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Tyrosine 136 phosphorylation of α-synuclein aggregates in the Lewy body dementia brain: involvement of serine 129 phosphorylation by casein kinase 2

Tyrosine 136 phosphorylation of α-synuclein aggregates in the Lewy body dementia brain:... Serine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replace- ment of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was signifi- cantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target. Keywords: α-Synuclein, Lewy body dementia, Y136 phosphorylation, S129 phosphorylation, Casein kinase 2 Introduction have not been fully elucidated, accumulation of LB, i.e., Lewy body (LB) diseases, including Lewy body demen- αSyn aggregates, is thought to play a significant role. tia (LBD) and Parkinson’s disease (PD), are progressive A significant proportion of αSyn accumulated within diseases characterized by extensive accumulation of LB is phosphorylated on the C-terminal serine 129 intracellular proteinaceous inclusions composed mainly (S129), while only a small fraction of αSyn is constitu- of aggregated α-synuclein (αSyn) in the brain called LB. tively phosphorylated at this residue in the brain with- Although the pathological mechanisms of LB disease out LB pathology. Earlier in vitro and vivo studies yielded contrasting results regarding the significance of S129 phosphorylation (pS129) for LB formation, showing facil- itatory [10, 32], inhibitory [6, 24], or no effect [17, 30] of *Correspondence: ksano@fukuoka-u.ac.jp Department of Physiology and Pharmacology, Faculty of Pharmaceutical phosphorylation on αSyn aggregation. In mice inoculated Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, with recombinant αSyn (r-αSyn) fibrils, the S129-phos - Fukuoka 814-0180, Japan phorylated and nonphosphorylated forms were shown Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sano et al. acta neuropathol commun (2021) 9:182 Page 2 of 17 to cause prion-like seeding and accumulation of endog- and immunoblotting analyses [9] and mass spectrometry enous αSyn in the brain, and the phosphorylated form [1]. pY125 has been reported to inhibit toxic oligomer showed greater potency to induce the pathology than the formation of αSyn in Drosophila [7], whereas an in vitro nonphosphorylated form [14]. Therefore, although pS129 study showed that pY125 had no influence on aggrega - is not necessarily required for LB formation, it appears to tion of synthetic αSyn [11, 30]. Therefore, αSyn appears induce more severe pathology. to be phosphorylated at Y125 in the human brain, but αSyn tyrosines Y125, Y133, and Y136 are located in the role of pY125 in αSyn aggregate formation has not close proximity to S129 at the C-terminus (Fig.  1a), been fully elucidated. Immunoblotting analysis indicated which raises questions regarding whether these tyros- the presence of phosphorylated Y133 (pY133) at similar ine residues are phosphorylated and whether there are levels in LBD, PD, and control brains [9], suggesting that interactions among the phosphorylated residues, includ- pY133 may not be crucial for LB pathology. In contrast, ing S129 and its association with αSyn aggregate for- there have been few studies regarding the presence of mation. Human r-αSyn incubated with spleen tyrosine phosphorylated Y136 (pY136) in the human brain and its kinase (Syk) [22] and αSyn in pervanadate-treated cul- physiological roles and implications in the pathogenesis tured human cells [8] have been reported to be phos- of LB disease. phorylated at Y125, Y133, and Y136. Several studies Casein kinase (CK) 1 and 2 are ubiquitous serine/ have demonstrated the presence of αSyn phosphorylated threonine protein kinases expressed in all eukaryotes. at Y125 (pY125) in the human brain. An immunohisto- Although both CK1 and CK2 have been shown to con- chemical study identified pY125 within LB in a case of stitutively phosphorylate S129 of αSyn in  vitro [23, 35], familial PD with G51D mutation [15]. Immunoblotting CK2 rather than CK1 was demonstrated to be the major analysis detected pY125 at similar levels in PD and con- enzyme in the brain that phosphorylates S129 of human trol brains [19] but at higher levels in control than LBD αSyn [13]. In a study using transgenic mice expressing brains [7]. Other studies showed that pY125 is not a com- human αSyn, S129-phosphorylated αSyn was shown to ponent of LB in LBD and PD by immunohistochemical be preferentially colocalized with CK2 rather than CK1 bc DN-LBD Li-LBD DN-LBD Li-LBD 1231 2 AD CJD 1231 2 AD CJD 1 MDVFMKGLSKAKEGVVAAAE 21 KTKQGVAEAAGKTKEGVLYV 41 GSKTKEGVVHGVATVAEKTK 37 37 61 EQVTNVGGAVVTGVTAVAQK 81 TVEGAGSIAAATGFVKKDQL GKNEEGAPQEGILEDMPVDP DNEAYEMPSEEGYQDYEPEA kDa αSyn kDa pS129 125 129 133 136 d ef DN-LBD Li-LBD DN-LBD Li-LBD DN-LBD Li-LBD 1231 2 AD CJD PC 1 2312 AD CJD PC 12312 AD CJD 250 250 100 100 37 37 20 20 kDa pY125 kDa pY133 kDapY136 Fig. 1 Insoluble αSyn was phosphorylated at Tyr136 as well as Ser129 in the DN-LBD brain. a Amino acid sequence of human αSyn. C-terminal phosphorylation sites are shown in bold and are underlined. b–f Brain lysates from DN-LBD (cases #1, #2, and #3), Li-LBD (cases #1 and #2), AD, and CJD patients were analyzed by SDS-PAGE followed by immunoblotting with b anti-αSyn antibody D119, c anti-pS129-αSyn antibody ab51253, d anti-pY125-αSyn antibody, e anti-pY133-αSyn antibody, and f anti-pY136-αSyn antibody. Molecular mass markers are indicated in kDa on the left side of each panel. Arrows indicate the top of the gel. In d and e, WT r-αSyn (40 μg) was incubated in the presence of 100 ng/mL Syk (S52-10G; SignalChem Pharmaceuticals) and 200 μM ATP in 100 μL of reaction buffer (20 mM Tris–HCl, pH 7.5, 50 mM KCl, and 10 mM MgCl ) at 37 °C for 1 h, and the sample containing WT r-αSyn (2 μg) was loaded as a positive control (PC) S ano et al. acta neuropathol commun (2021) 9:182 Page 3 of 17 in the nuclei [34]. Furthermore, the catalytic (α) and diagnosis. Three of these subjects had diffuse neocorti - regulatory (β) subunits of CK2 have been shown to be cal LBD (DN-LBD), and the remaining two cases had present in the insoluble fraction of the LBD brain [13] limbic LBD (Li-LBD) according to Braak staging. Brain and in LB in the PD brain [26], respectively. In contrast, tissues were obtained at autopsy from a patient with a CK1 isoform delta (CK1δ) was shown to be absent in LB neuropathological diagnosis of Alzheimer’s disease (AD) in the PD and LBD brain [31]. These studies suggested based on the presence of neurofibrillary tangles and neu - a role of CK2 in LB formation. CK2 has been shown to ritic plaque. The brain specimens showed pure AD with exhibit tyrosine kinase activity in cultured mamma- little or no coexisting LB disease. Prion disease brain tis- lian cells [3, 33]. In yeast, CK2 has also been reported sue specimens were obtained at autopsy from a patient to phosphorylate tyrosine 184 (Y184) of the nucleolar with sporadic Creutzfeldt–Jakob disease (CJD), which immunophilin, Fpr3, through prior phosphorylation at was diagnosed as the classical MM1 subtype according serine 186 (S186) at the + 2 position [37], suggesting that to the genotype at codon 129 of the PRNP gene and the phosphorylated serine residues play a key role in mediat- physicochemical properties of abnormal prion protein Sc ing phosphorylation of neighboring tyrosine residues. In (PrP ). For DN-LBD cases #1, #2, and #3, the individuals the case of αSyn, pS129 and pY125 were shown to have were 92, 80, and 91 years old at the time of death, respec- no effect on each other in Drosophila by immunoblotting tively. Li-LBD cases #1 and #2 were 80 and 87  years old analysis [7] and in vitro by immunoblotting and MALDI- at the time of death, respectively. The patients with AD TOF mass spectrometry (MS) [11], while a study using and CJD were 82 and 75  years old at the time of death, in  vitro NMR spectroscopy reported that pY125 primes respectively. All tissues were taken from the frontal cor- efficient phosphorylation of S129 by CK1 [16]. Therefore, tex and stored at − 80 °C. although tyrosines Y125, Y133, and Y136 are located in close proximity to S129 at the C-terminus of αSyn, it is Preparation of brain lysates not clear whether there is phosphorylation cross-talk Brain tissues were lysed with Triton-deoxycholate between these sites, and whether such cross-talk is func- (DOC) lysis buffer (50 mM Tris–HCl, pH 7.5, containing tionally relevant to the pathogenesis of LB disease. Here, 150  mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxy- we report that insoluble αSyn is highly phosphorylated cholate, 2 mM EDTA, and protease inhibitors) for 30 min at Y136 as well as S129 in the LBD brain. Furthermore, at 4  °C. After centrifugation for 2  min at 2000 × g, the experimental manipulation of the phosphorylation state supernatant was collected and stored at − 80 °C until use. demonstrated that phosphorylation of S129 by CK2 The total protein concentration of the lysates was meas - mediates phosphorylation of Y136 and thus suppresses ured using a bicinchoninic acid (BCA) protein assay kit the formation of αSyn aggregates. (23227; Pierce). Materials and methods Recombinant human α‑synuclein expression Reagents and purification Anti-αSyn polyclonal antibody (D119) and monoclonal The purification of recombinant human αSyn (r-αSyn) antibody (Syn204) were obtained from Bioworld Tech- and mutants was performed as described previously [29]. nology Inc. and Cell Signaling Technology, respectively, Briefly, plasmids carrying the DNA sequence encoding and were used for western blotting. Anti-pS129-αSyn N-terminal His-tagged human wild-type (WT) or non- polyclonal antibody (D1R1R) and anti-β-actin poly- phosphorylatable mutants with substitution of alanine clonal antibody (ab8227) were obtained from Cell Signal- for S129 (S129A) or Y136 (Y136A) were subcloned into ing Technology and Abcam, respectively, and were used the vector pET11a (69436-3; Novagen). The products for western blotting. Monoclonal antibody specific for encoded on the plasmid were overexpressed in compe- pS129-αSyn (ab51253) and polyclonal antibodies against tent BL21 DE3 Escherichia coli cells (DS250; BioDynam- pY125-αSyn (ab10789), pY133-αSyn (ab194910), and ics Laboratory) at 37  °C for 16  h using MagicMedia E. pY136-αSyn (ab194775) used for western blotting and coli Expression Medium (K6815; Invitrogen). The bacte - immunohistochemical staining were purchased from rial pellets were suspended in CelLytic B (B7435; Sigma- Abcam. Casein kinase 2 (CK2, P6010L) was obtained Aldrich) in the presence of 1 g/mL lysozyme (120-02674; from New England Biolabs Inc. ATP (A2383) was Wako) and 500 U/mL benzonase nuclease (70664-3; acquired from Sigma-Aldrich. Novagen). The lysate was centrifuged at 10,000  rpm for 30  min at 4  °C, and the supernatant was incubated with Brains of patients Ni–NTA Superflow resin (30430; Qiagen) at room tem - LBD brain tissues were obtained at autopsy from five perature for 30 min, and then loaded onto a gravity flow patients with histopathologically confirmed clinical column (Muromac mini-column; Muromachi Chemical Sano et al. acta neuropathol commun (2021) 9:182 Page 4 of 17 Inc.). The His-tagged proteins were eluted with a buffer antibody binding was detected by the labeled strepta- containing 300  mM NaCl, 50  mM Tris–HCl (pH 8.0), vidin–biotin method (DAKO). Peroxidase-conjugated and 250  mM imidazole, and dialyzed against 10  mM streptavidin was visualized with 3′3-diaminobenzidine phosphate buffer (pH 7.0) in cellulose dialysis tubing (7411-49-6; Wako) as the chromogen. Immunostained (68035; Thermo Scientific) at 4  °C overnight. Cleavage sections were lightly counterstained with Mayer’s of the His-tagged from the proteins followed by removal hematoxylin. of uncleaved His-tagged proteins was performed using the TAGZyme system (34300; Qiagen). The non-tagged Gel staining and in‑gel digestion proteins were then dialyzed against deionized distilled After separation of proteins by SDS-PAGE, the gels were water in cellulose dialysis tubing at 4 °C overnight and fil - stained at room temperature for 2 h with Coomassie Bril- tered with a 0.2-µm syringe filter (SLLGH25; Millipore). liant Blue solution (11642; Nacalai Tesque). The stained The purity of r-αSyn was ≥ 99.9% as estimated by SDS- bands near 16  kDa were excised and soaked in 50  mM PAGE and western blotting. After purification, aliquots Tris–HCl, pH 8.0, containing 50% acetonitrile for 30 min. of r-αSyn were stored at − 80 °C until use. The gel was dried in a Speed-Vac (Savant) and incubated in 50  mM triethylammonium bicarbonate containing In vitro phosphorylation of recombinant α‑synuclein proteomics grade trypsin (T7575; Sigma-Aldrich) at r-αSyn (40 μg) was incubated in the presence of 400 units 37  °C for 20  h. The digests were extracted from the gel of casein kinase 2 and 200 μM ATP in 100 μL of reac- with 100–200 μL of 0.1% TFA containing 60% acetoni- tion buffer (20  mM Tris–HCl, pH 7.5, 50  mM KCl, and trile. These extracts were evaporated in a Speed-Vac and 10 mM MgCl ) at 37 °C. stored at − 80 °C until assayed. Gel electrophoresis and immunoblotting Nano‑flow liquid chromatography‑ion trap mass For SDS-PAGE, samples were boiled for 5  min at 95  °C spectrometry (LC–MS/MS) with SDS loading buffer (62.5  mM Tris–HCl, pH 6.8, Peptides were resuspended in 0.1% formic acid contain- containing 5% 2-mercaptoethanol, 2% SDS, 5% sucrose, ing 2% acetonitrile. Measurements were performed on and 0.005% bromophenol blue) and were separated by a nano-flow high-performance liquid chromatography 15% SDS-PAGE. For BN-PAGE, samples were prepared (HPLC) system (EASY-nLC 1200; Thermo Fisher Sci - in a buffer containing 0.25% Coomassie Brilliant Blue entific). The samples were loaded onto packed nano- G-250 and separated by 4–16% Bis–Tris native PAGE capillary columns (0.075  mm I.D. × 125  mm L, particle (BN1004BOX; Invitrogen). The proteins were transferred diameter 3  μm, NTCC-360/75-3-123; Nikkyo Technos onto Immobilon-P membranes (IPVH304F0; Millipore) Co., Ltd.), which were eluted at a flow rate of 300 nL/min in transfer buffer containing 15% methanol followed by with a 2–80% linear gradient of acetonitrile for 80  min. blocking with 5% nonfat dry milk in TBST (10 mM Tris– Eluting peptides were detected with an ion trap mass HCl, pH 7.8, 100  mM NaCl, 0.1% Tween 20) for 2  h at spectrometer (QExactive HF; Thermo Fisher Scientific). 4  °C. Membranes were then probed with specific pri - For ionization, the spray voltage and capillary tempera- mary antibodies (1:2500) and appropriate horseradish ture were set to 2.0 kV and 250 °C, respectively. The mass peroxidase-conjugated secondary antibody (111-035-003 acquisition method consisted of one full MS survey scan or 115-035-003, 1:5000; Jackson ImmunoResearch Labs). with an Orbitrap resolution of 60,000 followed by mass Immunoreactive bands were visualized using Chemi- spectrometry (MS/MS) of the most abundant precursor Lumi One L (07880-70; Nacalai Tesque) or ECL prime ions from the survey scan with an Orbitrap resolution of Western Blotting Detection Reagents (PRN2232; GE 15,000. Dynamic exclusion for MS/MS was set to 30  s. Healthcare Life Sciences). MS was performed with a scan range of 350–1800  m/z in positive ion mode, followed by data-dependent MS/ Immunohistochemical staining MS using the HCD operating mode on the top 15 ions The brain tissues were fixed in 20% neutral buffered for - in order of abundance. The data were analyzed with Pro - malin, embedded in paraffin, cut into Sects.  8  μm thick teome Discoverer (Thermo Fisher Scientific) and Mascot with a microtome, and placed on glass slides. After software (Matrix Science). deparaffinization and rehydration, the tissue sections were heated for 40 min at 98 °C for antigen retrieval. All Transmission electron microscopy (TEM) sections were immersed in hydrogen peroxide (0.3%) Negative staining was performed on 400 mesh copper solution for 10  min to quench endogenous peroxidase grids with a carbon support film. Aliquots of the samples activity and incubated with specific primary antibodies were adsorbed onto the grids, and the residual solution (1:100) diluted in PBS containing 1% BSA for 2 h. Primary was carefully removed from the grid surface using filter S ano et al. acta neuropathol commun (2021) 9:182 Page 5 of 17 Preparation of lysate from cell culture paper. The grids were stained with 2% uranyl acetate. Cells were washed with PBS and harvested, and cellular Once dry, the samples were viewed with a transmission proteins were extracted with Triton-DOC lysis buffer. electron microscope (TEM) (JEM-2000FX; JEOL) at The total protein concentration of the lysates was meas - 200 kV. ured using a BCA protein assay kit (23227; Pierce). Thioflavin T (ThT) assay Statistics r-αSyn (40 μg) was prepared in 96-well optical black-bot- Statistical analyses were performed with OriginPro 2015 tomed plates (265301; Nunc) in 100 μL of reaction buffer (OriginLab). The 2-tailed Student’s t test was used for (20  mM Tris–HCl, pH 7.5, 50  mM KCl, 10  mM MgCl , comparisons between two groups. One-way analysis of and 10  μM thioflavin T [T Th ]). The 96-well plates were variance (ANOVA) followed by the Tukey–Kramer test covered with sealing tape (236366; Nunc) and incubated was used for comparisons among more than two groups. at 40 °C in a plate reader (FLUOstar Omega plate reader; In all analyses, P < 0.05 was taken to indicate statistical BMG Labtech) with intermittent shaking, consisting of significance. 30 s of double orbital shaking at 500 rpm and no shaking for 30 s. ThT fluorescence intensity on the bottom of the plates was measured every 10 min to monitor the kinet- ics of amyloid fibril formation using monochromators Results with excitation and emission wavelengths of 450 ± 10 and Insoluble aggregates of αSyn are predominantly 480 ± 10  nm, respectively. Lag phase was defined as the phosphorylated on Y136 as well as S129 time required to reach fluorescence greater than or equal within the C‑terminal region in the LBD brain to the mean fluorescence intensity for all samples within Brain lysates from LBD patients were analyzed by SDS- the first 24 h of reaction plus 4 × the standard deviation. PAGE followed by immunoblotting with anti-αSyn anti- body, and all exhibited a band at approximately 20  kDa Preparation of r‑αSyn seeds with another band just below 20  kDa likely correspond- r-αSyn (40  μg) was incubated in 20  mM Tris–HCl, pH ing to full-length and cleaved αSyn, respectively (Fig. 1b). 7.5, containing 50 mM KCl, and 10 mM MgCl , at 37 °C All lysates also exhibited an αSyn-positive band at under agitation at 2000  rpm for 3  days. As a control for approximately 50 kDa, which was presumably due to the agitated r-αSyn, the mix was prepared without incuba- oligomeric and/or ubiquitinated αSyn. Although these tion and agitation immediately before use. The morphol - αSyn-positive bands did not differ significantly between ogy of r-αSyn seeds was confirmed by TEM. cases, multimeric αSyn with molecular weight > 250 kDa was observed only in three cases of DN-LBD (Fig.  1b, Cell culture, treatment of r‑αSyn seeds, and transfection Additional file  1: Fig. S1). Immunostaining with an anti- of plasmids body against pS129-αSyn detected only a major band Human neuroblastoma SH-SY5Y cells were maintained at > 250  kDa in all cases of DN-LBD (Fig.  1c, Additional at 37  °C in 5% C O in DMEM/Ham’s F12 medium file  1: Fig. S1). A band of > 250  kDa was detected with (042-30795; Wako) containing 15% fetal bovine serum antibodies against αSyn and pS129-αSyn in Li-LBD case (SH30910; GE Healthcare Hyclone), penicillin–strep- #2, but not case #1, but the intensity was significantly tomycin (168-23191; Wako), and MEM Non-essential lower than in DN-LBD (Fig.  1b, c), indicating that mul- Amino Acids Solution (139-15651; Wako). Introduc- timer formation and pS129 of αSyn are relevant to dis- tion of r-αSyn seeds and/or pcDNA3.1 plasmid encod- ease progression. Consistent with our previous report ing human WT or Y136A mutant αSyn into cells [29], these results suggest that the detergent-insoluble was performed using Lipofectamine LTX (Invitro- multimer of αSyn highly phosphorylated at S129 with gen) according to the manufacturer’s instructions. molecular weight > 250  kDa is present in the DN-LBD Lipofectamine/r-αSyn seeds and/or plasmid complexes brain. We next examined the presence of αSyn phospho- were prepared in Optimem (31985062; Gibco) by mixing rylated at C-terminal tyrosine residues surrounding S129. Lipofectamine LTX reagent in the presence or absence of No signals were detected with antibodies against pY125- r-αSyn seeds (4 μg) or plasmid (5 μg). Cells were cultured αSyn and pY133-αSyn in any cases (Fig. 1d, e). A 50-kDa to 40–50% confluence in 6-well plates and treated with band was detected with an antibody against pY136-αSyn the complexes. For CK2 inhibitor treatment, cells were in all cases (Fig.  1f, Additional file  1: Fig. S1). Moreover, incubated with 10 or 100  nM 4,5,6,7-tetrabromobenzo- the three cases of DN-LBD showed a strong immunore- triazole (TBB, ab120988; Abcam) for 1 h before addition active band with mass > 250 kDa (Fig. 1f, Additional file  1: of the complexes. cells were incubated for 3 d after intro- Fig. S1). These results suggest that the insoluble aggre - duction of r-αSyn seeds and/or plasmid. gates of αSyn are predominantly phosphorylated on Y136 Sano et al. acta neuropathol commun (2021) 9:182 Page 6 of 17 as well as S129 within the C-terminal region in the LBD Consistent with our previous study [29], the deposits brain. showed positive staining for pS129-αSyn, and were also We next examined phosphorylation of the C-termi- positive for pY136-αSyn in both DN-LBD and Li-LBD nus of αSyn in sections of the frontal cortex of DN-LBD brains (Fig. 2a). The numbers of pS129-αSyn and pY136- and Li-LBD brains by immunohistochemical analysis. αSyn deposits were significantly greater in DN-LBD than pS129 pY125 pY133 pY136 DN-LBD pS129 pY125 pY133 pY136 Li-LBD 12 12 12 12 ** 10 10 10 10 *** 8 8 8 8 6 6 6 6 4 4 4 4 2 2 2 2 0 0 0 0 DN-LBD Li-LBD DN-LBD Li-LBD DN-LBD Li-LBD DN-LBD Li-LBD Fig. 2 Deposits of phosphorylated αSyn at C-terminal tyrosine residues are present in LBD brain. a Immunohistochemical staining using antibodies against pS129-αSyn ab51253, pY125-αSyn, pY133-αSyn, and pY136-αSyn in the frontal cortex from two patients with DN-LBD (case #3) and Li-LBD (case #2). Regions surrounded by white rectangles in the upper panels are magnified and shown in the lower panels. Typical pathological αSyn deposits are indicated by arrowheads. Scale bars, 50 μm. b The number of phosphorylated αSyn deposits > 2 μm was counted in 12 randomly selected areas of 10 mm in each tissue specimen using ImageJ. Data are presented as means ± standard deviation. Statistical significance was determined using the 2-tailed Student’s t test. **s < 0.01, ***P < 0.001 vs. Li-LBD Number of pS129-αSyn deposits Number of pY125-αSyn deposits Number of pY133-αSyn deposits Number of pY136-αSyn deposits S ano et al. acta neuropathol commun (2021) 9:182 Page 7 of 17 Li-LBD (Fig. 2b). Deposits were not detected in the brain 15 days revealed the formation of αSyn aggregates with a of a non-DLB patient by immunohistochemical analy- molecular weight of ~ 1048  kDa in addition to a band of sis using anti-pS129-αSyn antibody (Additional file  1: monomeric αSyn at approximately 60 kDa (Fig.  3d). The Fig. S2). Both DN-LBD and Li-LBD brains showed posi- aggregated WT r-αSyn was also formed more efficiently tive staining with anti-pY125-αSyn and anti-pY133-αSyn in the presence of both CK2 and ATP than in the absence antibodies (Fig.  2a), but pT125-αSyn and pY133-αSyn of either, suggesting that pS129 accelerates aggregate for- deposits were present at low levels in both DN-LBD and mation of αSyn (Fig. 3d, e). Li-LBD compared to pS129-αSyn and pY136-αSyn depos- its in DN-LBD (Fig. 2b). The lack of detectable pY125 and pY133 in the brain by immunoblotting was likely due to CK2 phosphorylates Y125 and Y136 of aggregated αSyn low levels of the deposits, and phosphorylated αSyn may through prior S129 phosphorylation in vitro not be detected by immunoblotting in brains with less WT r-αSyn or S129A r-αSyn was incubated with CK2 than two phosphorylated deposits per 10 mm of tissue. and ATP for 7 days, separated by SDS-PAGE, and the gel There were no significant differences in the numbers of portion corresponding to a molecular weight of ~ 16 kDa pY125-αSyn and pY133-αSyn deposits between the two was cut out as shown in Additional file  1: Fig. S5. The gel types of LBD (Fig. 2b). Thus, the results of immunohisto - slice was digested with trypsin and analyzed by LC–MS/ chemical analysis indicated the presence of pS129-αSyn- MS. As shown in Table  1, we identified 23 and seven and pY136-αSyn-positive deposits in larger amounts in phosphorylated peptides from WT and S129A r-αSyn, the brains of patients with DN-LBD than Li-LBD, and respectively. LC–MS/MS analysis detected pS129 in that pY125-αSyn and pY133-αSyn are present in relatively WT r-αSyn. Both WT r-αSyn and S129A r-αSyn con- small amounts in the brains of patients with both types tained pS87. WT r-αSyn showed phosphorylation at four of LBD. threonine residues, i.e., T44, T54, T59, and T64, while S129A r-αSyn showed phosphorylation at five threonine residues, i.e., T22, T44, T54, T59, and T75. There have Ser129 phosphorylation by CK2 promotes the formation been no previous reports of phosphorylation of αSyn at of insoluble αSyn aggregates in vitro T22, T44, T54, T59, T64, T75, and S87 by CK2, and the Analysis of WT r-αSyn by SDS-PAGE followed by number of peptides including these residues with phos- immunoblotting with anti-αSyn antibody revealed phorylation was very low in comparison to peptides detergent-insoluble aggregates > 250  kDa after 15  days with pS129 (Table  1). Therefore, CK2 may phosphoryl - of incubation, which were formed more efficiently in ate r-αSyn on these residues at very low levels in  vitro. the presence of both CK2 and ATP than in the absence Moreover, peptides with pY125 and pY136 were identi- of either (Additional file  1: Fig. S3). Insoluble aggregates fied in WT r-αSyn. The number of peptides with pY136 and 16-kDa WT r-αSyn monomer incubated with CK2 was comparable to that of peptides with pS129, and and ATP were detected by anti-pS129-αSyn antibody, only one peptide with pY125 was detected. In contrast, whereas no immunoreactivity was detected with anti- no peptides with C-terminal tyrosine phosphorylation pS129-αSyn antibody for WT r-αSyn incubated under were identified in S129A r-αSyn. After SDS-PAGE of WT other conditions (Additional file  1: Fig. S3). The insolu - r-αSyn, immunoblotting with anti-pY125-αSyn antibody ble aggregates of S129-phosphorylated WT r-αSyn were and anti-pY136-αSyn antibody detected detergent-insol- detected after 1  day of incubation (Fig.  3a, b). Mean- uble aggregates > 250  kDa after 7 and 3  days of incuba- while, S129A r-αSyn incubated with CK2 and ATP was tion with CK2 and ATP, respectively (Fig.  4a, b). No not phosphorylated at Ser129 and formed no insoluble immunoreactivity was detected on blots of S129A with aggregates (Fig.  3a, b). TEM revealed that WT r-αSyn anti-pY125-αSyn or anti-pY136-αSyn antibody (Fig.  4a, subjected to 15-day incubation with CK2 and ATP con- b). In BN-PAGE followed by immunoblotting analysis sisted exclusively of amorphous aggregates, but no such of WT r-αSyn incubated for 15  days, bands of approxi- aggregates were seen without incubation (Fig.  3c). The mately 1048  kDa were detected with antibodies against amorphous aggregates were present in relatively small pY125-αSyn and pY136-αSyn only in the presence of CK2 amounts in WT r-αSyn incubated in the absence of CK2 and ATP (Fig.  4c, d). Moreover, pS129 and pY136 were and ATP (Additional file  1: Fig. S4). These results were abolished and the formation of insoluble aggregates was consistent with a previous report [29] indicating that significantly inhibited by the addition of alkaline phos - CK2-induced pS129 accelerated aggregate formation of phatase in WT r-αSyn incubated with CK2 and ATP WT r-αSyn, and the insoluble aggregates were observed (Additional file  1: Fig. S6). These results suggested that in the mass range > 250 kDa as in the DN-LBD brain. Blue CK2 phosphorylates Y125 and Y136 of aggregated WT native (BN)-PAGE followed by immunoblotting analy- sis with anti-αSyn antibody of WT r-αSyn incubated for Sano et al. acta neuropathol commun (2021) 9:182 Page 8 of 17 Fig. 3 Ser129 phosphorylation by CK2 accelerates αSyn aggregate formation. a, b WT r-αSyn or S129A r-αSyn after 0–14 days of incubation with CK2 and ATP was analyzed by SDS-PAGE followed by immunoblotting with a anti-αSyn antibody D119 and b anti-pS129-αSyn antibody ab51253. c WT r-αSyn after 0 or 15 days of incubation with CK2 and ATP was examined by TEM. Bars, 100 nm. d, e WT r-αSyn after 0 or 15 days of incubation in the presence (+) or absence (−) of CK2 or ATP was analyzed by BN-PAGE followed by immunoblotting with d anti-αSyn antibody D119 and e anti-pS129-αSyn antibody ab51253. Molecular mass markers are indicated in kDa on the left side of each panel. Arrows indicate the top of the gel. In a, b, d and e, one representative blot from three independent experiments is shown r-αSyn in  vitro, and that the tyrosine phosphorylation is CK2 and ATP were detected by anti-pS129-αSyn anti- mediated through pS129. body (Additional file  1: Fig. S7), while no immunoreac- tivity was detected with anti-pY136-αSyn antibody in any Y136 phosphorylation prevents S129 phosphorylation samples (Additional file  1: Fig. S7). Although no αSyn- and aggregate formation of αSyn in vitro positive band > 250  kDa of WT r-αSyn or Y136A r-αSyn On SDS-PAGE followed by immunoblotting, Y136A was observed within 40 h of incubation in the absence of r-αSyn incubated for 14  days also exhibited detergent- CK2 and ATP (Additional file  1: Fig. S8), the band was insoluble aggregates > 250 kDa, which were most strongly detected after 8  h of incubation in the presence of CK2 detected in the presence of both CK2 and ATP (Addi- and ATP (Fig.  5a). The intensity of the αSyn-positive tional file  1: Fig. S7). Insoluble aggregates > 250  kDa as band > 250  kDa of Y136A r-αSyn was significantly well as 16 kDa monomer of Y136A r-αSyn incubated with greater than that of WT r-αSyn after 8  h of incubation S ano et al. acta neuropathol commun (2021) 9:182 Page 9 of 17 Table 1 List of identified peptides derived from r-αSyn incubated with CK2 and ATP for 7 days Query Start End Observed Mr(expt) Mr(calc) ppm Score Expect Peptide WT 5284 44 58 802.9034 1603.792 1603.797 − 2.97 65 3.5E−07 K.TpKEGVVHGVATVAEK.T 11012 46 80 1172.286 3513.836 3513.808 8.02 51 8.4 E−06 K.EGVVHGVATpVAEKTKEQVTN*VGGAVVTGVTAVAQK.T 8325 59 80 746.3851 2236.134 2236.147 − 5.77 47 0.000021 K.TKEQVTpNVGGAVVTGVTAVAQK.T 8336 59 80 1120.068 2238.122 2238.115 3.44 21 0.0083 K.TpKEQ*VTN*VGGAVVTGVTAVAQK.T 11170 59 96 924.9822 3695.9 3695.914 − 3.77 76 2.4 E−08 K.TpKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVK.K 5009 81 96 779.8777 1557.741 1557.744 − 1.95 88 1.6 E−09 K.TVEGAGSpIAAATGFVK.K 12198 98 140 1228.245 4908.951 4908.947 0.96 20 0.011 K.DQLGKN*EEGAPQEGILEDMPVDPDN*EAYEMPSpEEGYQDYEPEA 12214 98 140 1232.002 4923.98 4923.958 4.6 34 0.00044 K.DQLGKN*EEGAPQEGILEDMoPVDPDNEAYEMPSEEGYQDYpEPEA 12225 98 140 1235.754 4938.985 4938.969 3.36 21 0.01 K.DQLGKNEEGAPQEGILEDMoPVDPDNEAYEMoPSEEGYQDYpEPEA 12226 98 140 1235.756 4938.994 4938.969 5.24 30 0.0011 K.DQLGKNEEGAPQEGILEDMoPVDPDNEAYpEMoPSEEGYQDYEPEA 12228 98 140 1236.002 4939.979 4939.953 5.31 23 0.0073 K.DQLGKNEEGAPQEGILEDMoPVDPDN*EAYEMoPSEEGYQDYpEPEA 12229 98 140 1236.003 4939.982 4939.953 5.91 26 0.0032 K.DQ*LGKNEEGAPQEGILEDMoPVDPDNEAYEMoPSEEGYQDYpEPEA 11769 103 140 1092.433 4365.703 4365.693 2.34 21 0.0077 K.NEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYpEPEA 11770 103 140 1456.242 4365.704 4365.693 2.61 18 0.017 K.NEEGAPQEGILEDMPVDPDNEAYEMPSpEEGYQDYEPEA 11805 103 140 1461.57 4381.688 4381.688 0.11 24 0.004 K.NEEGAPQEGILEDMPVDPDNEAYEMoPSpEEGYQDYEPEA 11806 103 140 1461.572 4381.693 4381.688 1.19 22 0.0069 K.NEEGAPQEGILEDMPVDPDNEAYEMoPSpEEGYQDYEPEA 11807 103 140 1461.572 4381.694 4381.688 1.45 28 0.0015 K.NEEGAPQEGILEDMoPVDPDNEAYEMPSpEEGYQDYEPEA 11808 103 140 1096.431 4381.696 4381.688 1.93 25 0.0034 K.NEEGAPQEGILEDMPVDPDNEAYEMoPSpEEGYQDYEPEA 11809 103 140 1461.576 4381.707 4381.688 4.54 28 0.0017 K.NEEGAPQEGILEDMPVDPDNEAYEMoPSpEEGYQDYEPEA 11851 103 140 1100.425 4397.671 4397.682 − 2.69 31 0.00073 K.NEEGAPQEGILEDMoPVDPDNEAYEMoPSpEEGYQDYEPEA 11855 103 140 1466.906 4397.696 4397.682 3.13 31 0.00086 K.NEEGAPQEGILEDMoPVDPDNEAYEMoPSEEGYQDYpEPEA 11856 103 140 1466.908 4397.701 4397.682 4.29 24 0.004 K.NEEGAPQEGILEDMoPVDPDNEAYEMoPSEEGYQDYpEPEA 11870 103 140 1467.57 4399.687 4399.651 8.37 24 0.0039 K.NEEGAPQEGILEDMoPVDPDN*EAYEMoPSpEEGYQDYEPEA S129A 3243 11 23 691.3577 1380.701 1380.701 − 0.32 28 0.0014 K.AKEGVVAAAEKTpK.Q 1528 22 32 570.2711 1138.528 1138.538 − 9.45 46 0.000028 K.TpKQGVAEAAGK.T 4891 44 58 802.8981 1603.782 1603.797 − 9.67 60 9.1 E−07 K.TpKEGVVHGVATVAEK.T 3201 46 58 688.3341 1374.654 1374.654 − 0.57 42 0.00006 K.EGVVHGVATpVAEK.T 8000 59 80 1119.079 2236.144 2236.147 − 1.15 64 4.2 E−07 K.TpKEQVTNVGGAVVTGVTAVAQK.T 8001 59 80 746.3887 2236.144 2236.147 − 1.02 25 0.003 K.TKEQVTNVGGAVVTGVTpAVAQK.T 5372 81 97 843.9226 1685.831 1685.839 − 4.98 52 6.4 E−06 K.TVEGAGSpIAAATGFVKK.D p indicates phosphorylation sites; o indicates oxidation site *Indicates deamidation site (Fig.  5b). Moreover, ThT spectroscopic assay showed an presence of CK2 and ATP than other reactions (Fig.  5e). increase in fluorescence in reactions with Y136A r-αSyn The lag phase was significantly shorter in reactions with in the presence of CK2 and ATP, but not in their absence, Y136A r-αSyn in the presence of CK2 and ATP than whereas no increase in fluorescence was observed with other reactions (Fig. 5f ). These results suggest that block - WT r-αSyn regardless of the presence or absence of CK2 ing Y136 phosphorylation facilitates aggregation and and ATP within 7 days (Fig. 5c, Additional file  1: Fig. S9). amyloid fibril formation of αSyn. The insoluble aggre - Y136A r-αSyn amyloid fibrils were observed in reactions gates > 250  kDa were detected in WT r-αSyn, but not in the presence of CK2 and ATP on day 7 of incubation Y136A r-αSyn, with an antibody against pY136-αSyn and by TEM analysis (Fig.  5d, Additional file  1: Fig. S10). In in both WT and Y136A r-αSyn with an antibody against contrast, amorphous aggregates, but not fibrils, were pS129-αSyn after 8  h of incubation in the presence of exclusively observed in reactions with WT r-αSyn in CK2 and ATP (Fig.  6a, b). Interestingly, the intensity of the presence of CK2 and ATP (Fig.  5d, Additional file  1: the pS129-αSyn-positive band of Y136A r-αSyn was sig- Fig. S10). The maximal fluorescence intensity was sig - nificantly higher than that of WT r-αSyn at 8  h of incu - nificantly higher in reactions with Y136A r-αSyn in the bation (Fig.  6c). Unlike WT r-αSyn, immunoreactivity Sano et al. acta neuropathol commun (2021) 9:182 Page 10 of 17 Fig. 4 Ser129 phosphorylation by CK2 elicits Tyr125 and Tyr136 phosphorylation of αSyn. a, b WT r-αSyn or S129A r-αSyn after 0–14 days of incubation with CK2 and ATP was analyzed by SDS-PAGE followed by immunoblotting with a anti-pY125-αSyn antibody and b anti-pY136-αSyn antibody. c, d WT r-αSyn after 0 or 15 days of incubation in the presence (+) or absence (−) of CK2 or ATP was analyzed by BN-PAGE followed by immunoblotting with c anti-pY125-αSyn antibody and d anti-pY136-αSyn antibody. Molecular mass markers are indicated in kDa on the left side of each panel. Arrows indicate the top of the gel. In a–d, one representative blot from three independent experiments is shown with anti-pS129-αSyn antibody was more prominent in These observations indicated that blocking of Y136 phos - the insoluble aggregates with molecular weight > 250 kDa phorylation facilitated aggregate formation and phospho- than 16-kDa monomer of Y136A r-αSyn at 8–40  h of rylation at other C-terminal residues, especially S129, of incubation (Fig.  6b). The insoluble aggregates > 250  kDa r-αSyn. The results suggest that pY136 inhibits pS129 of were detected at relatively low levels in WT r-αSyn and αSyn and thereby prevents aggregate formation. Y136A r-αSyn with anti-pY125-αSyn antibody after 8  h of incubation in the presence of CK2 and ATP (Fig.  6d). Exogenous oligomeric αSyn converts endogenous The intensity of the pY125-αSyn-positive band of Y136A αSyn into insoluble aggregates with pS129 and pY136 r-αSyn was significantly higher than that of WT r-αSyn in a prion‑like manner and Y136 phosphorylation is at 8 h of incubation (Fig.  6e). Only a low level of insolu- involved in protecting against S129 phosphorylation and ble aggregates > 250  kDa was detected in Y136A r-αSyn aggregate formation of αSyn in cultured cells by anti-pY133-αSyn antibody after 32 and 40  h of incu- We reported previously that oligomeric r-αSyn produced bation in the presence of CK2 and ATP, whereas no by agitation shows potent prion-like seeding activity immunoreactivity was detected with anti-pY133-αSyn in  vitro by real-time quaking-induced conversion (RT- antibody for WT r-αSyn (Additional file  1: Fig. S11). QUIC) seeding assay [29]. We confirmed the contribution S ano et al. acta neuropathol commun (2021) 9:182 Page 11 of 17 WT Y136A WT Y136A a a 08 16 24 32 40 08 16 24 32 40 (h) 08 16 24 32 40 08 16 24 32 40 (h) kDa *** 15 kDa pY136 WT Y136A αSyn WT Y136A bc 08 16 24 32 40 08 16 24 32 40 (h) CK2+ATP c d WT WT *** CK2+ATP CK2+ATP CK2+ATP WT Y136A Y136A Y136A kDa 100000 50 60000 200 40000 20 07 123456 WT Y136A Time (d) pS129 WT Y136A de ** ** ef 816243240( 08 16 24 32 40 h) ** ** ** * ** 260000 kDa 250 300 20000 0 WT Y136A pY125 Fig. 6 Blocking Tyr136 phosphorylation promotes phosphorylation of αSyn at Ser129 and Tyr125 induced by CK2. WT r-αSyn or Fig. 5 Blocking Tyr136 phosphorylation promotes aggregation Y136A r-αSyn after 0–40 hours of incubation with CK2 and ATP and amyloid fibril formation of αSyn. a WT r-αSyn or Y136A r-αSyn was analyzed by SDS-PAGE followed by immunoblotting with a after 0–40 h of incubation with CK2 and ATP was analyzed by anti-pY136-αSyn antibody, b anti-pS129-αSyn antibody ab51253, and SDS-PAGE followed by immunoblotting with anti-αSyn antibody d anti-pY125-αSyn antibody. Molecular mass markers are indicated D119. Molecular mass markers are indicated in kDa on the left side in kDa on the left side of each panel. Arrows indicate the top of the of each panel. Arrows indicate the top of the gel. b Intensity ratios gel. Intensity ratios (%) of immunoreactive c pS129-αSyn, the sum (%) of immunoreactive > 250-kDa αSyn after 8 h of incubation were of > 250-kDa and 16-kDa forms, and e > 250-kDa pY125-αSyn after 8 h quantified in seven independent experiments. Data are presented as of incubation were quantified in seven independent experiments. means ± standard deviation. Statistical significance was determined Data are presented as means ± standard deviation. Statistical using the 2-tailed Student’s t test. ***P < 0.001 vs. W T r-αSyn. c ThT significance was determined using the 2-tailed Student’s t test. assays were performed in reactions with WT r-αSyn in the presence **P < 0.01, ***P < 0.001 versus W T r-αSyn CK2+ATP (WT ) or absence ( WT ) of CK2 and ATP or in reactions with CK2+ATP Y136A r-αSyn in the presence (Y136A ) or absence (Y136A) of CK2 and ATP. The results show the kinetics of ThT fluorescence from one representative of six replicate wells for each condition. d The showed that polymers of αSyn > 25  kDa in addition to CK2+ATP CK2+ATP end products from reactions with WT or Y136A were monomers accumulated in cells transfected with WT examined by TEM. Bars, 100 nm. Values of e maximal fluorescence intensities and f lag phase obtained in six individual wells in ThT r-αSyn or Y136A r-αSyn subjected to agitation, whereas assay are plotted. Data are presented as means ± standard deviation. no αSyn immunoreactivity was detected in cells trans- Statistical significance was determined using one-way ANOVA fected with WT r-αSyn or Y136A r-αSyn without agita- followed by Tukey–Kramer test. *P < 0.05, **P < 0.01 tion (Fig. 7b). The levels of accumulation of αSyn > 25 kDa were significantly higher in cells transfected with agitated Y136A r-αSyn than with agitated WT r-αSyn (Fig.  7c). of phosphorylation at S129 and Y136 to aggregate forma- Furthermore, the levels of αSyn accumulation in cells tion of αSyn in SH-SY5Y cells using oligomeric r-αSyn as transfected with agitated WT r-αSyn or Y136A r-αSyn seeds for prion-like propagation. WT r-αSyn and Y136A were significantly increased by overexpression of WT r-αSyn were converted into oligomeric forms by agitation αSyn or Y136A αSyn, respectively (Fig.  7d, e), indicat- (Fig.  7a). There were no significant differences in both ing the prion-like seeding activity of agitated WT r-αSyn forms. SDS-PAGE followed by immunoblotting analysis CK2+ATP WT CK2+ATP Y136A WT Y136A CK2+ATP WT CK2+ATP Y136A WT Y136A ThT fluorescence(arbitrary units) ThT fluorescence(arbitrary units) Lag phase (h) >250 kDa αSyn intensity ratio (%) pY125-αSyn intensity ratio (%) pS129-αSyn intensity ratio (%) Sano et al. acta neuropathol commun (2021) 9:182 Page 12 of 17 a bc d WT kDa kDa ** *** 100 100 αSyn αSyn 100 20 Y136A β-actin β-actin WT - -- -- - + + Plasmid 37 -agitation +agitation - -- -- + - + Y136A Seed Seed e f g h pS129 pY136 *** kDa kDa 250 * *** 400 0 20 -- - WT + 0 Plasmid - - - 15 WT + - Y136A + Plasmid WT - - - - + + - WT + + Y136A + Plasmid Plasmid - + - + - - Y136A Y136A + + Seed Seed +agitation +agitation Seed Seed Fig. 7 Tyr136 phosphorylation prevents aggregate formation and Ser129 phosphorylation of αSyn in cultured cells. a WT r-αSyn or Y136A r-αSyn subjected to agitation was examined by TEM. Bars, 100 nm. b–h WT r-αSyn or Y136A r-αSyn seed with (+ agitation) or without (− agitation) agitation was introduced into SH-SY5Y cells with (+) or without (−) pcDNA3.1 plasmid encoding WT or Y136A αSyn. The lysates from cells were analyzed by SDS-PAGE followed by immunoblotting with b, d anti-αSyn antibody Syn204, b, d anti-β-actin antibody, f anti-pS129-αSyn antibody D1R1R, and h anti-pY136-αSyn antibody. Molecular mass markers are indicated in kDa on the left side of each panel. Arrows indicate the top of the gel. Intensity ratios (%) of immunoreactive (c, e) αSyn > 25 kDa and g pS129-αSyn were quantified in at least three independent experiments. Data are presented as means ± standard deviation. Statistical significance was determined using c, e one-way ANOVA followed by Tukey–Kramer test and g 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001 and Y136A r-αSyn in cultured cells. Detergent-insoluble in WT αSyn-overexpressing cells transfected with agi- aggregates > 250  kDa were detected in cells overexpress- tated WT r-αSyn (Fig.  7h). Although antibodies against ing WT αSyn or Y136A αSyn transfected with agitated, pY125-αSyn and pY133-αSyn also detected bands in the but not non-agitated, WT r-αSyn or Y136A r-αSyn using mass range of around 15–100 kDa in cells, the intensities an antibody against pS129-αSyn, although 16-kDa mono- of the bands were unaffected by the introduction of WT mer was detected in all samples (Fig. 7f ). The intensity of r-αSyn or Y136A r-αSyn regardless of the form of seeds the pS129-αSyn-positive band was significantly higher in (i.e., agitated or non-agitated) (Additional file 1: Fig. S12). cells transfected with agitated Y136A r-αSyn than with These results suggest that oligomeric αSyn can convert agitated WT r-αSyn (Fig.  7g). Immunostaining with an native αSyn into insoluble aggregates that undergo phos- antibody against pY136-αSyn detected high molecular phorylation of S129 and Y136, and that blocking Y136 weight bands at > 250 kDa in addition to monomers only -agitation +agitation -agitation +agitation -agitation +agitation -agitation +agitation -agitation +agitation >25 kDa αSyn intensity ratio (%) WT Y136A WT Y136A WT Y136A WT Y136A >25 kDa αSyn intensity ratio (%) WT Y136A WT Y136A pS129-αSyn intensity ratio (%) WT WT Y136A Y136A WT Y136A WT Y136A WT WT Y136A Y136A WT WT Y136A Y136A S ano et al. acta neuropathol commun (2021) 9:182 Page 13 of 17 phosphorylation facilitates aggregate formation and S129 phosphorylation of αSyn in cultured cells. * a b kDa *** 120 *** CK2 inhibitor suppresses αSyn aggregate formation and S129 phosphorylation, and increases Y136 αSyn phosphorylation To examine whether CK2 is involved in aggregate forma- tion and C-terminal phosphorylation of αSyn in cells, we investigated the effects of the CK2 inhibitor, 4,5,6,7-tetra - Seed - + + + TBB (nM) 01 0 10 00 bromobenzotriazole (TBB), in SH-SY5Y cells exposed to β-actin agitated WT r-αSyn. TBB significantly reduced the levels Seed ++ + of accumulation of αSyn > 25  kDa in a dose-dependent TBB (nM) 00 10 100 manner in cells transfected with agitated WT r-αSyn ** c d (Fig.  8a, b). TBB treatment also significantly reduced kDa * *** the intensity of the pS129-αSyn-positive band of αSyn aggregates > 250  kDa in the cells in a dose-dependent 50 80 manner (Fig.  8c, d). Unexpectedly, the cells also showed pS129 an increase in the intensity of the pY136-αSyn-positive band of αSyn aggregates > 250  kDa with TBB treatment (Fig.  8e). The levels of pY136-αSyn were significantly increased by TBB in a dose-dependent manner (Fig.  8f ). Seed - + + + TBB (nM) 01 0 10 00 These results suggest that inhibition of CK2 suppresses Seed ++ + TBB (nM) 00 10 100 αSyn aggregate formation by reduction of S129 phospho- rylation and an unexpected increase in Y136 phospho- e f kDa rylation in cells. *** 300 * Discussion 50 pY136 The results of the present study showed that insoluble 200 αSyn is highly phosphorylated at both Y136 and S129 in the LBD brain. In addition, pY136-αSyn, which was presumed to be oligomeric and/or ubiquitinated, was 0 Seed - + + + found to be constitutively expressed in the brain regard- Seed ++ + TBB (nM) 01 0 10 00 TBB (nM)0 010100 less of the presence or absence of neocortical LB. Both Fig. 8 TBB suppresses αSyn aggregates through reduced Ser129 pY125-αSyn and pY133-αSyn were almost undetectable phosphorylation and increased Tyr136 phosphorylation in SH-SY5Y by immunoblotting and were detected at low levels by cells. SH-SY5Y cells treated with TBB (10 and 100 nM) or 0.05% DMSO immunohistochemical analysis, indicating that phospho- (0) as vehicle were transfected with (+) or without (−) agitated WT rylated αSyn is present in relatively small amounts in the r-αSyn as seed. The lysates from cells were analyzed by SDS-PAGE followed by immunoblotting with a anti-αSyn antibody Syn204, brain. Moreover, Y125 and Y133 phosphorylation were a anti-β-actin antibody, c anti-pS129-αSyn antibody D1R1R, and e unaffected by the formation of insoluble αSyn aggregates anti-pY136-αSyn antibody. Molecular mass markers are indicated in the brain. The level of pY125 was reported to be higher in kDa on the left side of each panel. Arrows indicate the top of in the control than LBD brain by immunoblotting, and the gel. Intensity ratios (%) of immunoreactive b αSyn > 25 kDa, d was shown to be reduced in the aging human brain [7]. pS129-αSyn, and f pY136-αSyn were quantified in four independent experiments. Data are presented as means ± standard deviation. Both control subjects and LBD patients were 65–86 years Statistical significance was determined using one-way ANOVA old in this previous study [7], while brain tissues from followed by Tukey–Kramer test. *P < 0.05, **P < 0.01, ***P < 0.001 patients 75–92  years of age were examined in the pre- sent study. The previous study also showed that the level of pY125 in heads from flies expressing WT human αSyn previous study was not clearly indicated. Although all decreased with increasing incubation time at room tem- tissues were stored at − 80  °C until use in this study, the perature [7]. Therefore, the lack of detectable pY125 in postmortem interval or freeze-thawing may have been the brain by immunoblotting was likely due to the age of involved in the lack of detectable pY125. the patients and the postmortem interval in the present Several protein kinases have been suggested to be study. Brain tissues were examined 8–12 years postmor- responsible for S129 phosphorylation of αSyn, including tem in this study, while the postmortem interval in the pY136-αSyn pS129-αSyn >25 kDa αSyn intensity ratio (%) intensity ratio (%) intensity ratio (%) Sano et al. acta neuropathol commun (2021) 9:182 Page 14 of 17 CK1, G protein-coupled receptor kinases (GRKs), and commonly recognized by CK2 for tyrosine phosphoryla- polo-like kinases (PLKs). We also showed that CK2 phos- tion, it is possible that Y136 is the C-terminal tyrosine phorylated αSyn at S129 in vitro, which was strongly cor- residue most susceptible to phosphorylation, consistent related with αSyn aggregate formation consistent with with our results, because the amino acid at positions n + 1 our previous report [29]. Although CK2 has generally and n + 3 from Y136 are the acidic residues, E137 and been classified as a serine/threonine protein kinase, sev - E139, respectively, while acidic residues are present at eral studies have demonstrated its tyrosine phosphoryla- positions n + 1 from Y125 and n + 2 from Y133, (E126 tion activity, suggesting that it acts as a dual-specificity and D135, respectively) (Fig.  1a). Two members of the kinase [3, 33, 37]. Indeed, we found that the C-terminal PLK family, PLK2 and PLK3, recognize an acidic residue tyrosine residues surrounding S129, Y125 and Y136, similar to CK2 [27]. An in  vitro study showed that PLK2, were phosphorylated by CK2, and that tyrosine phos- and to a lesser extent PLK3, phosphorylated S129 more phorylation was exclusively found in insoluble αSyn spe- efficiently than CK2 [28]. However, PLK2 knockout (KO) cies in vitro. In addition, insoluble αSyn aggregation was mice showed not over 50% decrease in pS129, while PLK3 accompanied by pY136, but not pY125, as well as pS129 KO had little effect on pS129 in various brain regions [4 ]. in cultured human cells that had taken up extracellu- The remaining pS129 levels were not reduced by treatment lar αSyn oligomers as seeds. These findings suggested with PLK1-3 inhibitor in PLK2 KO mice [4]. The results that pY136 is related in some way to the formation and suggest that S129 can be phosphorylated by multiple propagation of αSyn aggregates in cells. Phosphorylation kinases in vivo. Moreover, PLK2 KO has been reported to at certain residues of αSyn has been shown to affect sub - have no effect on pS129 in LB but not presynaptic termi - sequent phosphorylation events in neighboring residues. nals in mice [36]. Therefore, it is likely that other, non-PLK Mutation of Y125 to phenylalanine (Y125F), preventing kinases, including CK2, mediate phosphorylation of S129 phosphorylation at this site, has been shown to decrease of αSyn aggregates in  vivo. Although it remains unclear the levels of S129 phosphorylation by CK1 in  vitro [16]. why pS129 is essential for subsequent tyrosine phospho- Double mutation of Y133 and Y136 to phenylalanine rylation, pS129 increases the negative charge on the C-ter- 2− enhanced phosphorylation of Y125 by Lyn tyrosine minus by the addition of a PO group and may therefore kinase in  vitro [22]. In our in  vitro experiment, preven- lower the threshold for tyrosine phosphorylation by CK2. tion of S129 phosphorylation in S129A mutant blocked It has been reported that most of the phosphorylation sites Y125 and Y136 phosphorylation by CK2 with forma- are located in intrinsically disordered regions [12], and that tion of αSyn aggregates, suggesting that pS129 mediates phosphorylation induces folding of the intrinsically disor- phosphorylation of Y125 and Y136 and this plays a role dered protein [2, 18]. Indeed, phosphorylation of the papil- in αSyn aggregate formation. Prominent pS129 was seen lomavirus E2 protein by CK2 has been reported to induce within 1 day of incubation of WT αSyn with CK2, while a conformational change that leads to degradation of the pY125 and pY136 were observed after 7 and 3  days of protein [25]. It has been also reported that pS129 increases incubation, respectively. Analysis of the kinetic param- the conformational flexibility of αSyn [24]. Therefore, it eters for phosphorylation of tyrosine-containing peptides is possible that disorder-to-order conformational tran- by CK2 in  vitro demonstrated that tyrosine phospho- sitions occur in the intrinsically disordered C-terminal rylation is less favorable than serine/threonine phospho- region of αSyn by pS129, and the conformational changes rylation [20]. Therefore, these results suggest that CK2 also enable phosphorylation of tyrosine residues by CK2 preferentially catalyzes phosphorylation of S129, which is in addition to induction of αSyn aggregate formation. The essential for subsequent Y125 and Y136 phosphorylation affinity of anti-pS129-αSyn antibody could be enhanced by in αSyn. Y136A mutation, or the levels of pS129 may be increased The substrate specificity of CK2 is determined by one or by Y136A substitution itself. However, the levels of expres- more negatively charged residues, i.e., aspartic acid (D)/ sion of αSyn and the levels of pS129 were not significantly glutamic acid (E), surrounding the phosphorylatable serine affected by Y136A mutation in αSyn-overexpressing cells (S) and threonine (T) residues. The minimum consensus as determined by immunoblotting analysis (Additional sequence is S/T-X-X-D/E, where X can be any amino acid. file  1: Fig. S13). There were also no differences in the The most crucial acidic residue position for susceptibility expression pattern of αSyn between WT and Y136A αSyn- to phosphorylation by CK2 is n + 3 followed by n + 1 [21]. overexpressing cells (Additional file  1: Fig. S13). Moreover, In the case of αSyn, the amino acid at position n + 1 from insoluble aggregates and amyloid fibrils of nonphosphoryl - S129 is E130 (Fig. 1a), which is predicted to mostly act as a ated Y136A r-αSyn were not formed by incubation (Fig. 5c, specificity determinant for S129 phosphorylation by CK2. Additional file  1: Fig. S7a). Therefore, it is unlikely that Little is known about the substrate specificity of CK2 for anti-pS129-αSyn antibody binds Y136A αSyn with differ - tyrosine phosphorylation. If the consensus sequence is ent affinity than WT αSyn and that the increase in pS129 S ano et al. acta neuropathol commun (2021) 9:182 Page 15 of 17 Abbreviations and acceleration of aggregate formation of αSyn by Y136A AD: Alzheimer’s disease; CK1: Casein kinase 1; CK2: Casein kinase 2; CJD: mutation are due to Y136A substitution itself. Although Creutzfeldt–Jakob disease; LB: Lewy body; LBD: Lewy body dementia; LC–MS/ the 16-kDa monomer of WT αSyn was phosphorylated MS: Liquid chromatography-ion trap mass spectrometry; MS: Mass spec- trometry; PD: Parkinson’s disease; TBB: 4,5,6,7-Tetrabromobenzotriazole; TEM: at Y136, there was no significant difference in the level Transmission electron microscopy; ThT: Thioflavin T; αSyn: α-Synuclein; WT: of pS129 between WT and Y136A αSyn-overexpressing Wild-type. cells (Additional file  1: Fig. S13). These results suggest that pY136 has little effect on pS129 of αSyn monomer in non- Supplementary Information diseased cells. The online version contains supplementary material available at https:// doi. Preventing phosphorylation of Y136 by Y136A muta- org/ 10. 1186/ s40478- 021- 01281-9. tion facilitated aggregate formation and S129 phos- phorylation of r-αSyn and αSyn in cultured cells. Y136 Additional file1. Supplementary fgures. may also be one of the major phosphorylatable sites for CK2 in negatively charged αSyn aggregates formed Acknowledgements with increased pS129 and to protect against further We thank Fuyuki Kametani of Tokyo Metropolitan Institute of Medical Science for LC-MS/MS analysis, Shinya Dohgu of Fukuoka University for providing S129 phosphorylation and αSyn aggregate formation by SH-SY5Y cells, and Megumi Saiki, Kaori Hirakawa, Shota Hasegawa, Yuhei undergoing phosphorylation instead of S129. TBB signifi - Moriyama, Saki Ogami, Haruka Taketomi, Yumiko Hori, Kasumi Kubo, Manami cantly inhibited S129 phosphorylation and suppressed Noda, Dan Hokama, Ayumi Yamada, and Hinako Nonaka of Fukuoka University for technical assistance. αSyn aggregate formation in cultured cells. These results suggested that CK2 is the main protein kinase for S129 Authors’ contributions phosphorylation of αSyn and that phosphorylation of KS and KM designed the project. YI and KS collected clinical specimens. YI performed immunohistochemistry. YY, KI, MH, and KS performed in vitro S129 by CK2 is closely related to the formation of αSyn experiments and cell culture experiments. KS and KS performed immunoblot- aggregates in SH-SY5Y cells. Therefore, CK2 may be a ting of clinical specimens. YI, KS, KM, and KS analyzed the data. KS wrote the therapeutic target for LB disease. Unexpectedly, phos- manuscript. All authors read and approved the final manuscript. phorylation of Y136 in αSyn aggregates was significantly Funding increased by TBB in cultured cells, suggesting that CK2 This work was supported by a grant-in-aid for Scientific Research (C) (grant plays an inhibitory role against Y136 phosphorylation no. 19K07858) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. in cells. As CK2 phosphorylates hundreds of physiologi- cal substrates to control various cellular processes [5], Availability of data and materials its in  vitro and in  vivo functions may not be consistent. All data generated or analyzed during this study are included in this published article and its supplementary information files. CK2 has been reported to phosphorylate threonine resi- dues of Src family tyrosine kinases, thereby resulting in Declarations reduction of their activities in  vitro [38]. Therefore, the significant increase in pY136 in αSyn aggregates may be Ethics approval and consent to participate due to disinhibition of tyrosine kinases under conditions The study protocol was approved by the Institutional Review Board-Inde- pendent Ethics Committee of Fukuoka University (ID: 20-03-M2), the Ethics where CK2 is inhibited in cells, although the major pro- Committee of Nagasaki University (ID: 19083005-2) and the Ethics Committee tein kinases for phosphorylation of Y136 have yet to be of Aichi Medical University (ID: 15-017). Informed consent was obtained from elucidated, and may contribute to reduction in pS129 and the patients and/or their families. confer protection against αSyn aggregate formation. Consent for publication Consent for the use of the brain tissue for research purposes and for publica- tion was obtained from the patients and/or their families. Conclusions The findings of the present study provide the first evidence Competing interests that CK2 phosphorylates Y136 in αSyn aggregates by medi- The authors report no competing interests. ating S129 phosphorylation as a dual-specificity kinase and Author details that pY136 has a protective effect against αSyn aggregation. 1 Department of Physiology and Pharmacology, Faculty of Pharmaceutical Although the primary kinase for pY136 in  vivo is not yet Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. Department of Neuropathology, Institute for Medical Science clear, this study suggested that CK2 is a candidate kinase of Aging, Aichi Medical University, Aichi 480-1195, Japan. Depar tment responsible for pY136 and that it participates in regulating of Immunological and Molecular Pharmacology, Faculty of Pharmaceutical Sci- αSyn aggregate formation. Further studies are necessary ences, Fukuoka University, Fukuoka 814-0180, Japan. Department of Health Sciences, Unit of Medical and Dental Sciences, Nagasaki University Graduate to elucidate the potential roles of the interactions between School of Biomedical Sciences, Nagasaki 852-8523, Japan. CK2 and αSyn C-terminal phosphorylation, their involve- ment in the pathogenesis of LB diseases, and their potential Received: 31 July 2021 Accepted: 22 October 2021 for the development of novel therapeutic strategies. Sano et al. acta neuropathol commun (2021) 9:182 Page 16 of 17 References 17. Lee G, Tanaka M, Park K, Lee SS, Kim YM, Junn E, Lee SH, Mouradian MM 1. Anderson JP, Walker DE, Goldstein JM, de Laat R, Banducci K, Caccavello (2004) Casein kinase II-mediated phosphorylation regulates alpha-synu- RJ, Barbour R, Huang J, Kling K, Lee M et al (2006) Phosphorylation of clein/synphilin-1 interaction and inclusion body formation. J Biol Chem Ser-129 is the dominant pathological modification of alpha-synuclein in 279:6834–6839. https:// doi. org/ 10. 1074/ jbc. 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Tyrosine 136 phosphorylation of α-synuclein aggregates in the Lewy body dementia brain: involvement of serine 129 phosphorylation by casein kinase 2

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Abstract

Serine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replace- ment of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was signifi- cantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target. Keywords: α-Synuclein, Lewy body dementia, Y136 phosphorylation, S129 phosphorylation, Casein kinase 2 Introduction have not been fully elucidated, accumulation of LB, i.e., Lewy body (LB) diseases, including Lewy body demen- αSyn aggregates, is thought to play a significant role. tia (LBD) and Parkinson’s disease (PD), are progressive A significant proportion of αSyn accumulated within diseases characterized by extensive accumulation of LB is phosphorylated on the C-terminal serine 129 intracellular proteinaceous inclusions composed mainly (S129), while only a small fraction of αSyn is constitu- of aggregated α-synuclein (αSyn) in the brain called LB. tively phosphorylated at this residue in the brain with- Although the pathological mechanisms of LB disease out LB pathology. Earlier in vitro and vivo studies yielded contrasting results regarding the significance of S129 phosphorylation (pS129) for LB formation, showing facil- itatory [10, 32], inhibitory [6, 24], or no effect [17, 30] of *Correspondence: ksano@fukuoka-u.ac.jp Department of Physiology and Pharmacology, Faculty of Pharmaceutical phosphorylation on αSyn aggregation. In mice inoculated Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, with recombinant αSyn (r-αSyn) fibrils, the S129-phos - Fukuoka 814-0180, Japan phorylated and nonphosphorylated forms were shown 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:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sano et al. acta neuropathol commun (2021) 9:182 Page 2 of 17 to cause prion-like seeding and accumulation of endog- and immunoblotting analyses [9] and mass spectrometry enous αSyn in the brain, and the phosphorylated form [1]. pY125 has been reported to inhibit toxic oligomer showed greater potency to induce the pathology than the formation of αSyn in Drosophila [7], whereas an in vitro nonphosphorylated form [14]. Therefore, although pS129 study showed that pY125 had no influence on aggrega - is not necessarily required for LB formation, it appears to tion of synthetic αSyn [11, 30]. Therefore, αSyn appears induce more severe pathology. to be phosphorylated at Y125 in the human brain, but αSyn tyrosines Y125, Y133, and Y136 are located in the role of pY125 in αSyn aggregate formation has not close proximity to S129 at the C-terminus (Fig.  1a), been fully elucidated. Immunoblotting analysis indicated which raises questions regarding whether these tyros- the presence of phosphorylated Y133 (pY133) at similar ine residues are phosphorylated and whether there are levels in LBD, PD, and control brains [9], suggesting that interactions among the phosphorylated residues, includ- pY133 may not be crucial for LB pathology. In contrast, ing S129 and its association with αSyn aggregate for- there have been few studies regarding the presence of mation. Human r-αSyn incubated with spleen tyrosine phosphorylated Y136 (pY136) in the human brain and its kinase (Syk) [22] and αSyn in pervanadate-treated cul- physiological roles and implications in the pathogenesis tured human cells [8] have been reported to be phos- of LB disease. phorylated at Y125, Y133, and Y136. Several studies Casein kinase (CK) 1 and 2 are ubiquitous serine/ have demonstrated the presence of αSyn phosphorylated threonine protein kinases expressed in all eukaryotes. at Y125 (pY125) in the human brain. An immunohisto- Although both CK1 and CK2 have been shown to con- chemical study identified pY125 within LB in a case of stitutively phosphorylate S129 of αSyn in  vitro [23, 35], familial PD with G51D mutation [15]. Immunoblotting CK2 rather than CK1 was demonstrated to be the major analysis detected pY125 at similar levels in PD and con- enzyme in the brain that phosphorylates S129 of human trol brains [19] but at higher levels in control than LBD αSyn [13]. In a study using transgenic mice expressing brains [7]. Other studies showed that pY125 is not a com- human αSyn, S129-phosphorylated αSyn was shown to ponent of LB in LBD and PD by immunohistochemical be preferentially colocalized with CK2 rather than CK1 bc DN-LBD Li-LBD DN-LBD Li-LBD 1231 2 AD CJD 1231 2 AD CJD 1 MDVFMKGLSKAKEGVVAAAE 21 KTKQGVAEAAGKTKEGVLYV 41 GSKTKEGVVHGVATVAEKTK 37 37 61 EQVTNVGGAVVTGVTAVAQK 81 TVEGAGSIAAATGFVKKDQL GKNEEGAPQEGILEDMPVDP DNEAYEMPSEEGYQDYEPEA kDa αSyn kDa pS129 125 129 133 136 d ef DN-LBD Li-LBD DN-LBD Li-LBD DN-LBD Li-LBD 1231 2 AD CJD PC 1 2312 AD CJD PC 12312 AD CJD 250 250 100 100 37 37 20 20 kDa pY125 kDa pY133 kDapY136 Fig. 1 Insoluble αSyn was phosphorylated at Tyr136 as well as Ser129 in the DN-LBD brain. a Amino acid sequence of human αSyn. C-terminal phosphorylation sites are shown in bold and are underlined. b–f Brain lysates from DN-LBD (cases #1, #2, and #3), Li-LBD (cases #1 and #2), AD, and CJD patients were analyzed by SDS-PAGE followed by immunoblotting with b anti-αSyn antibody D119, c anti-pS129-αSyn antibody ab51253, d anti-pY125-αSyn antibody, e anti-pY133-αSyn antibody, and f anti-pY136-αSyn antibody. Molecular mass markers are indicated in kDa on the left side of each panel. Arrows indicate the top of the gel. In d and e, WT r-αSyn (40 μg) was incubated in the presence of 100 ng/mL Syk (S52-10G; SignalChem Pharmaceuticals) and 200 μM ATP in 100 μL of reaction buffer (20 mM Tris–HCl, pH 7.5, 50 mM KCl, and 10 mM MgCl ) at 37 °C for 1 h, and the sample containing WT r-αSyn (2 μg) was loaded as a positive control (PC) S ano et al. acta neuropathol commun (2021) 9:182 Page 3 of 17 in the nuclei [34]. Furthermore, the catalytic (α) and diagnosis. Three of these subjects had diffuse neocorti - regulatory (β) subunits of CK2 have been shown to be cal LBD (DN-LBD), and the remaining two cases had present in the insoluble fraction of the LBD brain [13] limbic LBD (Li-LBD) according to Braak staging. Brain and in LB in the PD brain [26], respectively. In contrast, tissues were obtained at autopsy from a patient with a CK1 isoform delta (CK1δ) was shown to be absent in LB neuropathological diagnosis of Alzheimer’s disease (AD) in the PD and LBD brain [31]. These studies suggested based on the presence of neurofibrillary tangles and neu - a role of CK2 in LB formation. CK2 has been shown to ritic plaque. The brain specimens showed pure AD with exhibit tyrosine kinase activity in cultured mamma- little or no coexisting LB disease. Prion disease brain tis- lian cells [3, 33]. In yeast, CK2 has also been reported sue specimens were obtained at autopsy from a patient to phosphorylate tyrosine 184 (Y184) of the nucleolar with sporadic Creutzfeldt–Jakob disease (CJD), which immunophilin, Fpr3, through prior phosphorylation at was diagnosed as the classical MM1 subtype according serine 186 (S186) at the + 2 position [37], suggesting that to the genotype at codon 129 of the PRNP gene and the phosphorylated serine residues play a key role in mediat- physicochemical properties of abnormal prion protein Sc ing phosphorylation of neighboring tyrosine residues. In (PrP ). For DN-LBD cases #1, #2, and #3, the individuals the case of αSyn, pS129 and pY125 were shown to have were 92, 80, and 91 years old at the time of death, respec- no effect on each other in Drosophila by immunoblotting tively. Li-LBD cases #1 and #2 were 80 and 87  years old analysis [7] and in vitro by immunoblotting and MALDI- at the time of death, respectively. The patients with AD TOF mass spectrometry (MS) [11], while a study using and CJD were 82 and 75  years old at the time of death, in  vitro NMR spectroscopy reported that pY125 primes respectively. All tissues were taken from the frontal cor- efficient phosphorylation of S129 by CK1 [16]. Therefore, tex and stored at − 80 °C. although tyrosines Y125, Y133, and Y136 are located in close proximity to S129 at the C-terminus of αSyn, it is Preparation of brain lysates not clear whether there is phosphorylation cross-talk Brain tissues were lysed with Triton-deoxycholate between these sites, and whether such cross-talk is func- (DOC) lysis buffer (50 mM Tris–HCl, pH 7.5, containing tionally relevant to the pathogenesis of LB disease. Here, 150  mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxy- we report that insoluble αSyn is highly phosphorylated cholate, 2 mM EDTA, and protease inhibitors) for 30 min at Y136 as well as S129 in the LBD brain. Furthermore, at 4  °C. After centrifugation for 2  min at 2000 × g, the experimental manipulation of the phosphorylation state supernatant was collected and stored at − 80 °C until use. demonstrated that phosphorylation of S129 by CK2 The total protein concentration of the lysates was meas - mediates phosphorylation of Y136 and thus suppresses ured using a bicinchoninic acid (BCA) protein assay kit the formation of αSyn aggregates. (23227; Pierce). Materials and methods Recombinant human α‑synuclein expression Reagents and purification Anti-αSyn polyclonal antibody (D119) and monoclonal The purification of recombinant human αSyn (r-αSyn) antibody (Syn204) were obtained from Bioworld Tech- and mutants was performed as described previously [29]. nology Inc. and Cell Signaling Technology, respectively, Briefly, plasmids carrying the DNA sequence encoding and were used for western blotting. Anti-pS129-αSyn N-terminal His-tagged human wild-type (WT) or non- polyclonal antibody (D1R1R) and anti-β-actin poly- phosphorylatable mutants with substitution of alanine clonal antibody (ab8227) were obtained from Cell Signal- for S129 (S129A) or Y136 (Y136A) were subcloned into ing Technology and Abcam, respectively, and were used the vector pET11a (69436-3; Novagen). The products for western blotting. Monoclonal antibody specific for encoded on the plasmid were overexpressed in compe- pS129-αSyn (ab51253) and polyclonal antibodies against tent BL21 DE3 Escherichia coli cells (DS250; BioDynam- pY125-αSyn (ab10789), pY133-αSyn (ab194910), and ics Laboratory) at 37  °C for 16  h using MagicMedia E. pY136-αSyn (ab194775) used for western blotting and coli Expression Medium (K6815; Invitrogen). The bacte - immunohistochemical staining were purchased from rial pellets were suspended in CelLytic B (B7435; Sigma- Abcam. Casein kinase 2 (CK2, P6010L) was obtained Aldrich) in the presence of 1 g/mL lysozyme (120-02674; from New England Biolabs Inc. ATP (A2383) was Wako) and 500 U/mL benzonase nuclease (70664-3; acquired from Sigma-Aldrich. Novagen). The lysate was centrifuged at 10,000  rpm for 30  min at 4  °C, and the supernatant was incubated with Brains of patients Ni–NTA Superflow resin (30430; Qiagen) at room tem - LBD brain tissues were obtained at autopsy from five perature for 30 min, and then loaded onto a gravity flow patients with histopathologically confirmed clinical column (Muromac mini-column; Muromachi Chemical Sano et al. acta neuropathol commun (2021) 9:182 Page 4 of 17 Inc.). The His-tagged proteins were eluted with a buffer antibody binding was detected by the labeled strepta- containing 300  mM NaCl, 50  mM Tris–HCl (pH 8.0), vidin–biotin method (DAKO). Peroxidase-conjugated and 250  mM imidazole, and dialyzed against 10  mM streptavidin was visualized with 3′3-diaminobenzidine phosphate buffer (pH 7.0) in cellulose dialysis tubing (7411-49-6; Wako) as the chromogen. Immunostained (68035; Thermo Scientific) at 4  °C overnight. Cleavage sections were lightly counterstained with Mayer’s of the His-tagged from the proteins followed by removal hematoxylin. of uncleaved His-tagged proteins was performed using the TAGZyme system (34300; Qiagen). The non-tagged Gel staining and in‑gel digestion proteins were then dialyzed against deionized distilled After separation of proteins by SDS-PAGE, the gels were water in cellulose dialysis tubing at 4 °C overnight and fil - stained at room temperature for 2 h with Coomassie Bril- tered with a 0.2-µm syringe filter (SLLGH25; Millipore). liant Blue solution (11642; Nacalai Tesque). The stained The purity of r-αSyn was ≥ 99.9% as estimated by SDS- bands near 16  kDa were excised and soaked in 50  mM PAGE and western blotting. After purification, aliquots Tris–HCl, pH 8.0, containing 50% acetonitrile for 30 min. of r-αSyn were stored at − 80 °C until use. The gel was dried in a Speed-Vac (Savant) and incubated in 50  mM triethylammonium bicarbonate containing In vitro phosphorylation of recombinant α‑synuclein proteomics grade trypsin (T7575; Sigma-Aldrich) at r-αSyn (40 μg) was incubated in the presence of 400 units 37  °C for 20  h. The digests were extracted from the gel of casein kinase 2 and 200 μM ATP in 100 μL of reac- with 100–200 μL of 0.1% TFA containing 60% acetoni- tion buffer (20  mM Tris–HCl, pH 7.5, 50  mM KCl, and trile. These extracts were evaporated in a Speed-Vac and 10 mM MgCl ) at 37 °C. stored at − 80 °C until assayed. Gel electrophoresis and immunoblotting Nano‑flow liquid chromatography‑ion trap mass For SDS-PAGE, samples were boiled for 5  min at 95  °C spectrometry (LC–MS/MS) with SDS loading buffer (62.5  mM Tris–HCl, pH 6.8, Peptides were resuspended in 0.1% formic acid contain- containing 5% 2-mercaptoethanol, 2% SDS, 5% sucrose, ing 2% acetonitrile. Measurements were performed on and 0.005% bromophenol blue) and were separated by a nano-flow high-performance liquid chromatography 15% SDS-PAGE. For BN-PAGE, samples were prepared (HPLC) system (EASY-nLC 1200; Thermo Fisher Sci - in a buffer containing 0.25% Coomassie Brilliant Blue entific). The samples were loaded onto packed nano- G-250 and separated by 4–16% Bis–Tris native PAGE capillary columns (0.075  mm I.D. × 125  mm L, particle (BN1004BOX; Invitrogen). The proteins were transferred diameter 3  μm, NTCC-360/75-3-123; Nikkyo Technos onto Immobilon-P membranes (IPVH304F0; Millipore) Co., Ltd.), which were eluted at a flow rate of 300 nL/min in transfer buffer containing 15% methanol followed by with a 2–80% linear gradient of acetonitrile for 80  min. blocking with 5% nonfat dry milk in TBST (10 mM Tris– Eluting peptides were detected with an ion trap mass HCl, pH 7.8, 100  mM NaCl, 0.1% Tween 20) for 2  h at spectrometer (QExactive HF; Thermo Fisher Scientific). 4  °C. Membranes were then probed with specific pri - For ionization, the spray voltage and capillary tempera- mary antibodies (1:2500) and appropriate horseradish ture were set to 2.0 kV and 250 °C, respectively. The mass peroxidase-conjugated secondary antibody (111-035-003 acquisition method consisted of one full MS survey scan or 115-035-003, 1:5000; Jackson ImmunoResearch Labs). with an Orbitrap resolution of 60,000 followed by mass Immunoreactive bands were visualized using Chemi- spectrometry (MS/MS) of the most abundant precursor Lumi One L (07880-70; Nacalai Tesque) or ECL prime ions from the survey scan with an Orbitrap resolution of Western Blotting Detection Reagents (PRN2232; GE 15,000. Dynamic exclusion for MS/MS was set to 30  s. Healthcare Life Sciences). MS was performed with a scan range of 350–1800  m/z in positive ion mode, followed by data-dependent MS/ Immunohistochemical staining MS using the HCD operating mode on the top 15 ions The brain tissues were fixed in 20% neutral buffered for - in order of abundance. The data were analyzed with Pro - malin, embedded in paraffin, cut into Sects.  8  μm thick teome Discoverer (Thermo Fisher Scientific) and Mascot with a microtome, and placed on glass slides. After software (Matrix Science). deparaffinization and rehydration, the tissue sections were heated for 40 min at 98 °C for antigen retrieval. All Transmission electron microscopy (TEM) sections were immersed in hydrogen peroxide (0.3%) Negative staining was performed on 400 mesh copper solution for 10  min to quench endogenous peroxidase grids with a carbon support film. Aliquots of the samples activity and incubated with specific primary antibodies were adsorbed onto the grids, and the residual solution (1:100) diluted in PBS containing 1% BSA for 2 h. Primary was carefully removed from the grid surface using filter S ano et al. acta neuropathol commun (2021) 9:182 Page 5 of 17 Preparation of lysate from cell culture paper. The grids were stained with 2% uranyl acetate. Cells were washed with PBS and harvested, and cellular Once dry, the samples were viewed with a transmission proteins were extracted with Triton-DOC lysis buffer. electron microscope (TEM) (JEM-2000FX; JEOL) at The total protein concentration of the lysates was meas - 200 kV. ured using a BCA protein assay kit (23227; Pierce). Thioflavin T (ThT) assay Statistics r-αSyn (40 μg) was prepared in 96-well optical black-bot- Statistical analyses were performed with OriginPro 2015 tomed plates (265301; Nunc) in 100 μL of reaction buffer (OriginLab). The 2-tailed Student’s t test was used for (20  mM Tris–HCl, pH 7.5, 50  mM KCl, 10  mM MgCl , comparisons between two groups. One-way analysis of and 10  μM thioflavin T [T Th ]). The 96-well plates were variance (ANOVA) followed by the Tukey–Kramer test covered with sealing tape (236366; Nunc) and incubated was used for comparisons among more than two groups. at 40 °C in a plate reader (FLUOstar Omega plate reader; In all analyses, P < 0.05 was taken to indicate statistical BMG Labtech) with intermittent shaking, consisting of significance. 30 s of double orbital shaking at 500 rpm and no shaking for 30 s. ThT fluorescence intensity on the bottom of the plates was measured every 10 min to monitor the kinet- ics of amyloid fibril formation using monochromators Results with excitation and emission wavelengths of 450 ± 10 and Insoluble aggregates of αSyn are predominantly 480 ± 10  nm, respectively. Lag phase was defined as the phosphorylated on Y136 as well as S129 time required to reach fluorescence greater than or equal within the C‑terminal region in the LBD brain to the mean fluorescence intensity for all samples within Brain lysates from LBD patients were analyzed by SDS- the first 24 h of reaction plus 4 × the standard deviation. PAGE followed by immunoblotting with anti-αSyn anti- body, and all exhibited a band at approximately 20  kDa Preparation of r‑αSyn seeds with another band just below 20  kDa likely correspond- r-αSyn (40  μg) was incubated in 20  mM Tris–HCl, pH ing to full-length and cleaved αSyn, respectively (Fig. 1b). 7.5, containing 50 mM KCl, and 10 mM MgCl , at 37 °C All lysates also exhibited an αSyn-positive band at under agitation at 2000  rpm for 3  days. As a control for approximately 50 kDa, which was presumably due to the agitated r-αSyn, the mix was prepared without incuba- oligomeric and/or ubiquitinated αSyn. Although these tion and agitation immediately before use. The morphol - αSyn-positive bands did not differ significantly between ogy of r-αSyn seeds was confirmed by TEM. cases, multimeric αSyn with molecular weight > 250 kDa was observed only in three cases of DN-LBD (Fig.  1b, Cell culture, treatment of r‑αSyn seeds, and transfection Additional file  1: Fig. S1). Immunostaining with an anti- of plasmids body against pS129-αSyn detected only a major band Human neuroblastoma SH-SY5Y cells were maintained at > 250  kDa in all cases of DN-LBD (Fig.  1c, Additional at 37  °C in 5% C O in DMEM/Ham’s F12 medium file  1: Fig. S1). A band of > 250  kDa was detected with (042-30795; Wako) containing 15% fetal bovine serum antibodies against αSyn and pS129-αSyn in Li-LBD case (SH30910; GE Healthcare Hyclone), penicillin–strep- #2, but not case #1, but the intensity was significantly tomycin (168-23191; Wako), and MEM Non-essential lower than in DN-LBD (Fig.  1b, c), indicating that mul- Amino Acids Solution (139-15651; Wako). Introduc- timer formation and pS129 of αSyn are relevant to dis- tion of r-αSyn seeds and/or pcDNA3.1 plasmid encod- ease progression. Consistent with our previous report ing human WT or Y136A mutant αSyn into cells [29], these results suggest that the detergent-insoluble was performed using Lipofectamine LTX (Invitro- multimer of αSyn highly phosphorylated at S129 with gen) according to the manufacturer’s instructions. molecular weight > 250  kDa is present in the DN-LBD Lipofectamine/r-αSyn seeds and/or plasmid complexes brain. We next examined the presence of αSyn phospho- were prepared in Optimem (31985062; Gibco) by mixing rylated at C-terminal tyrosine residues surrounding S129. Lipofectamine LTX reagent in the presence or absence of No signals were detected with antibodies against pY125- r-αSyn seeds (4 μg) or plasmid (5 μg). Cells were cultured αSyn and pY133-αSyn in any cases (Fig. 1d, e). A 50-kDa to 40–50% confluence in 6-well plates and treated with band was detected with an antibody against pY136-αSyn the complexes. For CK2 inhibitor treatment, cells were in all cases (Fig.  1f, Additional file  1: Fig. S1). Moreover, incubated with 10 or 100  nM 4,5,6,7-tetrabromobenzo- the three cases of DN-LBD showed a strong immunore- triazole (TBB, ab120988; Abcam) for 1 h before addition active band with mass > 250 kDa (Fig. 1f, Additional file  1: of the complexes. cells were incubated for 3 d after intro- Fig. S1). These results suggest that the insoluble aggre - duction of r-αSyn seeds and/or plasmid. gates of αSyn are predominantly phosphorylated on Y136 Sano et al. acta neuropathol commun (2021) 9:182 Page 6 of 17 as well as S129 within the C-terminal region in the LBD Consistent with our previous study [29], the deposits brain. showed positive staining for pS129-αSyn, and were also We next examined phosphorylation of the C-termi- positive for pY136-αSyn in both DN-LBD and Li-LBD nus of αSyn in sections of the frontal cortex of DN-LBD brains (Fig. 2a). The numbers of pS129-αSyn and pY136- and Li-LBD brains by immunohistochemical analysis. αSyn deposits were significantly greater in DN-LBD than pS129 pY125 pY133 pY136 DN-LBD pS129 pY125 pY133 pY136 Li-LBD 12 12 12 12 ** 10 10 10 10 *** 8 8 8 8 6 6 6 6 4 4 4 4 2 2 2 2 0 0 0 0 DN-LBD Li-LBD DN-LBD Li-LBD DN-LBD Li-LBD DN-LBD Li-LBD Fig. 2 Deposits of phosphorylated αSyn at C-terminal tyrosine residues are present in LBD brain. a Immunohistochemical staining using antibodies against pS129-αSyn ab51253, pY125-αSyn, pY133-αSyn, and pY136-αSyn in the frontal cortex from two patients with DN-LBD (case #3) and Li-LBD (case #2). Regions surrounded by white rectangles in the upper panels are magnified and shown in the lower panels. Typical pathological αSyn deposits are indicated by arrowheads. Scale bars, 50 μm. b The number of phosphorylated αSyn deposits > 2 μm was counted in 12 randomly selected areas of 10 mm in each tissue specimen using ImageJ. Data are presented as means ± standard deviation. Statistical significance was determined using the 2-tailed Student’s t test. **s < 0.01, ***P < 0.001 vs. Li-LBD Number of pS129-αSyn deposits Number of pY125-αSyn deposits Number of pY133-αSyn deposits Number of pY136-αSyn deposits S ano et al. acta neuropathol commun (2021) 9:182 Page 7 of 17 Li-LBD (Fig. 2b). Deposits were not detected in the brain 15 days revealed the formation of αSyn aggregates with a of a non-DLB patient by immunohistochemical analy- molecular weight of ~ 1048  kDa in addition to a band of sis using anti-pS129-αSyn antibody (Additional file  1: monomeric αSyn at approximately 60 kDa (Fig.  3d). The Fig. S2). Both DN-LBD and Li-LBD brains showed posi- aggregated WT r-αSyn was also formed more efficiently tive staining with anti-pY125-αSyn and anti-pY133-αSyn in the presence of both CK2 and ATP than in the absence antibodies (Fig.  2a), but pT125-αSyn and pY133-αSyn of either, suggesting that pS129 accelerates aggregate for- deposits were present at low levels in both DN-LBD and mation of αSyn (Fig. 3d, e). Li-LBD compared to pS129-αSyn and pY136-αSyn depos- its in DN-LBD (Fig. 2b). The lack of detectable pY125 and pY133 in the brain by immunoblotting was likely due to CK2 phosphorylates Y125 and Y136 of aggregated αSyn low levels of the deposits, and phosphorylated αSyn may through prior S129 phosphorylation in vitro not be detected by immunoblotting in brains with less WT r-αSyn or S129A r-αSyn was incubated with CK2 than two phosphorylated deposits per 10 mm of tissue. and ATP for 7 days, separated by SDS-PAGE, and the gel There were no significant differences in the numbers of portion corresponding to a molecular weight of ~ 16 kDa pY125-αSyn and pY133-αSyn deposits between the two was cut out as shown in Additional file  1: Fig. S5. The gel types of LBD (Fig. 2b). Thus, the results of immunohisto - slice was digested with trypsin and analyzed by LC–MS/ chemical analysis indicated the presence of pS129-αSyn- MS. As shown in Table  1, we identified 23 and seven and pY136-αSyn-positive deposits in larger amounts in phosphorylated peptides from WT and S129A r-αSyn, the brains of patients with DN-LBD than Li-LBD, and respectively. LC–MS/MS analysis detected pS129 in that pY125-αSyn and pY133-αSyn are present in relatively WT r-αSyn. Both WT r-αSyn and S129A r-αSyn con- small amounts in the brains of patients with both types tained pS87. WT r-αSyn showed phosphorylation at four of LBD. threonine residues, i.e., T44, T54, T59, and T64, while S129A r-αSyn showed phosphorylation at five threonine residues, i.e., T22, T44, T54, T59, and T75. There have Ser129 phosphorylation by CK2 promotes the formation been no previous reports of phosphorylation of αSyn at of insoluble αSyn aggregates in vitro T22, T44, T54, T59, T64, T75, and S87 by CK2, and the Analysis of WT r-αSyn by SDS-PAGE followed by number of peptides including these residues with phos- immunoblotting with anti-αSyn antibody revealed phorylation was very low in comparison to peptides detergent-insoluble aggregates > 250  kDa after 15  days with pS129 (Table  1). Therefore, CK2 may phosphoryl - of incubation, which were formed more efficiently in ate r-αSyn on these residues at very low levels in  vitro. the presence of both CK2 and ATP than in the absence Moreover, peptides with pY125 and pY136 were identi- of either (Additional file  1: Fig. S3). Insoluble aggregates fied in WT r-αSyn. The number of peptides with pY136 and 16-kDa WT r-αSyn monomer incubated with CK2 was comparable to that of peptides with pS129, and and ATP were detected by anti-pS129-αSyn antibody, only one peptide with pY125 was detected. In contrast, whereas no immunoreactivity was detected with anti- no peptides with C-terminal tyrosine phosphorylation pS129-αSyn antibody for WT r-αSyn incubated under were identified in S129A r-αSyn. After SDS-PAGE of WT other conditions (Additional file  1: Fig. S3). The insolu - r-αSyn, immunoblotting with anti-pY125-αSyn antibody ble aggregates of S129-phosphorylated WT r-αSyn were and anti-pY136-αSyn antibody detected detergent-insol- detected after 1  day of incubation (Fig.  3a, b). Mean- uble aggregates > 250  kDa after 7 and 3  days of incuba- while, S129A r-αSyn incubated with CK2 and ATP was tion with CK2 and ATP, respectively (Fig.  4a, b). No not phosphorylated at Ser129 and formed no insoluble immunoreactivity was detected on blots of S129A with aggregates (Fig.  3a, b). TEM revealed that WT r-αSyn anti-pY125-αSyn or anti-pY136-αSyn antibody (Fig.  4a, subjected to 15-day incubation with CK2 and ATP con- b). In BN-PAGE followed by immunoblotting analysis sisted exclusively of amorphous aggregates, but no such of WT r-αSyn incubated for 15  days, bands of approxi- aggregates were seen without incubation (Fig.  3c). The mately 1048  kDa were detected with antibodies against amorphous aggregates were present in relatively small pY125-αSyn and pY136-αSyn only in the presence of CK2 amounts in WT r-αSyn incubated in the absence of CK2 and ATP (Fig.  4c, d). Moreover, pS129 and pY136 were and ATP (Additional file  1: Fig. S4). These results were abolished and the formation of insoluble aggregates was consistent with a previous report [29] indicating that significantly inhibited by the addition of alkaline phos - CK2-induced pS129 accelerated aggregate formation of phatase in WT r-αSyn incubated with CK2 and ATP WT r-αSyn, and the insoluble aggregates were observed (Additional file  1: Fig. S6). These results suggested that in the mass range > 250 kDa as in the DN-LBD brain. Blue CK2 phosphorylates Y125 and Y136 of aggregated WT native (BN)-PAGE followed by immunoblotting analy- sis with anti-αSyn antibody of WT r-αSyn incubated for Sano et al. acta neuropathol commun (2021) 9:182 Page 8 of 17 Fig. 3 Ser129 phosphorylation by CK2 accelerates αSyn aggregate formation. a, b WT r-αSyn or S129A r-αSyn after 0–14 days of incubation with CK2 and ATP was analyzed by SDS-PAGE followed by immunoblotting with a anti-αSyn antibody D119 and b anti-pS129-αSyn antibody ab51253. c WT r-αSyn after 0 or 15 days of incubation with CK2 and ATP was examined by TEM. Bars, 100 nm. d, e WT r-αSyn after 0 or 15 days of incubation in the presence (+) or absence (−) of CK2 or ATP was analyzed by BN-PAGE followed by immunoblotting with d anti-αSyn antibody D119 and e anti-pS129-αSyn antibody ab51253. Molecular mass markers are indicated in kDa on the left side of each panel. Arrows indicate the top of the gel. In a, b, d and e, one representative blot from three independent experiments is shown r-αSyn in  vitro, and that the tyrosine phosphorylation is CK2 and ATP were detected by anti-pS129-αSyn anti- mediated through pS129. body (Additional file  1: Fig. S7), while no immunoreac- tivity was detected with anti-pY136-αSyn antibody in any Y136 phosphorylation prevents S129 phosphorylation samples (Additional file  1: Fig. S7). Although no αSyn- and aggregate formation of αSyn in vitro positive band > 250  kDa of WT r-αSyn or Y136A r-αSyn On SDS-PAGE followed by immunoblotting, Y136A was observed within 40 h of incubation in the absence of r-αSyn incubated for 14  days also exhibited detergent- CK2 and ATP (Additional file  1: Fig. S8), the band was insoluble aggregates > 250 kDa, which were most strongly detected after 8  h of incubation in the presence of CK2 detected in the presence of both CK2 and ATP (Addi- and ATP (Fig.  5a). The intensity of the αSyn-positive tional file  1: Fig. S7). Insoluble aggregates > 250  kDa as band > 250  kDa of Y136A r-αSyn was significantly well as 16 kDa monomer of Y136A r-αSyn incubated with greater than that of WT r-αSyn after 8  h of incubation S ano et al. acta neuropathol commun (2021) 9:182 Page 9 of 17 Table 1 List of identified peptides derived from r-αSyn incubated with CK2 and ATP for 7 days Query Start End Observed Mr(expt) Mr(calc) ppm Score Expect Peptide WT 5284 44 58 802.9034 1603.792 1603.797 − 2.97 65 3.5E−07 K.TpKEGVVHGVATVAEK.T 11012 46 80 1172.286 3513.836 3513.808 8.02 51 8.4 E−06 K.EGVVHGVATpVAEKTKEQVTN*VGGAVVTGVTAVAQK.T 8325 59 80 746.3851 2236.134 2236.147 − 5.77 47 0.000021 K.TKEQVTpNVGGAVVTGVTAVAQK.T 8336 59 80 1120.068 2238.122 2238.115 3.44 21 0.0083 K.TpKEQ*VTN*VGGAVVTGVTAVAQK.T 11170 59 96 924.9822 3695.9 3695.914 − 3.77 76 2.4 E−08 K.TpKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVK.K 5009 81 96 779.8777 1557.741 1557.744 − 1.95 88 1.6 E−09 K.TVEGAGSpIAAATGFVK.K 12198 98 140 1228.245 4908.951 4908.947 0.96 20 0.011 K.DQLGKN*EEGAPQEGILEDMPVDPDN*EAYEMPSpEEGYQDYEPEA 12214 98 140 1232.002 4923.98 4923.958 4.6 34 0.00044 K.DQLGKN*EEGAPQEGILEDMoPVDPDNEAYEMPSEEGYQDYpEPEA 12225 98 140 1235.754 4938.985 4938.969 3.36 21 0.01 K.DQLGKNEEGAPQEGILEDMoPVDPDNEAYEMoPSEEGYQDYpEPEA 12226 98 140 1235.756 4938.994 4938.969 5.24 30 0.0011 K.DQLGKNEEGAPQEGILEDMoPVDPDNEAYpEMoPSEEGYQDYEPEA 12228 98 140 1236.002 4939.979 4939.953 5.31 23 0.0073 K.DQLGKNEEGAPQEGILEDMoPVDPDN*EAYEMoPSEEGYQDYpEPEA 12229 98 140 1236.003 4939.982 4939.953 5.91 26 0.0032 K.DQ*LGKNEEGAPQEGILEDMoPVDPDNEAYEMoPSEEGYQDYpEPEA 11769 103 140 1092.433 4365.703 4365.693 2.34 21 0.0077 K.NEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYpEPEA 11770 103 140 1456.242 4365.704 4365.693 2.61 18 0.017 K.NEEGAPQEGILEDMPVDPDNEAYEMPSpEEGYQDYEPEA 11805 103 140 1461.57 4381.688 4381.688 0.11 24 0.004 K.NEEGAPQEGILEDMPVDPDNEAYEMoPSpEEGYQDYEPEA 11806 103 140 1461.572 4381.693 4381.688 1.19 22 0.0069 K.NEEGAPQEGILEDMPVDPDNEAYEMoPSpEEGYQDYEPEA 11807 103 140 1461.572 4381.694 4381.688 1.45 28 0.0015 K.NEEGAPQEGILEDMoPVDPDNEAYEMPSpEEGYQDYEPEA 11808 103 140 1096.431 4381.696 4381.688 1.93 25 0.0034 K.NEEGAPQEGILEDMPVDPDNEAYEMoPSpEEGYQDYEPEA 11809 103 140 1461.576 4381.707 4381.688 4.54 28 0.0017 K.NEEGAPQEGILEDMPVDPDNEAYEMoPSpEEGYQDYEPEA 11851 103 140 1100.425 4397.671 4397.682 − 2.69 31 0.00073 K.NEEGAPQEGILEDMoPVDPDNEAYEMoPSpEEGYQDYEPEA 11855 103 140 1466.906 4397.696 4397.682 3.13 31 0.00086 K.NEEGAPQEGILEDMoPVDPDNEAYEMoPSEEGYQDYpEPEA 11856 103 140 1466.908 4397.701 4397.682 4.29 24 0.004 K.NEEGAPQEGILEDMoPVDPDNEAYEMoPSEEGYQDYpEPEA 11870 103 140 1467.57 4399.687 4399.651 8.37 24 0.0039 K.NEEGAPQEGILEDMoPVDPDN*EAYEMoPSpEEGYQDYEPEA S129A 3243 11 23 691.3577 1380.701 1380.701 − 0.32 28 0.0014 K.AKEGVVAAAEKTpK.Q 1528 22 32 570.2711 1138.528 1138.538 − 9.45 46 0.000028 K.TpKQGVAEAAGK.T 4891 44 58 802.8981 1603.782 1603.797 − 9.67 60 9.1 E−07 K.TpKEGVVHGVATVAEK.T 3201 46 58 688.3341 1374.654 1374.654 − 0.57 42 0.00006 K.EGVVHGVATpVAEK.T 8000 59 80 1119.079 2236.144 2236.147 − 1.15 64 4.2 E−07 K.TpKEQVTNVGGAVVTGVTAVAQK.T 8001 59 80 746.3887 2236.144 2236.147 − 1.02 25 0.003 K.TKEQVTNVGGAVVTGVTpAVAQK.T 5372 81 97 843.9226 1685.831 1685.839 − 4.98 52 6.4 E−06 K.TVEGAGSpIAAATGFVKK.D p indicates phosphorylation sites; o indicates oxidation site *Indicates deamidation site (Fig.  5b). Moreover, ThT spectroscopic assay showed an presence of CK2 and ATP than other reactions (Fig.  5e). increase in fluorescence in reactions with Y136A r-αSyn The lag phase was significantly shorter in reactions with in the presence of CK2 and ATP, but not in their absence, Y136A r-αSyn in the presence of CK2 and ATP than whereas no increase in fluorescence was observed with other reactions (Fig. 5f ). These results suggest that block - WT r-αSyn regardless of the presence or absence of CK2 ing Y136 phosphorylation facilitates aggregation and and ATP within 7 days (Fig. 5c, Additional file  1: Fig. S9). amyloid fibril formation of αSyn. The insoluble aggre - Y136A r-αSyn amyloid fibrils were observed in reactions gates > 250  kDa were detected in WT r-αSyn, but not in the presence of CK2 and ATP on day 7 of incubation Y136A r-αSyn, with an antibody against pY136-αSyn and by TEM analysis (Fig.  5d, Additional file  1: Fig. S10). In in both WT and Y136A r-αSyn with an antibody against contrast, amorphous aggregates, but not fibrils, were pS129-αSyn after 8  h of incubation in the presence of exclusively observed in reactions with WT r-αSyn in CK2 and ATP (Fig.  6a, b). Interestingly, the intensity of the presence of CK2 and ATP (Fig.  5d, Additional file  1: the pS129-αSyn-positive band of Y136A r-αSyn was sig- Fig. S10). The maximal fluorescence intensity was sig - nificantly higher than that of WT r-αSyn at 8  h of incu - nificantly higher in reactions with Y136A r-αSyn in the bation (Fig.  6c). Unlike WT r-αSyn, immunoreactivity Sano et al. acta neuropathol commun (2021) 9:182 Page 10 of 17 Fig. 4 Ser129 phosphorylation by CK2 elicits Tyr125 and Tyr136 phosphorylation of αSyn. a, b WT r-αSyn or S129A r-αSyn after 0–14 days of incubation with CK2 and ATP was analyzed by SDS-PAGE followed by immunoblotting with a anti-pY125-αSyn antibody and b anti-pY136-αSyn antibody. c, d WT r-αSyn after 0 or 15 days of incubation in the presence (+) or absence (−) of CK2 or ATP was analyzed by BN-PAGE followed by immunoblotting with c anti-pY125-αSyn antibody and d anti-pY136-αSyn antibody. Molecular mass markers are indicated in kDa on the left side of each panel. Arrows indicate the top of the gel. In a–d, one representative blot from three independent experiments is shown with anti-pS129-αSyn antibody was more prominent in These observations indicated that blocking of Y136 phos - the insoluble aggregates with molecular weight > 250 kDa phorylation facilitated aggregate formation and phospho- than 16-kDa monomer of Y136A r-αSyn at 8–40  h of rylation at other C-terminal residues, especially S129, of incubation (Fig.  6b). The insoluble aggregates > 250  kDa r-αSyn. The results suggest that pY136 inhibits pS129 of were detected at relatively low levels in WT r-αSyn and αSyn and thereby prevents aggregate formation. Y136A r-αSyn with anti-pY125-αSyn antibody after 8  h of incubation in the presence of CK2 and ATP (Fig.  6d). Exogenous oligomeric αSyn converts endogenous The intensity of the pY125-αSyn-positive band of Y136A αSyn into insoluble aggregates with pS129 and pY136 r-αSyn was significantly higher than that of WT r-αSyn in a prion‑like manner and Y136 phosphorylation is at 8 h of incubation (Fig.  6e). Only a low level of insolu- involved in protecting against S129 phosphorylation and ble aggregates > 250  kDa was detected in Y136A r-αSyn aggregate formation of αSyn in cultured cells by anti-pY133-αSyn antibody after 32 and 40  h of incu- We reported previously that oligomeric r-αSyn produced bation in the presence of CK2 and ATP, whereas no by agitation shows potent prion-like seeding activity immunoreactivity was detected with anti-pY133-αSyn in  vitro by real-time quaking-induced conversion (RT- antibody for WT r-αSyn (Additional file  1: Fig. S11). QUIC) seeding assay [29]. We confirmed the contribution S ano et al. acta neuropathol commun (2021) 9:182 Page 11 of 17 WT Y136A WT Y136A a a 08 16 24 32 40 08 16 24 32 40 (h) 08 16 24 32 40 08 16 24 32 40 (h) kDa *** 15 kDa pY136 WT Y136A αSyn WT Y136A bc 08 16 24 32 40 08 16 24 32 40 (h) CK2+ATP c d WT WT *** CK2+ATP CK2+ATP CK2+ATP WT Y136A Y136A Y136A kDa 100000 50 60000 200 40000 20 07 123456 WT Y136A Time (d) pS129 WT Y136A de ** ** ef 816243240( 08 16 24 32 40 h) ** ** ** * ** 260000 kDa 250 300 20000 0 WT Y136A pY125 Fig. 6 Blocking Tyr136 phosphorylation promotes phosphorylation of αSyn at Ser129 and Tyr125 induced by CK2. WT r-αSyn or Fig. 5 Blocking Tyr136 phosphorylation promotes aggregation Y136A r-αSyn after 0–40 hours of incubation with CK2 and ATP and amyloid fibril formation of αSyn. a WT r-αSyn or Y136A r-αSyn was analyzed by SDS-PAGE followed by immunoblotting with a after 0–40 h of incubation with CK2 and ATP was analyzed by anti-pY136-αSyn antibody, b anti-pS129-αSyn antibody ab51253, and SDS-PAGE followed by immunoblotting with anti-αSyn antibody d anti-pY125-αSyn antibody. Molecular mass markers are indicated D119. Molecular mass markers are indicated in kDa on the left side in kDa on the left side of each panel. Arrows indicate the top of the of each panel. Arrows indicate the top of the gel. b Intensity ratios gel. Intensity ratios (%) of immunoreactive c pS129-αSyn, the sum (%) of immunoreactive > 250-kDa αSyn after 8 h of incubation were of > 250-kDa and 16-kDa forms, and e > 250-kDa pY125-αSyn after 8 h quantified in seven independent experiments. Data are presented as of incubation were quantified in seven independent experiments. means ± standard deviation. Statistical significance was determined Data are presented as means ± standard deviation. Statistical using the 2-tailed Student’s t test. ***P < 0.001 vs. W T r-αSyn. c ThT significance was determined using the 2-tailed Student’s t test. assays were performed in reactions with WT r-αSyn in the presence **P < 0.01, ***P < 0.001 versus W T r-αSyn CK2+ATP (WT ) or absence ( WT ) of CK2 and ATP or in reactions with CK2+ATP Y136A r-αSyn in the presence (Y136A ) or absence (Y136A) of CK2 and ATP. The results show the kinetics of ThT fluorescence from one representative of six replicate wells for each condition. d The showed that polymers of αSyn > 25  kDa in addition to CK2+ATP CK2+ATP end products from reactions with WT or Y136A were monomers accumulated in cells transfected with WT examined by TEM. Bars, 100 nm. Values of e maximal fluorescence intensities and f lag phase obtained in six individual wells in ThT r-αSyn or Y136A r-αSyn subjected to agitation, whereas assay are plotted. Data are presented as means ± standard deviation. no αSyn immunoreactivity was detected in cells trans- Statistical significance was determined using one-way ANOVA fected with WT r-αSyn or Y136A r-αSyn without agita- followed by Tukey–Kramer test. *P < 0.05, **P < 0.01 tion (Fig. 7b). The levels of accumulation of αSyn > 25 kDa were significantly higher in cells transfected with agitated Y136A r-αSyn than with agitated WT r-αSyn (Fig.  7c). of phosphorylation at S129 and Y136 to aggregate forma- Furthermore, the levels of αSyn accumulation in cells tion of αSyn in SH-SY5Y cells using oligomeric r-αSyn as transfected with agitated WT r-αSyn or Y136A r-αSyn seeds for prion-like propagation. WT r-αSyn and Y136A were significantly increased by overexpression of WT r-αSyn were converted into oligomeric forms by agitation αSyn or Y136A αSyn, respectively (Fig.  7d, e), indicat- (Fig.  7a). There were no significant differences in both ing the prion-like seeding activity of agitated WT r-αSyn forms. SDS-PAGE followed by immunoblotting analysis CK2+ATP WT CK2+ATP Y136A WT Y136A CK2+ATP WT CK2+ATP Y136A WT Y136A ThT fluorescence(arbitrary units) ThT fluorescence(arbitrary units) Lag phase (h) >250 kDa αSyn intensity ratio (%) pY125-αSyn intensity ratio (%) pS129-αSyn intensity ratio (%) Sano et al. acta neuropathol commun (2021) 9:182 Page 12 of 17 a bc d WT kDa kDa ** *** 100 100 αSyn αSyn 100 20 Y136A β-actin β-actin WT - -- -- - + + Plasmid 37 -agitation +agitation - -- -- + - + Y136A Seed Seed e f g h pS129 pY136 *** kDa kDa 250 * *** 400 0 20 -- - WT + 0 Plasmid - - - 15 WT + - Y136A + Plasmid WT - - - - + + - WT + + Y136A + Plasmid Plasmid - + - + - - Y136A Y136A + + Seed Seed +agitation +agitation Seed Seed Fig. 7 Tyr136 phosphorylation prevents aggregate formation and Ser129 phosphorylation of αSyn in cultured cells. a WT r-αSyn or Y136A r-αSyn subjected to agitation was examined by TEM. Bars, 100 nm. b–h WT r-αSyn or Y136A r-αSyn seed with (+ agitation) or without (− agitation) agitation was introduced into SH-SY5Y cells with (+) or without (−) pcDNA3.1 plasmid encoding WT or Y136A αSyn. The lysates from cells were analyzed by SDS-PAGE followed by immunoblotting with b, d anti-αSyn antibody Syn204, b, d anti-β-actin antibody, f anti-pS129-αSyn antibody D1R1R, and h anti-pY136-αSyn antibody. Molecular mass markers are indicated in kDa on the left side of each panel. Arrows indicate the top of the gel. Intensity ratios (%) of immunoreactive (c, e) αSyn > 25 kDa and g pS129-αSyn were quantified in at least three independent experiments. Data are presented as means ± standard deviation. Statistical significance was determined using c, e one-way ANOVA followed by Tukey–Kramer test and g 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001 and Y136A r-αSyn in cultured cells. Detergent-insoluble in WT αSyn-overexpressing cells transfected with agi- aggregates > 250  kDa were detected in cells overexpress- tated WT r-αSyn (Fig.  7h). Although antibodies against ing WT αSyn or Y136A αSyn transfected with agitated, pY125-αSyn and pY133-αSyn also detected bands in the but not non-agitated, WT r-αSyn or Y136A r-αSyn using mass range of around 15–100 kDa in cells, the intensities an antibody against pS129-αSyn, although 16-kDa mono- of the bands were unaffected by the introduction of WT mer was detected in all samples (Fig. 7f ). The intensity of r-αSyn or Y136A r-αSyn regardless of the form of seeds the pS129-αSyn-positive band was significantly higher in (i.e., agitated or non-agitated) (Additional file 1: Fig. S12). cells transfected with agitated Y136A r-αSyn than with These results suggest that oligomeric αSyn can convert agitated WT r-αSyn (Fig.  7g). Immunostaining with an native αSyn into insoluble aggregates that undergo phos- antibody against pY136-αSyn detected high molecular phorylation of S129 and Y136, and that blocking Y136 weight bands at > 250 kDa in addition to monomers only -agitation +agitation -agitation +agitation -agitation +agitation -agitation +agitation -agitation +agitation >25 kDa αSyn intensity ratio (%) WT Y136A WT Y136A WT Y136A WT Y136A >25 kDa αSyn intensity ratio (%) WT Y136A WT Y136A pS129-αSyn intensity ratio (%) WT WT Y136A Y136A WT Y136A WT Y136A WT WT Y136A Y136A WT WT Y136A Y136A S ano et al. acta neuropathol commun (2021) 9:182 Page 13 of 17 phosphorylation facilitates aggregate formation and S129 phosphorylation of αSyn in cultured cells. * a b kDa *** 120 *** CK2 inhibitor suppresses αSyn aggregate formation and S129 phosphorylation, and increases Y136 αSyn phosphorylation To examine whether CK2 is involved in aggregate forma- tion and C-terminal phosphorylation of αSyn in cells, we investigated the effects of the CK2 inhibitor, 4,5,6,7-tetra - Seed - + + + TBB (nM) 01 0 10 00 bromobenzotriazole (TBB), in SH-SY5Y cells exposed to β-actin agitated WT r-αSyn. TBB significantly reduced the levels Seed ++ + of accumulation of αSyn > 25  kDa in a dose-dependent TBB (nM) 00 10 100 manner in cells transfected with agitated WT r-αSyn ** c d (Fig.  8a, b). TBB treatment also significantly reduced kDa * *** the intensity of the pS129-αSyn-positive band of αSyn aggregates > 250  kDa in the cells in a dose-dependent 50 80 manner (Fig.  8c, d). Unexpectedly, the cells also showed pS129 an increase in the intensity of the pY136-αSyn-positive band of αSyn aggregates > 250  kDa with TBB treatment (Fig.  8e). The levels of pY136-αSyn were significantly increased by TBB in a dose-dependent manner (Fig.  8f ). Seed - + + + TBB (nM) 01 0 10 00 These results suggest that inhibition of CK2 suppresses Seed ++ + TBB (nM) 00 10 100 αSyn aggregate formation by reduction of S129 phospho- rylation and an unexpected increase in Y136 phospho- e f kDa rylation in cells. *** 300 * Discussion 50 pY136 The results of the present study showed that insoluble 200 αSyn is highly phosphorylated at both Y136 and S129 in the LBD brain. In addition, pY136-αSyn, which was presumed to be oligomeric and/or ubiquitinated, was 0 Seed - + + + found to be constitutively expressed in the brain regard- Seed ++ + TBB (nM) 01 0 10 00 TBB (nM)0 010100 less of the presence or absence of neocortical LB. Both Fig. 8 TBB suppresses αSyn aggregates through reduced Ser129 pY125-αSyn and pY133-αSyn were almost undetectable phosphorylation and increased Tyr136 phosphorylation in SH-SY5Y by immunoblotting and were detected at low levels by cells. SH-SY5Y cells treated with TBB (10 and 100 nM) or 0.05% DMSO immunohistochemical analysis, indicating that phospho- (0) as vehicle were transfected with (+) or without (−) agitated WT rylated αSyn is present in relatively small amounts in the r-αSyn as seed. The lysates from cells were analyzed by SDS-PAGE followed by immunoblotting with a anti-αSyn antibody Syn204, brain. Moreover, Y125 and Y133 phosphorylation were a anti-β-actin antibody, c anti-pS129-αSyn antibody D1R1R, and e unaffected by the formation of insoluble αSyn aggregates anti-pY136-αSyn antibody. Molecular mass markers are indicated in the brain. The level of pY125 was reported to be higher in kDa on the left side of each panel. Arrows indicate the top of in the control than LBD brain by immunoblotting, and the gel. Intensity ratios (%) of immunoreactive b αSyn > 25 kDa, d was shown to be reduced in the aging human brain [7]. pS129-αSyn, and f pY136-αSyn were quantified in four independent experiments. Data are presented as means ± standard deviation. Both control subjects and LBD patients were 65–86 years Statistical significance was determined using one-way ANOVA old in this previous study [7], while brain tissues from followed by Tukey–Kramer test. *P < 0.05, **P < 0.01, ***P < 0.001 patients 75–92  years of age were examined in the pre- sent study. The previous study also showed that the level of pY125 in heads from flies expressing WT human αSyn previous study was not clearly indicated. Although all decreased with increasing incubation time at room tem- tissues were stored at − 80  °C until use in this study, the perature [7]. Therefore, the lack of detectable pY125 in postmortem interval or freeze-thawing may have been the brain by immunoblotting was likely due to the age of involved in the lack of detectable pY125. the patients and the postmortem interval in the present Several protein kinases have been suggested to be study. Brain tissues were examined 8–12 years postmor- responsible for S129 phosphorylation of αSyn, including tem in this study, while the postmortem interval in the pY136-αSyn pS129-αSyn >25 kDa αSyn intensity ratio (%) intensity ratio (%) intensity ratio (%) Sano et al. acta neuropathol commun (2021) 9:182 Page 14 of 17 CK1, G protein-coupled receptor kinases (GRKs), and commonly recognized by CK2 for tyrosine phosphoryla- polo-like kinases (PLKs). We also showed that CK2 phos- tion, it is possible that Y136 is the C-terminal tyrosine phorylated αSyn at S129 in vitro, which was strongly cor- residue most susceptible to phosphorylation, consistent related with αSyn aggregate formation consistent with with our results, because the amino acid at positions n + 1 our previous report [29]. Although CK2 has generally and n + 3 from Y136 are the acidic residues, E137 and been classified as a serine/threonine protein kinase, sev - E139, respectively, while acidic residues are present at eral studies have demonstrated its tyrosine phosphoryla- positions n + 1 from Y125 and n + 2 from Y133, (E126 tion activity, suggesting that it acts as a dual-specificity and D135, respectively) (Fig.  1a). Two members of the kinase [3, 33, 37]. Indeed, we found that the C-terminal PLK family, PLK2 and PLK3, recognize an acidic residue tyrosine residues surrounding S129, Y125 and Y136, similar to CK2 [27]. An in  vitro study showed that PLK2, were phosphorylated by CK2, and that tyrosine phos- and to a lesser extent PLK3, phosphorylated S129 more phorylation was exclusively found in insoluble αSyn spe- efficiently than CK2 [28]. However, PLK2 knockout (KO) cies in vitro. In addition, insoluble αSyn aggregation was mice showed not over 50% decrease in pS129, while PLK3 accompanied by pY136, but not pY125, as well as pS129 KO had little effect on pS129 in various brain regions [4 ]. in cultured human cells that had taken up extracellu- The remaining pS129 levels were not reduced by treatment lar αSyn oligomers as seeds. These findings suggested with PLK1-3 inhibitor in PLK2 KO mice [4]. The results that pY136 is related in some way to the formation and suggest that S129 can be phosphorylated by multiple propagation of αSyn aggregates in cells. Phosphorylation kinases in vivo. Moreover, PLK2 KO has been reported to at certain residues of αSyn has been shown to affect sub - have no effect on pS129 in LB but not presynaptic termi - sequent phosphorylation events in neighboring residues. nals in mice [36]. Therefore, it is likely that other, non-PLK Mutation of Y125 to phenylalanine (Y125F), preventing kinases, including CK2, mediate phosphorylation of S129 phosphorylation at this site, has been shown to decrease of αSyn aggregates in  vivo. Although it remains unclear the levels of S129 phosphorylation by CK1 in  vitro [16]. why pS129 is essential for subsequent tyrosine phospho- Double mutation of Y133 and Y136 to phenylalanine rylation, pS129 increases the negative charge on the C-ter- 2− enhanced phosphorylation of Y125 by Lyn tyrosine minus by the addition of a PO group and may therefore kinase in  vitro [22]. In our in  vitro experiment, preven- lower the threshold for tyrosine phosphorylation by CK2. tion of S129 phosphorylation in S129A mutant blocked It has been reported that most of the phosphorylation sites Y125 and Y136 phosphorylation by CK2 with forma- are located in intrinsically disordered regions [12], and that tion of αSyn aggregates, suggesting that pS129 mediates phosphorylation induces folding of the intrinsically disor- phosphorylation of Y125 and Y136 and this plays a role dered protein [2, 18]. Indeed, phosphorylation of the papil- in αSyn aggregate formation. Prominent pS129 was seen lomavirus E2 protein by CK2 has been reported to induce within 1 day of incubation of WT αSyn with CK2, while a conformational change that leads to degradation of the pY125 and pY136 were observed after 7 and 3  days of protein [25]. It has been also reported that pS129 increases incubation, respectively. Analysis of the kinetic param- the conformational flexibility of αSyn [24]. Therefore, it eters for phosphorylation of tyrosine-containing peptides is possible that disorder-to-order conformational tran- by CK2 in  vitro demonstrated that tyrosine phospho- sitions occur in the intrinsically disordered C-terminal rylation is less favorable than serine/threonine phospho- region of αSyn by pS129, and the conformational changes rylation [20]. Therefore, these results suggest that CK2 also enable phosphorylation of tyrosine residues by CK2 preferentially catalyzes phosphorylation of S129, which is in addition to induction of αSyn aggregate formation. The essential for subsequent Y125 and Y136 phosphorylation affinity of anti-pS129-αSyn antibody could be enhanced by in αSyn. Y136A mutation, or the levels of pS129 may be increased The substrate specificity of CK2 is determined by one or by Y136A substitution itself. However, the levels of expres- more negatively charged residues, i.e., aspartic acid (D)/ sion of αSyn and the levels of pS129 were not significantly glutamic acid (E), surrounding the phosphorylatable serine affected by Y136A mutation in αSyn-overexpressing cells (S) and threonine (T) residues. The minimum consensus as determined by immunoblotting analysis (Additional sequence is S/T-X-X-D/E, where X can be any amino acid. file  1: Fig. S13). There were also no differences in the The most crucial acidic residue position for susceptibility expression pattern of αSyn between WT and Y136A αSyn- to phosphorylation by CK2 is n + 3 followed by n + 1 [21]. overexpressing cells (Additional file  1: Fig. S13). Moreover, In the case of αSyn, the amino acid at position n + 1 from insoluble aggregates and amyloid fibrils of nonphosphoryl - S129 is E130 (Fig. 1a), which is predicted to mostly act as a ated Y136A r-αSyn were not formed by incubation (Fig. 5c, specificity determinant for S129 phosphorylation by CK2. Additional file  1: Fig. S7a). Therefore, it is unlikely that Little is known about the substrate specificity of CK2 for anti-pS129-αSyn antibody binds Y136A αSyn with differ - tyrosine phosphorylation. If the consensus sequence is ent affinity than WT αSyn and that the increase in pS129 S ano et al. acta neuropathol commun (2021) 9:182 Page 15 of 17 Abbreviations and acceleration of aggregate formation of αSyn by Y136A AD: Alzheimer’s disease; CK1: Casein kinase 1; CK2: Casein kinase 2; CJD: mutation are due to Y136A substitution itself. Although Creutzfeldt–Jakob disease; LB: Lewy body; LBD: Lewy body dementia; LC–MS/ the 16-kDa monomer of WT αSyn was phosphorylated MS: Liquid chromatography-ion trap mass spectrometry; MS: Mass spec- trometry; PD: Parkinson’s disease; TBB: 4,5,6,7-Tetrabromobenzotriazole; TEM: at Y136, there was no significant difference in the level Transmission electron microscopy; ThT: Thioflavin T; αSyn: α-Synuclein; WT: of pS129 between WT and Y136A αSyn-overexpressing Wild-type. cells (Additional file  1: Fig. S13). These results suggest that pY136 has little effect on pS129 of αSyn monomer in non- Supplementary Information diseased cells. The online version contains supplementary material available at https:// doi. Preventing phosphorylation of Y136 by Y136A muta- org/ 10. 1186/ s40478- 021- 01281-9. tion facilitated aggregate formation and S129 phos- phorylation of r-αSyn and αSyn in cultured cells. Y136 Additional file1. Supplementary fgures. may also be one of the major phosphorylatable sites for CK2 in negatively charged αSyn aggregates formed Acknowledgements with increased pS129 and to protect against further We thank Fuyuki Kametani of Tokyo Metropolitan Institute of Medical Science for LC-MS/MS analysis, Shinya Dohgu of Fukuoka University for providing S129 phosphorylation and αSyn aggregate formation by SH-SY5Y cells, and Megumi Saiki, Kaori Hirakawa, Shota Hasegawa, Yuhei undergoing phosphorylation instead of S129. TBB signifi - Moriyama, Saki Ogami, Haruka Taketomi, Yumiko Hori, Kasumi Kubo, Manami cantly inhibited S129 phosphorylation and suppressed Noda, Dan Hokama, Ayumi Yamada, and Hinako Nonaka of Fukuoka University for technical assistance. αSyn aggregate formation in cultured cells. These results suggested that CK2 is the main protein kinase for S129 Authors’ contributions phosphorylation of αSyn and that phosphorylation of KS and KM designed the project. YI and KS collected clinical specimens. YI performed immunohistochemistry. YY, KI, MH, and KS performed in vitro S129 by CK2 is closely related to the formation of αSyn experiments and cell culture experiments. KS and KS performed immunoblot- aggregates in SH-SY5Y cells. Therefore, CK2 may be a ting of clinical specimens. YI, KS, KM, and KS analyzed the data. KS wrote the therapeutic target for LB disease. Unexpectedly, phos- manuscript. All authors read and approved the final manuscript. phorylation of Y136 in αSyn aggregates was significantly Funding increased by TBB in cultured cells, suggesting that CK2 This work was supported by a grant-in-aid for Scientific Research (C) (grant plays an inhibitory role against Y136 phosphorylation no. 19K07858) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. in cells. As CK2 phosphorylates hundreds of physiologi- cal substrates to control various cellular processes [5], Availability of data and materials its in  vitro and in  vivo functions may not be consistent. All data generated or analyzed during this study are included in this published article and its supplementary information files. CK2 has been reported to phosphorylate threonine resi- dues of Src family tyrosine kinases, thereby resulting in Declarations reduction of their activities in  vitro [38]. Therefore, the significant increase in pY136 in αSyn aggregates may be Ethics approval and consent to participate due to disinhibition of tyrosine kinases under conditions The study protocol was approved by the Institutional Review Board-Inde- pendent Ethics Committee of Fukuoka University (ID: 20-03-M2), the Ethics where CK2 is inhibited in cells, although the major pro- Committee of Nagasaki University (ID: 19083005-2) and the Ethics Committee tein kinases for phosphorylation of Y136 have yet to be of Aichi Medical University (ID: 15-017). Informed consent was obtained from elucidated, and may contribute to reduction in pS129 and the patients and/or their families. confer protection against αSyn aggregate formation. Consent for publication Consent for the use of the brain tissue for research purposes and for publica- tion was obtained from the patients and/or their families. Conclusions The findings of the present study provide the first evidence Competing interests that CK2 phosphorylates Y136 in αSyn aggregates by medi- The authors report no competing interests. ating S129 phosphorylation as a dual-specificity kinase and Author details that pY136 has a protective effect against αSyn aggregation. 1 Department of Physiology and Pharmacology, Faculty of Pharmaceutical Although the primary kinase for pY136 in  vivo is not yet Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. Department of Neuropathology, Institute for Medical Science clear, this study suggested that CK2 is a candidate kinase of Aging, Aichi Medical University, Aichi 480-1195, Japan. Depar tment responsible for pY136 and that it participates in regulating of Immunological and Molecular Pharmacology, Faculty of Pharmaceutical Sci- αSyn aggregate formation. Further studies are necessary ences, Fukuoka University, Fukuoka 814-0180, Japan. 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Weston LJ, Stackhouse TL, Spinelli KJ, Boutros SW, Rose EP, Osterberg Springer Nature remains neutral with regard to jurisdictional claims in pub- VR, Luk KC, Raber J, Weissman TA, Unni VK (2021) Genetic deletion of lished maps and institutional affiliations. Polo-like kinase 2 reduces alpha-synuclein serine-129 phosphorylation Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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Acta Neuropathologica CommunicationsSpringer Journals

Published: Nov 12, 2021

Keywords: α-Synuclein; Lewy body dementia; Y136 phosphorylation; S129 phosphorylation; Casein kinase 2

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