Acetylation of tau protein at Lys274 in argyrophilic grain disease and Alzheimer's disease Goran Šimić1*, Mirjana Babić Leko1, Danira Bažadona2, Vesna Benković3, Mirta Boban1, Fran Borovečki2, Domagoj Đikić3, Željka Petelin Gadže2, Maja Jazvinšćak Jembrek4, Nada Oršolić3, Patrick R. Hof5 1 Croatian Institute for Brain Research, University of Zagreb School of Medicine, Croatia 2 Clinic of Neurology, Clinical Hospital Center Zagreb, Croatia 3 Faculty of Science, University of Zagreb, Croatia 4 Institute Ruđer Bošković, Zagreb, Croatia 5 Fishberg Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, USA Abstract number: FENS-1615 Poster Board: C010, 5 July 2016 Croatian Science Foundation Grant no. IP-2014-09-9730 Abstract   Argyrophilic grain disease (AGD) is a common sporadic 4R tauopathy. The term ‘argyrophilic grains’ is derived from their strong staining with the Gallyas silver iodide method, although not all silver methods permit their visualization. In combination with Alzheimer’s disease (AD) or alone, AGD significantly contributes to dementia in older age subjects and alone accounts for about 5% of all dementia cases. By using a novel monoclonal antibody specifically immunoreactive to acetylated tau at Lys274 (MAb 359), it has been shown that acetylation of tau protein is an early change in AD brains, which occurs even before tangles are detectable. Interestingly, AGD differs from AD and other tauopathies by lacking tau acetylation at Lys274. By using MAb 359 developed to detect acetylation at Lys274 (a gift from Dr. Li Gan, Gladstone Institute of Neurological Disease, UCSF) we compared immunohistochemically temporal and entorhinal cortex and the hippocampal formation in an AD brain, a brain with pathological changes of AD and AGD, and a brain with AGD alone. Our detailed analysis confirms and expands previous findings and supports further investigations of tau acetylation as a potential new therapeutic target in AD, AGD, and other tauopathies. Introduction Materials and Methods Results The role of acetylation in tau pathology has been controversial. First, it has been proposed that tau protein acetylation may be responsible for tau aggregation in AD. On the contrary, however, it was recently shown that the acetylation of tau on KXGS motifs inhibits phosphorylation of the same motif, consequently also preventing tau polymerization and aggregation. Namely, using a site-specific antibody to detect acetylation of KXGS motifs, it has been found that these sites are hypoacetylated in AD patients as well as in a mouse tauopathy model, suggesting that loss of acetylation on KXGS motifs may be an early event in AD (and that augmenting acetylation of the KXGS motifs would probably decrease tau seeding capacity). The first antibody developed to detect acetylation of tau at Lys280 showed that tau acetylated at this epitope colocalized with other classical markers of tau pathology, and is therefore rather a response to than a cause of the disease process. Strikingly, subsequent usage of the second antibody developed to dectect acetylation at Lys274 residue of tau, has shown that acetylation of this epitope is a very early change in AD brains, which occurs even before tangles are detectable. Interestingly enough, acetylation of tau at Lys274 was detected in all tauopathies (both primary and secondary), except in AgD. Due to the fact that AGD pathological changes are mostly confined to the CA1 subfield of the cornu ammonis, entorhinal and transentorhinal cortices, the amygdala, and the hypothalamic lateral tuberal nuclei, it has been hypothesized that tau acetylation at Lys274 could also promote spreading of tau pathology (whereas in AGD it could have a protective role in this respect).The acetylation of tau protein, however, seems to be much more complex than described here due to the fact that, besides Lys274 and Lys280, there are many self-acetylation sites in addition to sites acetylated by the CBP (cAMP response element binding protein) and p300 acetyltransferase. Lysine residues acetylated by CBP may be deacetylated by histone deacetylase 6 (HDAC6), whereas p300 acetyltransferase sites can be deacetylated by sirtuin 1 (SIRT1). Depending on the sites involved, the acetylation of tau could both inhibit (lysine residues 163, 280, 281, and 369) or facilitate its degradation, at the same time suppressing its phosphorylation and aggregation (lysine residues within the KXGS motifs 259, 290, 321, 353, according to the numbering of the longest isoform; acetylation of these sites is reduced in AD and rTg4510 transgenic mice). Targeting specific lysine residues through specific binding of the molecular tweezer molecule CLR01 has been shown to inhibit both tau and A aggregation and fibrillogenesis in vitro. Brain sample Gen-der Age at onset (y) Age at death (y) Braak stage PMI (h) Cause of death AD 406 M 78  81  C / V < 24  Bronchopneumonia AD 200 F 83.5 88 B / IV < 24 Carcinoma AD + AGD 260  80.5 84 B / IV  < 48  Cardiovascular failure AGD 003  76 77 0 / I/II  Myocardial infarction HC 330  - 89 0 / I  < 12  Hyperpyrexia after urinary infection 240x 960x 960x 240x AGD 003, entorhinal cortex layer II, Gallyas silver staining + Darrow red AGD 003, CA1 region of the hippocampus, Gallyas silver staining + Darrow red AD + AGD 260, CA1 region of the hippocampus, Gallyas silver staining + Darrow red 60x Histochemistry was performed using Gallyas silver stain + light Darrow red (double staining). Immunocytochemistry was performed using MAb 359 - antibody specific for acetylated Lys274, affinity-purified polyclonal antibody specific for acetylated Lys280, as well as anti-tau R3 and anti-tau R4 monoclonal antibodies. 120x AD 406, entorhinal cortex, MAb 359, 1:250 AD 406, CA1, MAb 359, 1:250 AD 200, hippocampus, R3 Mab, 1:3000 References Conclusions Acknowledgements We thank Dr. Li Gan from the Gladstone Institute of Neurological Disease, UCSF, USA, for generously providing us with Mab 359 and Rohan de Silva (UCL, UK) for anti-tau R3 and anti-tau R4 antibodies. The work is funded by the Croatian Science Foundation grant no. 09/16 “Detection and tracking of biological markers for early therapeutic intervention in sporadic Alzheimer’s disease” to G.Š., and by the Croatian Science Foundation grant no. IP-2014-09-9730 ”Tau protein hyperphosphorylation, aggregation and trans-synaptic transfer in Alzheimer’s disease: cerebrospinal fluid analysis and assessment of potential neuroprotective compounds” to G.Š., and in part by NIH grant P50 AG005138 to P.R.H. The authors declare no conflict of interest. To determine the significance of tau acetylation, we performed immunohistochemical and immunocytochemical analysis to define the regional and cellular distribution of acetylated K274 and K280 immunoreactive tau pathology in two AD brain, AD comorbid with AGD, ‘pure’ AGD and one control elderly brain. We also tried to characterize the emergence and temporal course of acetylated K274 and K280-immunoreactive tangles in relation to those neurofibrillary changes induced by misfolded and hyperphosphorylated tau (Braak stages) and R3 and R4 tau. We concluded that: 1. in AGD neurofibrillary changes do not develop or develop very slowly, possibly due to lack of acetylation at K274, 2. acetylation at K274 is present in AD brains, but more cases, including those with mild cognitive impairment (MCI) are needed to determine the tempral course of neurofibrillary changes and their dependence on total tau acetylation and phosphorylation patterns. Cook, C., Carlomagno, Y., Gendron, T.F., Dunmore, J., Scheffel, K., Stetler, C., Davis, M., Dickson, D., Jarpe, M., DeTure, M., Petrucelli, L., 2014. Acetylation of the KXGS motifs in tau is a critical determinant in modulation of tau aggregation and clearance. Hum. Mol. Genet. 23, 104-116. Ferrer, I., Santpere, G., van Leeuwen F.W., 2008. Argyrophilic grain disease. Brain 131, 1416-1432. Grinberg, L.T., Wang, X., Wang, C., Sohn, P.D., Theofilas, P., Sidhu, M., Arevalo, J.B., Heinsen, H., Huang, E.J., Rosen, H., Miller, B.L., Gan, L., Seeley, W.W., 2013. Argyrophilic grain disease differs from other tauopathies by lacking tau acetylation. Acta Neuropathol. 125, 581-593. Irwin, D.J., Cohen, T.J., Grossman, M., Arnold, S.E., Xie, S.X., Lee, V.M., Trojanowski, J. Q., 2012. Acetylated tau, a novel pathological signature in Alzheimer’s disease and other tauopathies. Brain 135, 807-818. Luo, Y., Ma, B., Nussinov, R., Wei, G., 2014. Structural insight into tau protein’s paradox of intrinsically disordered behavior, self-acetylation activity, and aggregation. J. Phys. Chem. Lett. 5, 3026-3031. Min, S.W., Chen, X., Tracy, T.E., Li, Y., Zhou, Y., Wang, C., Shirakawa, K., Minami, S.S., Defensor, E., Mok, S.A., Sohn, P.D., Schilling, B., Cong, X., Ellerby, L., Gibson, B.W., Johnson, J., Krogan, N., Shamloo, M., Gestwicki, J., Masliah, E., Verdin, E., Gan, L., 2015. Critical roles of acetylation in tau-mediated neurodegeneration and cognitive deficits. Nat. Med. 21, 1154-1162.