controlled room with a 12: intraneuronally in detergent-insoluble aggregates with fi- 12 h dark/light cycle with ad libitum access to water and brillar morphology, termed neurofibrillary tangles (NFT). food. All animal experiments were performed in full Mass-spectrometry (MS) based studies show that Tau in compliance with the Principles of Laboratory Animal NFTs is heavily modified, predominantly by hyperpho- Care (NIH publication No. 86–23, revised 1985) in an sphorylation and truncation [8, 10–14]. However, it has AAALAC accredited program where veterinary care and become clear that misfolded Tau species can cause signifi- oversight was provided to ensure appropriate animal cant neuronal dysfunction before the appearance of ma- care. All animal studies were approved by the govern- ture NFT [15, 16]. Soluble Tau isolated from human ment of Rhineland Palatinate (Landesuntersuchungsamt patient brains exhibits properties associated with path- Koblenz) and conducted in accordance with the directive ology and induces the aggregation of endogenous Tau in a 2010/63/EU of the European Parliament and of the primary neuron model system [17]. Tau species capable of Council on the protection of animals used for scientific propagating Tau pathology have also been isolated from purpose, the ordinance on the protection of animals the interstitial fluid (ISF) of Tau transgenic mice, confirm- used for experimental or scientific purposes (German ing their presence in the extracellular space [18, 19]. Sol- implementation of EU directive 2010/63; BGBl. I S. uble Tau species may thus be a driver in the spreading of 3125, 3126), the Commission Recommendation 2007/ Tau pathology throughout the brain as observed in AD. 526/EC on guidelines for the accommodation and care Nevertheless, the biochemical properties and PTM pat- of animals used for experimental and other scientific terns that differentiate soluble, pathogenic Tau from purposes, the German Animal Welfare Act (BGBl. I S. physiological species remain unknown. 1206, 1313) amended by Art. 1 G from 17 December Here we demonstrate using an unbiased mass spec- 2018 I 2586. Tg4510 mice express 0N4R human P301L trometry (MS)-based method [20] that lysine mono- mutant hTau under the CaMKIIα promoter. For this methylation is a highly prevalent PTM on soluble Tau. study, mice were culled and hippocampi and cortices We identified six novel methylation sites, two of which were harvested from 8-, 16- and 30-week old heterozy- are enriched in the soluble fraction of AD brain lysate. gous rTg4510 mice and 16 week old WT mice and Monomethylation at specific lysines increases with age stored at − 80 °C until use. in the brain of a mouse model of tauopathy, and is enriched in a pool of soluble Tau. We also find that the Brain lysate preparation protein lysine methyltransferase (PKMT) SETD7 regu- Human and mouse brain samples were homogenized in Tri- lates Tau methylation at two sites in close vicinity, and ton lysis buffer (150 mM NaCl, 20 mM Tris pH 7.5, 1 mM these modified Tau species preferentially localize to nu- EDTA, 1 mM EGTA, 1% Triton-X100 and protease, phos- clear fractions in hiPSC-derived neurons as well as in phatase, demethylase (500 μMIOX1,2μM Daminozide, the brain of rTg4510 mice. 10 μM Paragyline Hydrochloride), deacetylase (10 μMTri- chostatin A, 5 mM Nicotinamide), O-GlcNAcase (1 μM Materials and methods Thiamet-G) inhibitors) with a Dounce homogenizer (Carl Human brain samples Roth). The tubes were twice scraped over a hard surface with Anonymized human post-mortem tissue was obtained in-between