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α-Synuclein arginylation in the human brain

α-Synuclein arginylation in the human brain Background: Alpha-synuclein (α-syn) exhibits pathological misfolding in many human neurodegenerative disorders. We previously showed that α-syn is arginylated in the mouse brain and that lack of arginylation leads to neurodegen- eration in mice. Methods: Here, we tested α-syn arginylation in human brain pathology using newly derived antibodies in combina- tion with Western blotting, biochemical assays, and experiments in live neurons. Results: We found that α-syn was arginylated in the human brain on E46 and E83, two sites previously implicated in α-syn pathology and familial cases of Parkinson’s disease. The levels of arginylation in different brain samples ranged between ~ 3% and ~ 50% of the total α-syn pool, and this arginylation nearly exclusively concentrated in the subcellu- lar α-syn fraction that sedimented at low centrifugation speeds and appeared to be simultaneously targeted by multi- ple posttranslational modifications. Arginylated α-syn was less susceptible to S129 phosphorylation and pathological aggregation in neurons. The arginylation level inversely correlated with the overall α-syn levels and with patient age, suggesting a possible causal relationship between arginylation decline and α-syn-dependent neuropathology. Conclusion: We propose that α-syn arginylation constitutes a potential neuroprotective mechanism that prevents its abnormal accumulation during neurodegeneration and aging in the human brain. Keywords: Arginylation, Neurodegeneration, Aging, α-Synuclein Background the exact role of this and other PTMs in regulating α-syn Alpha-synuclein (α-syn) exhibits pathological misfolding misfolding and aggregation is not well understood. and aggregation in human neurodegenerative disorders Protein arginylation mediated by arginyltransferase that are collectively called synucleinopathies [1]. One of ATE1 is a PTM of emerging biological significance that the most prevalent diseases in this group is Parkinson’s consists of transfer ribonucleic acid (tRNA)-dependent disease (PD), which is characterized by the formation transfer of Arg (R) to proteins and has been implicated of Lewy neurites and Lewy bodies (LBs) – large α-syn in many key physiological events in vivo [6, 7]. ATE1 can aggregates that contribute to neuronal loss, dementia, modify proteins either N-terminally or on the side chains and death [2]. It is well established that LB formation and of the acidic residues, i.e., Asp (D) or Glu (E) [8]. Previ- maturation are associated with multiple posttranslational ous work from our lab uncovered that α-syn is a highly modifications (PTMs) [3, 4], including, most promi- efficient target for ATE1 in vitro, and that in mouse brain nently, phosphorylation of α-syn at S129, which is gener- α-syn is arginylated at E46 and E83 [9], two sites that ally believed to be involved in PD pathology [5]. However, have been previously implicated in α-syn function and PD pathology in human patients [10, 11]. Mice lacking ATE1 in the brain develop symptoms of neurodegenera- *Correspondence: akashina@upenn.edu tion, suggesting that arginylation plays a role in normal Department of Biomedical Sciences, University of Pennsylvania School brain function and may act specifically via α-syn [9]. of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA Full list of author information is available at the end of the article © The Author(s) 2022. 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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. Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 2 of 13 Here, we tested whether E46 and E83 arginylation tar- washed with NaHCO and brine. The organic layer was gets α-syn in the human brain, and whether this argin- dried over M gSO and evaporated to dryness. Further ylation correlates with any physiological changes in the purification using silica gel gave rise to 375  mg pure patients, such as disease status or age. We also tested the Fmoc-Glu(Arg(Pbf)-OtBu)-OH with a total yield of potential interplay between α-syn arginylation and S129 45%. phosphorylation, another PTM previously implicated The compounds were verified by matrix assisted laser in PD pathology. Our results uncover a potential new desorption/ionization (MALDI) to determine their mechanism of α-syn regulation in the human brain and mass, as follows: Fmoc-Glu(Arg(Pbf)-OtBu)-OAll: + + propose functional implications of arginylation in neuro- [MH] 874.29; Fmoc-Glu(Arg(Pbf )-OtBu)-OH: [MH] degeneration and aging. 834.3750. Materials and methods Synthesis of E46‑ and E83‑arginylated α‑syn peptides used Materials as antigen for antibody generation Frozen postmortem brain samples of human frontal The arginylated peptides CVGSKTKE GVVH and cortex were from patient brain donors who underwent CAVAQKTVE GAG were synthesized manually using autopsy at the Center for Neurodegenerative Disease standard Fmoc-based strategy with modification as Research (CNDR) at the University of Pennsylvania reported in [13]. Briefly, 20  mg 2-chlorotrityl resin between 1992 and 2016 [12]. Detailed clinical character- (100–200 mesh, 1.5  mmol substitution/g) was swelled istics (age, sex, diagnosis) are listed in Additional file  1: in dry DCM for 1  h. Then the DCM was removed and Table  S1. All antibodies used in this project are listed in the first Fmoc-amino acid (1 equiv.) in 2  ml DCM and Additional file 1: Table S2. DIPEA (4 equiv.) were added to the resin. Upon mix- ing for 40  min, the resin was washed with DMF for Synthesis of fluorenylmethyloxycarbonyl four times. The resin was then capped by washing 3 (Fmoc)‑Glu(Arg(Pbf )‑OtBu)‑OH times with DCM/MeOH/DIPEA (volume ratio 17:2:1), To generate branched peptrides mimicking arginylated 3 times with DCM, and 3 times with DMF. The Fmoc sites on α-syn, we synthesized Fmoc-Glu(Arg(Pbf)- group was removed by adding 2 ml of 20% peperidine/ OtBu)-OH, an arginylated Glu derivative to be used to DMF and stirring for 20  min. The amount of coupled synthesize the Glu-arginylated peptides. To do this we amino acid was evaluated by the Fmoc content in the first generated a precursor, Fmoc-Glu(Arg(Pbf )-OtBu)- deprotection solution using absorbance at 300  nm OAll. The procedure for precursor generation and final −1 −1 (extinction coefficient: 7800  cm  M ). Subsequent synthesis was as follows. Two hundred and five milli - amino acids were coupled by adding Fmoc-amino acid grams of Fmoc-Glu-OAll (0.5  mmol) were dissolved (5 equiv.) that was activated by HBTU (5 equiv.) and in 15 ml tetrahydrofuran (THF) in a flask and the flask DIPEA (10 equiv.) and stirring for 30  min. For the on- was cooled to -20  °C. Next, 60  μl of N-methylmor- resin coupling of Fmoc-Glu(Arg(Pbf)-OtBu)-OH, 2 pholine (0.5  mmol) was added to this flask, followed equiv. of Fmoc-Glu(Arg(Pbf )-OtBu)-OH in DMF was by dropwise addition of 65  μl isobutyl chlorofor- activated by 2 equiv. of HBTU and 4 equiv. of DIPEA mate (0.5  mmol). The mixture was stirred for 10  min, and then mixed with resin for 2 h. After further elonga- and then 259  mg Arg(Pbf )-OtBu·HCl (0.5  mmol) and tion, the peptides were cleaved from resin by treatment additional 60  μl of N-methylmorpholine were added. with 90%TFA/5%TIPS/5%DCM for 1.5  h. The cleavage The resulting slurry was stirred at − 20  °C for 1  h and solution was pooled into cold ether and the precipi- then at room temperature for 3  h. The precipitate tate was collected and purified by reverse-phase HPLC was filtered off and the filtrate was evaporated under using VyDAC C18 column and 0.1% TFA/0.1% acetoni- reduced pressure. The residue was dissolved in 30  ml trile as the mobile phase. The purity was checked by EtOAc and then washed with N aHCO and brine. The analytic HPLC (Additional file  2: Dataset 1) and iden- organic layer was dried over M gSO and evaporated tity was confirmed by mass spectrometry (Additional to give rise to 460  mg oil, which was used directly for file 2 : Dataset 1). the next step. The obtained Fmoc-Glu(Arg(Pbf/OtBu)- MALDI: OAll (0.5  mmol) was dissolved in 5  ml dichlorometh- ane (DCM). To this flask was added 15  mg Pd(PPh3)4 R + CVGSKTKE GVVH: Calcd. [MH] 1399.75, found. (0.0125  mmol, 2.5% mol) and 617  μl PhSiH3 (5  mmol) 1399.69; under argon. The mixture was stirred for 4  h at room R + CAVAQKTVE GAG: Calcd. [MH] 1189.66, found. temperature and then evaporated under reduced pres- 1289.93. sure. The residue was re-dissolved in 20  ml DCM and Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 3 of 13 Generation of synthetic arginylated α‑syn variants After completion of the reaction, α-syn phosphoryla- Synthetic arginylated full-length α-syn variants were tion was detected by Western blots using the S129 generated as described in [13]. antibody. α‑Syn aggregation assays in cultured primary neurons Immunoblotting Primary neurons were isolated from C57-Bl6 mouse Frozen tissues were grounded in liquid nitrogen using neonatal brain as described [14], and incubated with a mortar and pestle until fine powder was formed. The non-arginylated α-syn or its arginylated variants (E46, pulverized tissue powder was then weighed and lysed E83, and double) as described [15]. Detection of α-syn directly in 4 × SDS loading buffer (1:10 w /v ratio), fol- inclusions was performed using the S129 antibody, and lowed by boiling for 10 min. Then 2.5 μl of each sample the inclusion fluorescence and shape were quantified was loaded on 15% SDS–polyacrylamide gels (PAGE) using the “integrated morphometry analysis” function in and transferred to a 0.2-μm nitrocellulose membrane the Metamorph imaging software (Molecular Devices, at 250  mA for 20  min. Blots were blocked in 3% BSA Downington, PA). The original images were uniformly in TBST, then incubated with primary antibodies processed using the “background subtraction” function (Additional file  1: Table S2) at 4 °C overnight. Then the in the Metamorph imaging software. membranes were incubated with secondary antibodies (1:5000 dilution) conjugated to IRDye800 or IRDye700 and images were acquired using the Odyssey Imag- Negative staining electron microscopy ing System. For comparison of arginylated α-syn lev- Non-arginylated or 5%-arginylated α-syn fibrils (E46, els across different human brain samples, GAPDH was E83, and double) were deposited on formvar-coated used as an internal loading control. grids, stained with 2% w/v uranyl acetate in water, and visualized by transmission electron microscopy. Fractionation of soluble and insoluble α‑syn Image analysis and quantification The tissue powder was lysed in reaction buffer (con - Western blots were quantified by direct measurements taining 100  mM HEPES, pH 7.2, 50  mM KCl, 1  mM of the secondary antibody fluorescence using Odyssey MgCl , 1 mM EGTA, and 1 mM DTT, with Sigma pro- gel imager (LiCor, Lincoln, NE). Quantification of the tease inhibitor cocktail added before use) at 1:10 w/v band intensity over the background was calculated using ratio, and then centrifuged at 13,000 r/min in a table- the Odyssey gel imaging software integrated with the top microfuge at 4  °C for 30  min. The supernatant and instrument. pellet were collected as soluble and insoluble pool for For description of intracellular S129-positive aggre- Western blotting analysis. The supernatant was mixed gates, the following parameters were measured: at 1:1 ratio with 4× SDS loading buffer. The pellet was resuspended in 4× SDS loading buffer equal to the 1) “total intensity” (following the command name in the volume of the original extract, and both samples were Metamorph imaging software) measures the total boiled for 10 min prior to loading on the gel. Then 6 µl gray level of each aggregate, thresholded against the of pellet and 20  µl of supernatant were loaded on 10% background; this parameter directly depends on the bis–tris gels for mass spectrometry or 15% SDS-PAGE area and density of the aggregate and thus quantita- for Western blot. tively reflects the aggregate size. 2) “average intensity” (following the command name in In vitro phosphorylation of α‑syn using Polo‑like kinase 2 the Metamorph imaging software) measures the gray (PLK2) levels (equaling fluorescence intensity) per unit area, For each reaction, PLK2 solution (Catalog Number: and this reflects the density of the aggregate, as more PV4204, ThermoFisher Scientific, Waltham, MA) was densely packed aggregate would have higher fluores - mixed with recombinant α-syn at a ratio of 2  µl PLK2 cence per unit area. per 72  µg α-syn in the reaction buffer containing 25  mM HEPES, pH 7.2, 50  mM NaCl, 20  mM MgCl , 2 mM DTT, and 1 mM ATP freshly added from a frozen Statistical analysis stock. For the reaction, components were mixed on ice Calculations for Figs. 1, 3, S3, S4, and S5 were performed in the following order: final reaction buffer, α-syn, fol - using MATLAB software, and for the other figures lowed by PLK2, and the mixture was then transferred using the Graphpad Prism software package (version 8.0). to 30  °C for 2  h. For negative controls, ATP or PLK2 For the Graphpad Prism analysis, the data are presented was omitted from the reaction, as indicated elsewhere. as mean ± standard deviation (SD), unless otherwise Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 4 of 13 Fig. 1 E46- and E83-arginylated α-syn are present in the human frontal cortex and show varied levels in PD patients and healthy controls. a Representative immunoblots of total α-syn, as well as α-syn modified by phosphorylation at S129 or arginylation at E46 or E83. b Quantification of α-syn signal from all antibodies in all samples; for total α-syn, signals from both bands of the doublet in the ~ 14 kDa range were added, and the upper and lower bands of the doublet were also quantified separately. Error bars represent SEM from triplicate runs of the same samples. C, control; PD, Parkinson’s disease; PDD, Parkinson disease with dementia. P-value comparisons between control and PD (P12), control and PDD (P13), as well as PD and PDD (P23) are shown on top of each panel, calculated by two-tailed Welsh t-test. See Table S1 for further details on the patient samples indicated,  and statistical comparisons between differ - analysis, followed by two-tailed student’s t-test between ent groups were first performed using one-way ANOVA two groups with the P-value corrected by Bonferroni test. Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 5 of 13 E46 and E83 arginylation targets the α‑syn pool Results that sediments at lower centrifugation speeds and is also α‑Syn is arginylated at E46 and E83 in the human brain enriched in S129 phosphorylation Our previous work showed that α-syn is arginylated in During the experiments described above, we noticed the mouse brain on two conserved sites, E46 and E83, that the single band on the SDS-PAGE recognized by which are targeted for arginylation within the intact non- the antibodies for arginylated and S129-phosphoryl- proteolyzed α-syn protein [9]. Both of these sites are con- ated α-syn ran somewhat higher on the gel than the served between mouse and human α-syn, and both have expected 14 kDa. At the same time, the total α-syn anti- previously been implicated in α-syn pathology, including bodies recognized a doublet in the ~ 14 kDa range, with the familial E46K mutation in PD patients [3, 11]. To test the upper band in the doublet matching the position of whether E46 and E83 arginylation occur in human synu- the band positive for both arginylated and phosphoryl- cleinopathy and to dissect their potential roles in patho- ated α-syn (Fig.  2a). Since SDS gel shifts often happen genesis, we raised rabbit polyclonal antibodies against after proteins have undergoen PTMs, including phos- synthetic peptides corresponding to the α-syn side-chain phorylation [16], we hypothesized that the upper band arginylated at E46 and E83 (Additional file  1: Fig. S1a). represents a subset of α-syn that is simultaneously tar- These antibodies were specific for the corresponding geted by multiple PTMs. arginylated peptides and full-length synthetic arginylated First, to test if this upper band indeed represents α-syn, and showed no cross-reactivity with each other, or α-syn, we excised bands of matching molecular weight with non-arginylated α-syn protein and peptides (Addi- from an SDS gel and identified protein composition in tional file 1: Fig. S1a, b). these bands by mass spectrometry to confirm the pres - We next used these antibodies to probe extracts from ence of α-syn as a major protein in both bands (Addi- the frontal cortex samples of patients, obtained from the tional file  1: Fig. S2). Next, to test whether modified and biobank at the Center for Neurodegenerative Disease unmodified α-syn are enriched in the same subcellular Research (CNDR) at the University of Pennsylvania. We fractions, we fractionated human brain extracts by cen- analyzed healthy controls, as well as patients with PD trifugation at 13,000  g and tested the supernatant and and PD-related dementia (PDD, characterized by severe pellet from this step by Western blot. Strikingly, the neuronal loss and pathological α-syn aggregation). Strik- majority of the upper band, detectable by pS129, E46- ingly, all brain samples showed prominent reactivity with Arg, and E83-Arg antibodies, was found in the pellet, both E46 and E83 antibodies (Fig.  1), suggesting that the while the lower band remained in the supernatants human α-syn is arginylated in vivo at both E46 and E83. (Fig. 2B). Thus, all three PTMs nearly exclusively target Several interesting trends were observed by compari- the α-syn pool that sediments at lower speeds, a subcel- sons of the patient samples (Fig. 1a, b). First, the levels of lular fraction that typically contains intracellular orga- both E46 and E83 arginylation strongly varied between nelles and larger protein aggregates. samples, suggesting that arginylation in different individu - During PD, pathologically misfolded insoluble α-syn als can target vastly different fractions of the intracellular often incorporates into LBs, large α-syn-rich protein α-syn pool. Second, the levels of arginylation of E46 and aggregates that are known to be targeted by multiple E83 showed a similar trend, with  a comparatively  higher PTMs as a hallmark feature of PD pathology [2]. Pre- or a lower antibody signal  in the same samples, suggest- vious studies have associated LBs with pS129 staining ing that α-syn arginylation in these samples is high or [17], even though the exact role of S129 phosphoryla- low as a whole, without apparent selectivity between the tion of α-syn in PD pathology remains controversial two arginylation sites. Notably, the same trends were also [8, 18]. To test whether E46 and E83 arginylation tar- observed with antibodies against α-syn phosphorylated gets LBs, we co-stained brain sections from human PD at S129, suggesting that all three modifications co-elevate patients with antibodies to total α-syn and either E46- in some samples compared to others and may potentially Arg or E83-Arg α-syn (Fig.  2c). Total α-syn staining interact with each other. revealed the presence of large aggregates clearly visible in different areas of the sections. However, these aggre - gates did not show any enrichment in E83-Arg stain- ing. In the case of E46-Arg, most of the aggregates were also not stained, but some of the larger aggregates, with morphology characteristic for the later stages of LB Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 6 of 13 Fig. 2 E46 and E83 arginylation targets the α-syn pool that sediments at lower centrifugation speeds in extracts from the frontal cortex, but does not strongly colocalize with Lewy bodies in the frontal cortex sections from human PD patients. a Magnification of a representative Western blot of the sample C1 probed with the total and modified α-syn antibodies, showing the doublet α-syn bands recognized by the total α-syn antibody and the relative position of the single band preferentially recognized by the antibodies to the modified α-syn variants as the upper band in this doublet. b Representative Western blots from the total brain extract, pellet, and supernatant fractions. Bands of heavily modified α-syn were preferentially found in the pellet, indicating its incorporation into the insoluble α-syn pool. c Representative images of human brain sections showing prominent LB formation, stained with antibodies to total α-syn, as well as the antibodies against E46- and E83-arginylated variants. LBs were prominently seen as large green dots in the top set of images stained with total α-syn antibodies. Most of these LBs show no specific staining with E83, and only occasional aggregates were visualized with E46 antibodies (short arrows). Both E46 and E83 antibodies also stained fibrillar aggregates (long arrows) Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 7 of 13 formation, were somewhat highlighted with E46-Arg, the decrease in the upper band of the doublet, while the suggesting that E46-arginylated α-syn penetrates the lower band followed the total α-syn trends (Additional LBs over time, possibly during LB maturation. Nota- file  1: Fig. S4). Interestingly, within the samples we tested, bly, both E46-Arg and E83-Arg antibodies also showed no significant correlation was seen between the levels of some fibrillar staining patterns, suggesting that these each PTM and the PD or PDD diagnosis (with P values modifications target α-syn-containing structures in the for comparison between the groups ranging at 0.5 and brain other than LBs. It is unclear if this LB-independ- above, Fig.  1b), suggesting that brain aging, rather than ent α-syn pool is involved in normal brain function PD diagnosis, is the primary factor in α-syn arginylation. and/or plays a neuroprotective, rather than pathologi- Both arginylation and S129 phosphorylation cal, role. inversely correlated with the total α-syn levels in dif- ferent patients (Fig.  3, bottom row), suggesting that α‑Syn E46 and E83 arginylation in the human brain all three modifications are antagonistic to α-syn inversely correlates with the total α‑syn levels accumulation in the brain, a proposed major fac- and prominently decreases with age tor in α-syn-driven neurodegeneration [19, 20]. All Next, we used the quantification data (Fig.  1) to analyze three modifications showed a strong positive correla- correlations between the Western blot signals for E46/ tion with the upper band in the doublet and a strong E83 arginylation, S129 phosphorylation, and total α-syn inverse correlation with the lower band (Additional in different patients. We quantified total α-syn in each file 1: Fig. S3). Consistent with this, all three modifica- sample as the sum of the upper and lower bands in the tions showed similar correlation trends, strongly sug- doublet (Fig.  3), and the upper and lower bands sepa- gesting that they were positively correlated with each rately, corresponding to the modified and unmodified other (Additional file 1: Fig. S3). All these changes may α-syn fractions, respectively (Additional file 1: Fig. S3). potentially reflect functional interactions between This analysis revealed several interesting trends. First, arginylation, phosphorylation, and changes of α-syn while the total α-syn levels increased with the patient age, states in the brain. Of note, none of these changes E46/E83 arginylation as well as S129 phosphorylation correlated with a total change in ATE1 level in these showed a prominent decrease in older patients (Fig.  3, samples (Additional file  1: Fig. S5), suggesting that top row). This decrease was due almost exclusively to the changes in arginylation of α-syn at E46 and E83 in Fig. 3 E46 and E83 arginylation in the human frontal cortex inversely correlates with α-syn levels and decreases with patient age. Correlation plots between different antibody signals calculated from the same datasets as those shown in Fig. 1. Spearman correlation coefficients and P values are shown on top of each plot. See Additional file 1: Fig. S3 and S4 for additional plots Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 8 of 13 Fig. 4 Quantification of E83 arginylation as a fraction of the total α-syn levels in the human frontal cortex. a Representative Western blot images (left) and calibration curves (right), of standard unmodified and E83-arginylated α-syn. b Quantification of the fraction of E83-arginylated α-syn as the percentage of the upper band (U, marked with arrows in both gels) and of the total α-syn in the preparation (the sum of upper and lower bands marked with arrow and asterisk, respectively), calculated by running the calibrations. Percentage of arginylated α-syn to the upper band (U) and the total are shown both as a table (left) and as a chart (right) different brain specimens are not a direct consequence Quantification of α‑syn arginylation in the human brain of the altered availability of arginyltransferase. To quantify the percentage of arginylated α-syn in the These data collectively demonstrate that α-syn E46 human brain in individual samples, we used synthetic and E83 arginylation targets a specific α-syn pool in the E83-Arg α-syn as a protein standard to calibrate the human brain in a manner antagonistic to overall α-syn signal, alongside the unmodified α-syn. We first ran accumulation and correlates with changes in the brain’s calibration curves to directly quantify the amount of physiological state during aging. modified and unmodified α-syn in the standard lanes as Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 9 of 13 the function of the Western blot signal and determine up to ~ 50% of total α-syn. This is, by far, the highest frac - the linear range (Fig. 4a). Next, we ran some of these lin- tion of arginylation ever observed for any protein in vivo. ear range standards at known loads alongside the human patient brain samples to directly measure the actual E46 and E83 arginylation prevents S129 phosphorylation amount of total and E83-Arg α-syn in each brain sample. Our data so far strongly suggest that arginylation at E46 These measurements enabled us to calculate the percent - and E83 targets the same pool of α-syn in the brain as age of E83-arginylated α-syn in each sample. S129 phosphorylation, with the possible exception of the While these percentages varied across different sam - LBs. While the role of S129 phosphorylation on α-syn ples, they were remarkably high overall (Fig. 4b), ranging is still debated in the literature, some lines of evidence from several percent in some samples to up to ~ 70% of suggest that this modification is associated with PD the upper gel band containing the highly modified α-syn pathology [17]. To determine whether arginylation acts pool (calculated from quantifying the signal in the upper synergistically with S129 phosphorylation, or whether band only and comparing it to the protein standard) and these two modifications are antagonistic, we used Fig. 5 E46 and E83 arginylation prevents S129 phosphorylation. Representative Western blot images (left) and quantification (right) of phosphorylation of unmodified (U) and arginylated α-syn protein standards after the phosphorylation reaction with the addition of components shown on top. a α-Syn variants, either unmodified (U) or 100% arginylated at E46 or E83, were incubated with PLK2 kinase in the presence or absence of ATP. Arginylation greatly reduced the efficiency of PLK2 phosphorylation. b Phosphorylation of α-syn variants, either unmodified or arginylated at varied percentages as indicated, incubated with or without PLK2 kinase (PLK) as indicated on top. Arginylation interfered with PLK2 phosphorylation in a dose-dependent manner. Band intensities of S129 signal normalized to α-syn load are plotted. Error bars represent SEM, n = 3 Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 10 of 13 Arginylation at E46 and E83 decreases intracellular α‑syn synthetic arginylated α-syn standards for in  vitro phos- aggregation phorylation assays with Polo-like kinase 2 (PLK2), which To test directly whether α-syn arginylation has a neuro- mediates α-syn S129 phosphorylation in vivo [18, 21]. protective role, we tested the ability of arginylated α-syn First, we compared the phosphorylation efficiency of at E46 or E83, or the E46/E83 double arginylated vari- unmodified α-syn with that of α-syn 100% arginylated at ant, to seed pathological inclusions in cultured primary E46 or E83. Both types of arginylation greatly reduced the neurons freshly isolated from mouse brain. This assay amount of α-syn capable of undergoing phosphorylation was previously developed to model the formation of LBs (Fig.  5a). Next, we tested the phosphorylation efficiency and has been shown to be capable of testing the potential on partially arginylated α-syn preparations, obtained by pathological properties of α-syn preparations in seeding mixing unmodified and fully arginylated α-syn at ratios LB-like intracellular inclusions that are detectable with comparable to physiological level (5% and 50%). These S129 antibody [15]. assays showed that a higher percentage of arginylation While fibrils prepared from unmodified α-syn formed led to a lower phosphorylation efficiency, suggesting prominent intracellular pS129-positive inclusions in that the negative effect of arginylation on phosphoryla - the soma and neurites, all of the single and double argi- tion was dose-dependent (Fig. 5b). Thus, arginylation and nylated α-syn variants formed much smaller inclu- phosphorylation of α-syn have an antagonistic relation- sions, less abundant than those in control (Fig.  6, and ship and arginylation at either E46 or E83 could interfere Additional file  1: Fig. S6). The total pS129 fluorescence with α-syn phosphorylation at S129. intensity of these inclusions (indicative of their size) was Fig. 6 E46 and E83 arginylation decreases intracellular α-syn aggregation. Mouse neurons were incubated with unmodified ( WT ) α-syn or α-syn arginylated at E46, E83, or both E46/E83 (double), and the formation of LB-like inclusions was examined with S129 antibody. a Quantification of the intensity, length, and width of S129-positive inclusions in neuronal cultures, plotted as mean with 95% confidence interval. n = 50 independent fields of view. P values were calculated by unpaired two-tailed Student’s t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. See Additional file 1: Fig. S6 for scatter plots of the data shown in the left panels, plotted as individual data points correlated to relative measurements of the same aggregates. b Representative images of inclusions revealed by S129 staining. Scale bar, 10 µm. See Additional file 1: Fig. S7 for broader-field-of-view images ofthe same aggregates. Images were processed by uniform background subtraction using Metamorph imaging software Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 11 of 13 overall ~ 30% lower in arginylated variants compared to penetrates the misfolding and aggregating α-syn pool unmodified α-syn (Fig.  6a, top left). The average fluo - with the potential to reverse its pathology and restore rescence intensity (calculated as the total fluorescence its normal function. While direct experiments to test intensity divided by the inclusion area, indicative of the this hypothesis are impossible at present, this idea is density of pS129 monomers within the inclusions) was supported by our data that arginylation was enriched in not changed (Fig. 6a, bottom left). In addition, the inclu- the S129-positive insoluble α-syn pool, where it could sions seeded by unmodified α-syn appeared more elon - potentially act as a mechanism to reduce and outcom- gated, while those seeded by arginylated α-syn variants pete the pathological S129 phosphorylation. Also, we were shorter and more dot-like, a substantial change found that in the brain, arginylation was primarily pre- from the typical morphology observed at the timepoints sent on fibril-like α-syn structures outside LBs, and used (Fig. 6a, right panels). This was confirmed by visual only E46-arginylated α-syn appeared to enter the espe- observations (Fig.  6b, and Additional file  1: Fig. S7). We cially mature LBs on occasion, possibly to counter their also confirmed that the fibrils pre-added to cells were not growth and toxicity to neurons. The pathological prop - substantially different in morphology or aggregation sta - erties of α-syn fibrils versus other misfolded aggregates tus (Additional file  1: Fig. S8). Thus, arginylation at E46 (oligomers) are still debated in the literature [27], and and E83 interferes with the ability of α-syn to seed LB- some reports suggest that fibrils, as opposed to other like intracellular aggregates and induce neuropathology aggregates, actually serve a neuroprotective function in cultured neurons. [28]. We speculate that arginylation may be enriched in such neuroprotective fibrillar aggregates, rather than in the pathology-inducing α-syn pool, and this hypothesis Discussion will be elucidated in our future studies. Our work demonstrated that α-syn was arginylated in Interestingly, neither arginylation nor phosphoryla- the human brain on two conserved, functionally impor- tion in  vitro, separately or together, caused a change in tant sites – E46, previously implicated in familial PD, and α-syn apparent molecular weight on SDS-PAGE. At the E83, previously found to be critically important for α-syn same time, the majority of arginylated and phosphoryl- pathology [22–24]. We found that E46 and E83 arginyla- ated α-syn in the human brain samples appears to cause a tion targeted a different percentage of α-syn in different gel shift (as seen by comparing the molecular weights of patients, ranging from a few percent to nearly 50% of the α-syn S129-positive band in Figs.  2 and 5). This strongly total α-syn pool. Notably, α-syn arginylation on these suggests that the arginylated/phosphorylated α-syn in two sites strongly decreased with age, and it negatively the brain is also targeted by additional PTMs, possibly impacted pathological intracellular aggregation of α-syn, ubiquitination, as well as potentially others, that collec- suggesting a neuroprotective role of this arginylation. tively cause a visible change in α-syn apparent molecu- The familial mutation E46K correlates with PD [22, 23]. lar weight. Identifying these PTMs and elucidating their Since K is a positively charged amino acid residue that role in α-syn physiology constitute an exciting direction is closest to R in structure and chemical property, one of future research. would expect that arginylation on the same site would While our understanding of protein arginylation as a mimic the effect of this mutation. However, previous PTM is still in its early stage, prior studies from our group studies showed that E46K α-syn is more degradation- and others suggest that only a small fraction of each resistant and aggregation-prone than wild-type protein protein is arginylated in  vivo at any particular time. For [25, 26], and a lack of E46 arginylation also induces α-syn example, less than 1% of β-actin can be arginylated [29], intracellular accumulation [9]. Clearly, E46K mutation is even though this arginylation is likely locally enriched to sufficiently different from E46 arginylation, which pro - facilitate its functions in cell migration [30]. In compari- duce nearly opposite effects on the protein. Given this son, our current study shows that 3%–50% of total α-syn knowledge, it appears possible that the E46K mutation can be arginylated in different human patients. This con - may exert at least some of its biological effects by pre - stitutes, by far, the highest fraction of arginylation ever venting arginylation on this site. observed on any protein in vivo. While our data suggest that arginylation at E46 and Previous studies from our group showed that α-syn E83 targets the same α-syn pool as phosphorylation at enzymatically arginylated in  vitro has a reduced ability S129, which has been previously proposed to be linked to aggregate in cells [9], and that synthetic arginylated to neuropathology [17], our data also indicate that α-syn constructs have reduced aggregation properties arginylation prevents or diminishes S129 phosphoryla- in  vitro [13]. Our current data expand on this find - tion. It is attractive to suggest that arginylation is an ing, showing that arginylation specifically at E46 and antagonistic mechanism in  vivo that counters patho- E83, individually or together, reduces the formation of logical phosphorylation, and that arginylated α-syn Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 12 of 13 intraneuronal inclusions. Notably, this assay measures brain decreases with patient age. Fig. S5: E46 and E83 arginylation levels the seeding capacity of the α-syn fibrils added to the show no correlation with ATE1 levels. Fig. S6: E46 and E83 arginylation alters the morphology and size of intracellular inclusions seeded by α-syn cells, which induce aggregation of intracellular α-syn. in cultured neurons. Fig. S7: E46 and E83 arginylation alters the morphol- Thus, a reduction in the aggregate size following the ogy and size of intracellular inclusions seeded by a-syn in cultured neu- seeding can in principle result not only from a reduc- rons. Fig. S8: E46 and E83 5% arginylated a-syn fibrils are morphologically similar to wild type. tion of the seeding capacity of arginylated α-syn, but Additional file 2. Dataset 1: HPLC conditions and analysis for the syn- also from a reduction in its cellular uptake. Investigat- thetic peptides used for antibody generation ing this additional possibility constitutes an exciting direction of further studies. Acknowledgements It is possible that arginylation also targets other α-syn We are grateful to Dr. Junling Wang for helpful discussion, technical assistance, sites, which may facilitate this effect in vivo and/or serve critical reading of the manuscript, and performing some of the initial tests of additional or different physiological roles. However, the arginylated α-syn antibodies, and to Brittany MacTaggart and Drs. Irem Avcilar-Kucukgoze, Li Chen, and Pavan Vedula for helpful discussions and criti- in  vivo detection of arginylation by mass spectrometry cal reading of the manuscript. We thank Dr. Hsin-Yao Tang and Wistar Institute still presents a challenge, even in the cases where other Proteomics Facility for mass spectrometry. This work was supported by the methods suggest the presence of highly arginylated pro- NIH grants R01NS102435 to AK and EJP and R35GM122505 to AK. Instruments supported by the NIH and the NSF include: NMR (NSF CHE-1827457), HRMS tein fraction, making it difficult to definitively test this (NIH RR-023444), and MALDI MS (NSF MRI-0820996). B.P. thanks the University hypothesis. This phenomenon, and the potential exist - of Pennsylvania for support through a Dissertation Completion Fellowship. ence and role of other arginylation sites in the brain, M.S. thanks the Nakajima Foundation for scholarship funding. require further investigation. Author contributions PTMs are an emerging field, and very little is known JZ, BP, MF, MS: Performed the experiments and analyzed data; YH: designed about PTM hierarchy and their potential interactions and synthesized unique reagents for the experiments; DWD: analyzed data; KCL, ER, EJP, and AK designed the experiments and analyzed data; JZ and AK with each other. Our work sheds light on such interac- wrote the manuscript. All authors read and approved the final manuscript. tions by showing that the same protein pool is modified by multiple PTMs and that these PTMs can compete Funding This work was supported by the NIH grants R01NS102435 to AK and EJP with each other in a hierarchical manner. and R35GM122505 to AK. Instruments supported by the NIH and the NSF include: NMR (NSF CHE-1827457), HRMS (NIH RR-023444), and MALDI MS (NSF MRI-0820996). Center for Neurodegeneration Disease Research Brain Bank is supported by NIH grants P30AG072979 and U19AG062418. ER is supported by Conclusions the NIH grant R01NS120625. BP thanks the University of Pennsylvania for sup- In conclusion, the present study found that (1) α-syn is port through a Dissertation Completion Fellowship. M. S. thanks the Nakajima arginylated in the human brain on two conserved, func- Foundation for scholarship funding. tionally important sites – E46, previously implicated in Availability of data and materials familial PD, and E83, previously found to be critically All data and materials are included with the manuscript. important to α-syn pathology; (2) E46 and E83 arginyla- tion targets a different percentage of α-syn in different Declarations patients, ranging from a few percent to nearly 50% of the Ethics approval and consent to participate total α-syn pool; (3) α-syn arginylation on these two sites The research was performed with IRB approval according to the ethics guide- strongly decreases with age, and negatively impacts α-syn lines using de-identified human patient samples. pathological intracellular aggregation; and (4) α-syn argi- Consent for publication nylation counteracts S129 phosphorylation. All involved parties consent to publication. Competing interests Abbreviations The authors declare that they have no competing interests. α-syn: α-Synuclein; PD: Parkinson’s disease; LB: Lewy bodies; PTM: Posttransla- tional modifications; ATE1: Arginyl transfer enzyme 1. Author details Department of Biomedical Sciences, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA. Depar t- Supplementary Information ment of Chemistry, University of Pennsylvania School of Arts and Sciences, The online version contains supplementary material available at https:// doi. Philadelphia, Pennsylvania 19104, USA. Center for Neurodegenerative Disease org/ 10. 1186/ s40035- 022- 00295-0. Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA. Additional file 1. Table S1: Clinical profiles of donors with and without PD. Table S2: Primary antibodies used for western blot. Fig. S1: Characteri- Received: 18 November 2021 Accepted: 23 March 2022 zation of E46- and E83-arginylated α-syn antibodies. Fig. S2: Both bands in the doublet recognized with α-syn antibodies contain α-syn. Fig. S3: S129 phosphorylation and E46 and E83 arginylation shows negative correlation with total α-syn levels. Fig. S4: E46 and E83 arginylation in the human Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 13 of 13 References 24. Luk KC, Covell DJ, Kehm VM, Zhang B, Song IY, Byrne MD, et al. Molecular 1. Bennett MC. The role of alpha-synuclein in neurodegenerative diseases. and biological compatibility with host alpha-synuclein influences fibril Pharmacol Ther. 2005;105(3):311–31. pathogenicity. Cell Rep. 2016;16(12):3373–87. 2. Kim WS, Kagedal K, Halliday GM. Alpha-synuclein biology in Lewy body 25. Fredenburg RA, Rospigliosi C, Meray RK, Kessler JC, Lashuel HA, Eliezer diseases. Alzheimers Res Ther. 2014;6(5):73. D, et al. 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α-Synuclein arginylation in the human brain

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Abstract

Background: Alpha-synuclein (α-syn) exhibits pathological misfolding in many human neurodegenerative disorders. We previously showed that α-syn is arginylated in the mouse brain and that lack of arginylation leads to neurodegen- eration in mice. Methods: Here, we tested α-syn arginylation in human brain pathology using newly derived antibodies in combina- tion with Western blotting, biochemical assays, and experiments in live neurons. Results: We found that α-syn was arginylated in the human brain on E46 and E83, two sites previously implicated in α-syn pathology and familial cases of Parkinson’s disease. The levels of arginylation in different brain samples ranged between ~ 3% and ~ 50% of the total α-syn pool, and this arginylation nearly exclusively concentrated in the subcellu- lar α-syn fraction that sedimented at low centrifugation speeds and appeared to be simultaneously targeted by multi- ple posttranslational modifications. Arginylated α-syn was less susceptible to S129 phosphorylation and pathological aggregation in neurons. The arginylation level inversely correlated with the overall α-syn levels and with patient age, suggesting a possible causal relationship between arginylation decline and α-syn-dependent neuropathology. Conclusion: We propose that α-syn arginylation constitutes a potential neuroprotective mechanism that prevents its abnormal accumulation during neurodegeneration and aging in the human brain. Keywords: Arginylation, Neurodegeneration, Aging, α-Synuclein Background the exact role of this and other PTMs in regulating α-syn Alpha-synuclein (α-syn) exhibits pathological misfolding misfolding and aggregation is not well understood. and aggregation in human neurodegenerative disorders Protein arginylation mediated by arginyltransferase that are collectively called synucleinopathies [1]. One of ATE1 is a PTM of emerging biological significance that the most prevalent diseases in this group is Parkinson’s consists of transfer ribonucleic acid (tRNA)-dependent disease (PD), which is characterized by the formation transfer of Arg (R) to proteins and has been implicated of Lewy neurites and Lewy bodies (LBs) – large α-syn in many key physiological events in vivo [6, 7]. ATE1 can aggregates that contribute to neuronal loss, dementia, modify proteins either N-terminally or on the side chains and death [2]. It is well established that LB formation and of the acidic residues, i.e., Asp (D) or Glu (E) [8]. Previ- maturation are associated with multiple posttranslational ous work from our lab uncovered that α-syn is a highly modifications (PTMs) [3, 4], including, most promi- efficient target for ATE1 in vitro, and that in mouse brain nently, phosphorylation of α-syn at S129, which is gener- α-syn is arginylated at E46 and E83 [9], two sites that ally believed to be involved in PD pathology [5]. However, have been previously implicated in α-syn function and PD pathology in human patients [10, 11]. Mice lacking ATE1 in the brain develop symptoms of neurodegenera- *Correspondence: akashina@upenn.edu tion, suggesting that arginylation plays a role in normal Department of Biomedical Sciences, University of Pennsylvania School brain function and may act specifically via α-syn [9]. of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA Full list of author information is available at the end of the article © The Author(s) 2022. 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. Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 2 of 13 Here, we tested whether E46 and E83 arginylation tar- washed with NaHCO and brine. The organic layer was gets α-syn in the human brain, and whether this argin- dried over M gSO and evaporated to dryness. Further ylation correlates with any physiological changes in the purification using silica gel gave rise to 375  mg pure patients, such as disease status or age. We also tested the Fmoc-Glu(Arg(Pbf)-OtBu)-OH with a total yield of potential interplay between α-syn arginylation and S129 45%. phosphorylation, another PTM previously implicated The compounds were verified by matrix assisted laser in PD pathology. Our results uncover a potential new desorption/ionization (MALDI) to determine their mechanism of α-syn regulation in the human brain and mass, as follows: Fmoc-Glu(Arg(Pbf)-OtBu)-OAll: + + propose functional implications of arginylation in neuro- [MH] 874.29; Fmoc-Glu(Arg(Pbf )-OtBu)-OH: [MH] degeneration and aging. 834.3750. Materials and methods Synthesis of E46‑ and E83‑arginylated α‑syn peptides used Materials as antigen for antibody generation Frozen postmortem brain samples of human frontal The arginylated peptides CVGSKTKE GVVH and cortex were from patient brain donors who underwent CAVAQKTVE GAG were synthesized manually using autopsy at the Center for Neurodegenerative Disease standard Fmoc-based strategy with modification as Research (CNDR) at the University of Pennsylvania reported in [13]. Briefly, 20  mg 2-chlorotrityl resin between 1992 and 2016 [12]. Detailed clinical character- (100–200 mesh, 1.5  mmol substitution/g) was swelled istics (age, sex, diagnosis) are listed in Additional file  1: in dry DCM for 1  h. Then the DCM was removed and Table  S1. All antibodies used in this project are listed in the first Fmoc-amino acid (1 equiv.) in 2  ml DCM and Additional file 1: Table S2. DIPEA (4 equiv.) were added to the resin. Upon mix- ing for 40  min, the resin was washed with DMF for Synthesis of fluorenylmethyloxycarbonyl four times. The resin was then capped by washing 3 (Fmoc)‑Glu(Arg(Pbf )‑OtBu)‑OH times with DCM/MeOH/DIPEA (volume ratio 17:2:1), To generate branched peptrides mimicking arginylated 3 times with DCM, and 3 times with DMF. The Fmoc sites on α-syn, we synthesized Fmoc-Glu(Arg(Pbf)- group was removed by adding 2 ml of 20% peperidine/ OtBu)-OH, an arginylated Glu derivative to be used to DMF and stirring for 20  min. The amount of coupled synthesize the Glu-arginylated peptides. To do this we amino acid was evaluated by the Fmoc content in the first generated a precursor, Fmoc-Glu(Arg(Pbf )-OtBu)- deprotection solution using absorbance at 300  nm OAll. The procedure for precursor generation and final −1 −1 (extinction coefficient: 7800  cm  M ). Subsequent synthesis was as follows. Two hundred and five milli - amino acids were coupled by adding Fmoc-amino acid grams of Fmoc-Glu-OAll (0.5  mmol) were dissolved (5 equiv.) that was activated by HBTU (5 equiv.) and in 15 ml tetrahydrofuran (THF) in a flask and the flask DIPEA (10 equiv.) and stirring for 30  min. For the on- was cooled to -20  °C. Next, 60  μl of N-methylmor- resin coupling of Fmoc-Glu(Arg(Pbf)-OtBu)-OH, 2 pholine (0.5  mmol) was added to this flask, followed equiv. of Fmoc-Glu(Arg(Pbf )-OtBu)-OH in DMF was by dropwise addition of 65  μl isobutyl chlorofor- activated by 2 equiv. of HBTU and 4 equiv. of DIPEA mate (0.5  mmol). The mixture was stirred for 10  min, and then mixed with resin for 2 h. After further elonga- and then 259  mg Arg(Pbf )-OtBu·HCl (0.5  mmol) and tion, the peptides were cleaved from resin by treatment additional 60  μl of N-methylmorpholine were added. with 90%TFA/5%TIPS/5%DCM for 1.5  h. The cleavage The resulting slurry was stirred at − 20  °C for 1  h and solution was pooled into cold ether and the precipi- then at room temperature for 3  h. The precipitate tate was collected and purified by reverse-phase HPLC was filtered off and the filtrate was evaporated under using VyDAC C18 column and 0.1% TFA/0.1% acetoni- reduced pressure. The residue was dissolved in 30  ml trile as the mobile phase. The purity was checked by EtOAc and then washed with N aHCO and brine. The analytic HPLC (Additional file  2: Dataset 1) and iden- organic layer was dried over M gSO and evaporated tity was confirmed by mass spectrometry (Additional to give rise to 460  mg oil, which was used directly for file 2 : Dataset 1). the next step. The obtained Fmoc-Glu(Arg(Pbf/OtBu)- MALDI: OAll (0.5  mmol) was dissolved in 5  ml dichlorometh- ane (DCM). To this flask was added 15  mg Pd(PPh3)4 R + CVGSKTKE GVVH: Calcd. [MH] 1399.75, found. (0.0125  mmol, 2.5% mol) and 617  μl PhSiH3 (5  mmol) 1399.69; under argon. The mixture was stirred for 4  h at room R + CAVAQKTVE GAG: Calcd. [MH] 1189.66, found. temperature and then evaporated under reduced pres- 1289.93. sure. The residue was re-dissolved in 20  ml DCM and Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 3 of 13 Generation of synthetic arginylated α‑syn variants After completion of the reaction, α-syn phosphoryla- Synthetic arginylated full-length α-syn variants were tion was detected by Western blots using the S129 generated as described in [13]. antibody. α‑Syn aggregation assays in cultured primary neurons Immunoblotting Primary neurons were isolated from C57-Bl6 mouse Frozen tissues were grounded in liquid nitrogen using neonatal brain as described [14], and incubated with a mortar and pestle until fine powder was formed. The non-arginylated α-syn or its arginylated variants (E46, pulverized tissue powder was then weighed and lysed E83, and double) as described [15]. Detection of α-syn directly in 4 × SDS loading buffer (1:10 w /v ratio), fol- inclusions was performed using the S129 antibody, and lowed by boiling for 10 min. Then 2.5 μl of each sample the inclusion fluorescence and shape were quantified was loaded on 15% SDS–polyacrylamide gels (PAGE) using the “integrated morphometry analysis” function in and transferred to a 0.2-μm nitrocellulose membrane the Metamorph imaging software (Molecular Devices, at 250  mA for 20  min. Blots were blocked in 3% BSA Downington, PA). The original images were uniformly in TBST, then incubated with primary antibodies processed using the “background subtraction” function (Additional file  1: Table S2) at 4 °C overnight. Then the in the Metamorph imaging software. membranes were incubated with secondary antibodies (1:5000 dilution) conjugated to IRDye800 or IRDye700 and images were acquired using the Odyssey Imag- Negative staining electron microscopy ing System. For comparison of arginylated α-syn lev- Non-arginylated or 5%-arginylated α-syn fibrils (E46, els across different human brain samples, GAPDH was E83, and double) were deposited on formvar-coated used as an internal loading control. grids, stained with 2% w/v uranyl acetate in water, and visualized by transmission electron microscopy. Fractionation of soluble and insoluble α‑syn Image analysis and quantification The tissue powder was lysed in reaction buffer (con - Western blots were quantified by direct measurements taining 100  mM HEPES, pH 7.2, 50  mM KCl, 1  mM of the secondary antibody fluorescence using Odyssey MgCl , 1 mM EGTA, and 1 mM DTT, with Sigma pro- gel imager (LiCor, Lincoln, NE). Quantification of the tease inhibitor cocktail added before use) at 1:10 w/v band intensity over the background was calculated using ratio, and then centrifuged at 13,000 r/min in a table- the Odyssey gel imaging software integrated with the top microfuge at 4  °C for 30  min. The supernatant and instrument. pellet were collected as soluble and insoluble pool for For description of intracellular S129-positive aggre- Western blotting analysis. The supernatant was mixed gates, the following parameters were measured: at 1:1 ratio with 4× SDS loading buffer. The pellet was resuspended in 4× SDS loading buffer equal to the 1) “total intensity” (following the command name in the volume of the original extract, and both samples were Metamorph imaging software) measures the total boiled for 10 min prior to loading on the gel. Then 6 µl gray level of each aggregate, thresholded against the of pellet and 20  µl of supernatant were loaded on 10% background; this parameter directly depends on the bis–tris gels for mass spectrometry or 15% SDS-PAGE area and density of the aggregate and thus quantita- for Western blot. tively reflects the aggregate size. 2) “average intensity” (following the command name in In vitro phosphorylation of α‑syn using Polo‑like kinase 2 the Metamorph imaging software) measures the gray (PLK2) levels (equaling fluorescence intensity) per unit area, For each reaction, PLK2 solution (Catalog Number: and this reflects the density of the aggregate, as more PV4204, ThermoFisher Scientific, Waltham, MA) was densely packed aggregate would have higher fluores - mixed with recombinant α-syn at a ratio of 2  µl PLK2 cence per unit area. per 72  µg α-syn in the reaction buffer containing 25  mM HEPES, pH 7.2, 50  mM NaCl, 20  mM MgCl , 2 mM DTT, and 1 mM ATP freshly added from a frozen Statistical analysis stock. For the reaction, components were mixed on ice Calculations for Figs. 1, 3, S3, S4, and S5 were performed in the following order: final reaction buffer, α-syn, fol - using MATLAB software, and for the other figures lowed by PLK2, and the mixture was then transferred using the Graphpad Prism software package (version 8.0). to 30  °C for 2  h. For negative controls, ATP or PLK2 For the Graphpad Prism analysis, the data are presented was omitted from the reaction, as indicated elsewhere. as mean ± standard deviation (SD), unless otherwise Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 4 of 13 Fig. 1 E46- and E83-arginylated α-syn are present in the human frontal cortex and show varied levels in PD patients and healthy controls. a Representative immunoblots of total α-syn, as well as α-syn modified by phosphorylation at S129 or arginylation at E46 or E83. b Quantification of α-syn signal from all antibodies in all samples; for total α-syn, signals from both bands of the doublet in the ~ 14 kDa range were added, and the upper and lower bands of the doublet were also quantified separately. Error bars represent SEM from triplicate runs of the same samples. C, control; PD, Parkinson’s disease; PDD, Parkinson disease with dementia. P-value comparisons between control and PD (P12), control and PDD (P13), as well as PD and PDD (P23) are shown on top of each panel, calculated by two-tailed Welsh t-test. See Table S1 for further details on the patient samples indicated,  and statistical comparisons between differ - analysis, followed by two-tailed student’s t-test between ent groups were first performed using one-way ANOVA two groups with the P-value corrected by Bonferroni test. Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 5 of 13 E46 and E83 arginylation targets the α‑syn pool Results that sediments at lower centrifugation speeds and is also α‑Syn is arginylated at E46 and E83 in the human brain enriched in S129 phosphorylation Our previous work showed that α-syn is arginylated in During the experiments described above, we noticed the mouse brain on two conserved sites, E46 and E83, that the single band on the SDS-PAGE recognized by which are targeted for arginylation within the intact non- the antibodies for arginylated and S129-phosphoryl- proteolyzed α-syn protein [9]. Both of these sites are con- ated α-syn ran somewhat higher on the gel than the served between mouse and human α-syn, and both have expected 14 kDa. At the same time, the total α-syn anti- previously been implicated in α-syn pathology, including bodies recognized a doublet in the ~ 14 kDa range, with the familial E46K mutation in PD patients [3, 11]. To test the upper band in the doublet matching the position of whether E46 and E83 arginylation occur in human synu- the band positive for both arginylated and phosphoryl- cleinopathy and to dissect their potential roles in patho- ated α-syn (Fig.  2a). Since SDS gel shifts often happen genesis, we raised rabbit polyclonal antibodies against after proteins have undergoen PTMs, including phos- synthetic peptides corresponding to the α-syn side-chain phorylation [16], we hypothesized that the upper band arginylated at E46 and E83 (Additional file  1: Fig. S1a). represents a subset of α-syn that is simultaneously tar- These antibodies were specific for the corresponding geted by multiple PTMs. arginylated peptides and full-length synthetic arginylated First, to test if this upper band indeed represents α-syn, and showed no cross-reactivity with each other, or α-syn, we excised bands of matching molecular weight with non-arginylated α-syn protein and peptides (Addi- from an SDS gel and identified protein composition in tional file 1: Fig. S1a, b). these bands by mass spectrometry to confirm the pres - We next used these antibodies to probe extracts from ence of α-syn as a major protein in both bands (Addi- the frontal cortex samples of patients, obtained from the tional file  1: Fig. S2). Next, to test whether modified and biobank at the Center for Neurodegenerative Disease unmodified α-syn are enriched in the same subcellular Research (CNDR) at the University of Pennsylvania. We fractions, we fractionated human brain extracts by cen- analyzed healthy controls, as well as patients with PD trifugation at 13,000  g and tested the supernatant and and PD-related dementia (PDD, characterized by severe pellet from this step by Western blot. Strikingly, the neuronal loss and pathological α-syn aggregation). Strik- majority of the upper band, detectable by pS129, E46- ingly, all brain samples showed prominent reactivity with Arg, and E83-Arg antibodies, was found in the pellet, both E46 and E83 antibodies (Fig.  1), suggesting that the while the lower band remained in the supernatants human α-syn is arginylated in vivo at both E46 and E83. (Fig. 2B). Thus, all three PTMs nearly exclusively target Several interesting trends were observed by compari- the α-syn pool that sediments at lower speeds, a subcel- sons of the patient samples (Fig. 1a, b). First, the levels of lular fraction that typically contains intracellular orga- both E46 and E83 arginylation strongly varied between nelles and larger protein aggregates. samples, suggesting that arginylation in different individu - During PD, pathologically misfolded insoluble α-syn als can target vastly different fractions of the intracellular often incorporates into LBs, large α-syn-rich protein α-syn pool. Second, the levels of arginylation of E46 and aggregates that are known to be targeted by multiple E83 showed a similar trend, with  a comparatively  higher PTMs as a hallmark feature of PD pathology [2]. Pre- or a lower antibody signal  in the same samples, suggest- vious studies have associated LBs with pS129 staining ing that α-syn arginylation in these samples is high or [17], even though the exact role of S129 phosphoryla- low as a whole, without apparent selectivity between the tion of α-syn in PD pathology remains controversial two arginylation sites. Notably, the same trends were also [8, 18]. To test whether E46 and E83 arginylation tar- observed with antibodies against α-syn phosphorylated gets LBs, we co-stained brain sections from human PD at S129, suggesting that all three modifications co-elevate patients with antibodies to total α-syn and either E46- in some samples compared to others and may potentially Arg or E83-Arg α-syn (Fig.  2c). Total α-syn staining interact with each other. revealed the presence of large aggregates clearly visible in different areas of the sections. However, these aggre - gates did not show any enrichment in E83-Arg stain- ing. In the case of E46-Arg, most of the aggregates were also not stained, but some of the larger aggregates, with morphology characteristic for the later stages of LB Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 6 of 13 Fig. 2 E46 and E83 arginylation targets the α-syn pool that sediments at lower centrifugation speeds in extracts from the frontal cortex, but does not strongly colocalize with Lewy bodies in the frontal cortex sections from human PD patients. a Magnification of a representative Western blot of the sample C1 probed with the total and modified α-syn antibodies, showing the doublet α-syn bands recognized by the total α-syn antibody and the relative position of the single band preferentially recognized by the antibodies to the modified α-syn variants as the upper band in this doublet. b Representative Western blots from the total brain extract, pellet, and supernatant fractions. Bands of heavily modified α-syn were preferentially found in the pellet, indicating its incorporation into the insoluble α-syn pool. c Representative images of human brain sections showing prominent LB formation, stained with antibodies to total α-syn, as well as the antibodies against E46- and E83-arginylated variants. LBs were prominently seen as large green dots in the top set of images stained with total α-syn antibodies. Most of these LBs show no specific staining with E83, and only occasional aggregates were visualized with E46 antibodies (short arrows). Both E46 and E83 antibodies also stained fibrillar aggregates (long arrows) Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 7 of 13 formation, were somewhat highlighted with E46-Arg, the decrease in the upper band of the doublet, while the suggesting that E46-arginylated α-syn penetrates the lower band followed the total α-syn trends (Additional LBs over time, possibly during LB maturation. Nota- file  1: Fig. S4). Interestingly, within the samples we tested, bly, both E46-Arg and E83-Arg antibodies also showed no significant correlation was seen between the levels of some fibrillar staining patterns, suggesting that these each PTM and the PD or PDD diagnosis (with P values modifications target α-syn-containing structures in the for comparison between the groups ranging at 0.5 and brain other than LBs. It is unclear if this LB-independ- above, Fig.  1b), suggesting that brain aging, rather than ent α-syn pool is involved in normal brain function PD diagnosis, is the primary factor in α-syn arginylation. and/or plays a neuroprotective, rather than pathologi- Both arginylation and S129 phosphorylation cal, role. inversely correlated with the total α-syn levels in dif- ferent patients (Fig.  3, bottom row), suggesting that α‑Syn E46 and E83 arginylation in the human brain all three modifications are antagonistic to α-syn inversely correlates with the total α‑syn levels accumulation in the brain, a proposed major fac- and prominently decreases with age tor in α-syn-driven neurodegeneration [19, 20]. All Next, we used the quantification data (Fig.  1) to analyze three modifications showed a strong positive correla- correlations between the Western blot signals for E46/ tion with the upper band in the doublet and a strong E83 arginylation, S129 phosphorylation, and total α-syn inverse correlation with the lower band (Additional in different patients. We quantified total α-syn in each file 1: Fig. S3). Consistent with this, all three modifica- sample as the sum of the upper and lower bands in the tions showed similar correlation trends, strongly sug- doublet (Fig.  3), and the upper and lower bands sepa- gesting that they were positively correlated with each rately, corresponding to the modified and unmodified other (Additional file 1: Fig. S3). All these changes may α-syn fractions, respectively (Additional file 1: Fig. S3). potentially reflect functional interactions between This analysis revealed several interesting trends. First, arginylation, phosphorylation, and changes of α-syn while the total α-syn levels increased with the patient age, states in the brain. Of note, none of these changes E46/E83 arginylation as well as S129 phosphorylation correlated with a total change in ATE1 level in these showed a prominent decrease in older patients (Fig.  3, samples (Additional file  1: Fig. S5), suggesting that top row). This decrease was due almost exclusively to the changes in arginylation of α-syn at E46 and E83 in Fig. 3 E46 and E83 arginylation in the human frontal cortex inversely correlates with α-syn levels and decreases with patient age. Correlation plots between different antibody signals calculated from the same datasets as those shown in Fig. 1. Spearman correlation coefficients and P values are shown on top of each plot. See Additional file 1: Fig. S3 and S4 for additional plots Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 8 of 13 Fig. 4 Quantification of E83 arginylation as a fraction of the total α-syn levels in the human frontal cortex. a Representative Western blot images (left) and calibration curves (right), of standard unmodified and E83-arginylated α-syn. b Quantification of the fraction of E83-arginylated α-syn as the percentage of the upper band (U, marked with arrows in both gels) and of the total α-syn in the preparation (the sum of upper and lower bands marked with arrow and asterisk, respectively), calculated by running the calibrations. Percentage of arginylated α-syn to the upper band (U) and the total are shown both as a table (left) and as a chart (right) different brain specimens are not a direct consequence Quantification of α‑syn arginylation in the human brain of the altered availability of arginyltransferase. To quantify the percentage of arginylated α-syn in the These data collectively demonstrate that α-syn E46 human brain in individual samples, we used synthetic and E83 arginylation targets a specific α-syn pool in the E83-Arg α-syn as a protein standard to calibrate the human brain in a manner antagonistic to overall α-syn signal, alongside the unmodified α-syn. We first ran accumulation and correlates with changes in the brain’s calibration curves to directly quantify the amount of physiological state during aging. modified and unmodified α-syn in the standard lanes as Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 9 of 13 the function of the Western blot signal and determine up to ~ 50% of total α-syn. This is, by far, the highest frac - the linear range (Fig. 4a). Next, we ran some of these lin- tion of arginylation ever observed for any protein in vivo. ear range standards at known loads alongside the human patient brain samples to directly measure the actual E46 and E83 arginylation prevents S129 phosphorylation amount of total and E83-Arg α-syn in each brain sample. Our data so far strongly suggest that arginylation at E46 These measurements enabled us to calculate the percent - and E83 targets the same pool of α-syn in the brain as age of E83-arginylated α-syn in each sample. S129 phosphorylation, with the possible exception of the While these percentages varied across different sam - LBs. While the role of S129 phosphorylation on α-syn ples, they were remarkably high overall (Fig. 4b), ranging is still debated in the literature, some lines of evidence from several percent in some samples to up to ~ 70% of suggest that this modification is associated with PD the upper gel band containing the highly modified α-syn pathology [17]. To determine whether arginylation acts pool (calculated from quantifying the signal in the upper synergistically with S129 phosphorylation, or whether band only and comparing it to the protein standard) and these two modifications are antagonistic, we used Fig. 5 E46 and E83 arginylation prevents S129 phosphorylation. Representative Western blot images (left) and quantification (right) of phosphorylation of unmodified (U) and arginylated α-syn protein standards after the phosphorylation reaction with the addition of components shown on top. a α-Syn variants, either unmodified (U) or 100% arginylated at E46 or E83, were incubated with PLK2 kinase in the presence or absence of ATP. Arginylation greatly reduced the efficiency of PLK2 phosphorylation. b Phosphorylation of α-syn variants, either unmodified or arginylated at varied percentages as indicated, incubated with or without PLK2 kinase (PLK) as indicated on top. Arginylation interfered with PLK2 phosphorylation in a dose-dependent manner. Band intensities of S129 signal normalized to α-syn load are plotted. Error bars represent SEM, n = 3 Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 10 of 13 Arginylation at E46 and E83 decreases intracellular α‑syn synthetic arginylated α-syn standards for in  vitro phos- aggregation phorylation assays with Polo-like kinase 2 (PLK2), which To test directly whether α-syn arginylation has a neuro- mediates α-syn S129 phosphorylation in vivo [18, 21]. protective role, we tested the ability of arginylated α-syn First, we compared the phosphorylation efficiency of at E46 or E83, or the E46/E83 double arginylated vari- unmodified α-syn with that of α-syn 100% arginylated at ant, to seed pathological inclusions in cultured primary E46 or E83. Both types of arginylation greatly reduced the neurons freshly isolated from mouse brain. This assay amount of α-syn capable of undergoing phosphorylation was previously developed to model the formation of LBs (Fig.  5a). Next, we tested the phosphorylation efficiency and has been shown to be capable of testing the potential on partially arginylated α-syn preparations, obtained by pathological properties of α-syn preparations in seeding mixing unmodified and fully arginylated α-syn at ratios LB-like intracellular inclusions that are detectable with comparable to physiological level (5% and 50%). These S129 antibody [15]. assays showed that a higher percentage of arginylation While fibrils prepared from unmodified α-syn formed led to a lower phosphorylation efficiency, suggesting prominent intracellular pS129-positive inclusions in that the negative effect of arginylation on phosphoryla - the soma and neurites, all of the single and double argi- tion was dose-dependent (Fig. 5b). Thus, arginylation and nylated α-syn variants formed much smaller inclu- phosphorylation of α-syn have an antagonistic relation- sions, less abundant than those in control (Fig.  6, and ship and arginylation at either E46 or E83 could interfere Additional file  1: Fig. S6). The total pS129 fluorescence with α-syn phosphorylation at S129. intensity of these inclusions (indicative of their size) was Fig. 6 E46 and E83 arginylation decreases intracellular α-syn aggregation. Mouse neurons were incubated with unmodified ( WT ) α-syn or α-syn arginylated at E46, E83, or both E46/E83 (double), and the formation of LB-like inclusions was examined with S129 antibody. a Quantification of the intensity, length, and width of S129-positive inclusions in neuronal cultures, plotted as mean with 95% confidence interval. n = 50 independent fields of view. P values were calculated by unpaired two-tailed Student’s t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. See Additional file 1: Fig. S6 for scatter plots of the data shown in the left panels, plotted as individual data points correlated to relative measurements of the same aggregates. b Representative images of inclusions revealed by S129 staining. Scale bar, 10 µm. See Additional file 1: Fig. S7 for broader-field-of-view images ofthe same aggregates. Images were processed by uniform background subtraction using Metamorph imaging software Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 11 of 13 overall ~ 30% lower in arginylated variants compared to penetrates the misfolding and aggregating α-syn pool unmodified α-syn (Fig.  6a, top left). The average fluo - with the potential to reverse its pathology and restore rescence intensity (calculated as the total fluorescence its normal function. While direct experiments to test intensity divided by the inclusion area, indicative of the this hypothesis are impossible at present, this idea is density of pS129 monomers within the inclusions) was supported by our data that arginylation was enriched in not changed (Fig. 6a, bottom left). In addition, the inclu- the S129-positive insoluble α-syn pool, where it could sions seeded by unmodified α-syn appeared more elon - potentially act as a mechanism to reduce and outcom- gated, while those seeded by arginylated α-syn variants pete the pathological S129 phosphorylation. Also, we were shorter and more dot-like, a substantial change found that in the brain, arginylation was primarily pre- from the typical morphology observed at the timepoints sent on fibril-like α-syn structures outside LBs, and used (Fig. 6a, right panels). This was confirmed by visual only E46-arginylated α-syn appeared to enter the espe- observations (Fig.  6b, and Additional file  1: Fig. S7). We cially mature LBs on occasion, possibly to counter their also confirmed that the fibrils pre-added to cells were not growth and toxicity to neurons. The pathological prop - substantially different in morphology or aggregation sta - erties of α-syn fibrils versus other misfolded aggregates tus (Additional file  1: Fig. S8). Thus, arginylation at E46 (oligomers) are still debated in the literature [27], and and E83 interferes with the ability of α-syn to seed LB- some reports suggest that fibrils, as opposed to other like intracellular aggregates and induce neuropathology aggregates, actually serve a neuroprotective function in cultured neurons. [28]. We speculate that arginylation may be enriched in such neuroprotective fibrillar aggregates, rather than in the pathology-inducing α-syn pool, and this hypothesis Discussion will be elucidated in our future studies. Our work demonstrated that α-syn was arginylated in Interestingly, neither arginylation nor phosphoryla- the human brain on two conserved, functionally impor- tion in  vitro, separately or together, caused a change in tant sites – E46, previously implicated in familial PD, and α-syn apparent molecular weight on SDS-PAGE. At the E83, previously found to be critically important for α-syn same time, the majority of arginylated and phosphoryl- pathology [22–24]. We found that E46 and E83 arginyla- ated α-syn in the human brain samples appears to cause a tion targeted a different percentage of α-syn in different gel shift (as seen by comparing the molecular weights of patients, ranging from a few percent to nearly 50% of the α-syn S129-positive band in Figs.  2 and 5). This strongly total α-syn pool. Notably, α-syn arginylation on these suggests that the arginylated/phosphorylated α-syn in two sites strongly decreased with age, and it negatively the brain is also targeted by additional PTMs, possibly impacted pathological intracellular aggregation of α-syn, ubiquitination, as well as potentially others, that collec- suggesting a neuroprotective role of this arginylation. tively cause a visible change in α-syn apparent molecu- The familial mutation E46K correlates with PD [22, 23]. lar weight. Identifying these PTMs and elucidating their Since K is a positively charged amino acid residue that role in α-syn physiology constitute an exciting direction is closest to R in structure and chemical property, one of future research. would expect that arginylation on the same site would While our understanding of protein arginylation as a mimic the effect of this mutation. However, previous PTM is still in its early stage, prior studies from our group studies showed that E46K α-syn is more degradation- and others suggest that only a small fraction of each resistant and aggregation-prone than wild-type protein protein is arginylated in  vivo at any particular time. For [25, 26], and a lack of E46 arginylation also induces α-syn example, less than 1% of β-actin can be arginylated [29], intracellular accumulation [9]. Clearly, E46K mutation is even though this arginylation is likely locally enriched to sufficiently different from E46 arginylation, which pro - facilitate its functions in cell migration [30]. In compari- duce nearly opposite effects on the protein. Given this son, our current study shows that 3%–50% of total α-syn knowledge, it appears possible that the E46K mutation can be arginylated in different human patients. This con - may exert at least some of its biological effects by pre - stitutes, by far, the highest fraction of arginylation ever venting arginylation on this site. observed on any protein in vivo. While our data suggest that arginylation at E46 and Previous studies from our group showed that α-syn E83 targets the same α-syn pool as phosphorylation at enzymatically arginylated in  vitro has a reduced ability S129, which has been previously proposed to be linked to aggregate in cells [9], and that synthetic arginylated to neuropathology [17], our data also indicate that α-syn constructs have reduced aggregation properties arginylation prevents or diminishes S129 phosphoryla- in  vitro [13]. Our current data expand on this find - tion. It is attractive to suggest that arginylation is an ing, showing that arginylation specifically at E46 and antagonistic mechanism in  vivo that counters patho- E83, individually or together, reduces the formation of logical phosphorylation, and that arginylated α-syn Zhao et al. Translational Neurodegeneration (2022) 11:20 Page 12 of 13 intraneuronal inclusions. Notably, this assay measures brain decreases with patient age. Fig. S5: E46 and E83 arginylation levels the seeding capacity of the α-syn fibrils added to the show no correlation with ATE1 levels. Fig. S6: E46 and E83 arginylation alters the morphology and size of intracellular inclusions seeded by α-syn cells, which induce aggregation of intracellular α-syn. in cultured neurons. Fig. S7: E46 and E83 arginylation alters the morphol- Thus, a reduction in the aggregate size following the ogy and size of intracellular inclusions seeded by a-syn in cultured neu- seeding can in principle result not only from a reduc- rons. Fig. S8: E46 and E83 5% arginylated a-syn fibrils are morphologically similar to wild type. tion of the seeding capacity of arginylated α-syn, but Additional file 2. Dataset 1: HPLC conditions and analysis for the syn- also from a reduction in its cellular uptake. Investigat- thetic peptides used for antibody generation ing this additional possibility constitutes an exciting direction of further studies. Acknowledgements It is possible that arginylation also targets other α-syn We are grateful to Dr. Junling Wang for helpful discussion, technical assistance, sites, which may facilitate this effect in vivo and/or serve critical reading of the manuscript, and performing some of the initial tests of additional or different physiological roles. However, the arginylated α-syn antibodies, and to Brittany MacTaggart and Drs. Irem Avcilar-Kucukgoze, Li Chen, and Pavan Vedula for helpful discussions and criti- in  vivo detection of arginylation by mass spectrometry cal reading of the manuscript. We thank Dr. Hsin-Yao Tang and Wistar Institute still presents a challenge, even in the cases where other Proteomics Facility for mass spectrometry. This work was supported by the methods suggest the presence of highly arginylated pro- NIH grants R01NS102435 to AK and EJP and R35GM122505 to AK. Instruments supported by the NIH and the NSF include: NMR (NSF CHE-1827457), HRMS tein fraction, making it difficult to definitively test this (NIH RR-023444), and MALDI MS (NSF MRI-0820996). B.P. thanks the University hypothesis. This phenomenon, and the potential exist - of Pennsylvania for support through a Dissertation Completion Fellowship. ence and role of other arginylation sites in the brain, M.S. thanks the Nakajima Foundation for scholarship funding. require further investigation. Author contributions PTMs are an emerging field, and very little is known JZ, BP, MF, MS: Performed the experiments and analyzed data; YH: designed about PTM hierarchy and their potential interactions and synthesized unique reagents for the experiments; DWD: analyzed data; KCL, ER, EJP, and AK designed the experiments and analyzed data; JZ and AK with each other. Our work sheds light on such interac- wrote the manuscript. All authors read and approved the final manuscript. tions by showing that the same protein pool is modified by multiple PTMs and that these PTMs can compete Funding This work was supported by the NIH grants R01NS102435 to AK and EJP with each other in a hierarchical manner. and R35GM122505 to AK. Instruments supported by the NIH and the NSF include: NMR (NSF CHE-1827457), HRMS (NIH RR-023444), and MALDI MS (NSF MRI-0820996). Center for Neurodegeneration Disease Research Brain Bank is supported by NIH grants P30AG072979 and U19AG062418. ER is supported by Conclusions the NIH grant R01NS120625. BP thanks the University of Pennsylvania for sup- In conclusion, the present study found that (1) α-syn is port through a Dissertation Completion Fellowship. M. S. thanks the Nakajima arginylated in the human brain on two conserved, func- Foundation for scholarship funding. tionally important sites – E46, previously implicated in Availability of data and materials familial PD, and E83, previously found to be critically All data and materials are included with the manuscript. important to α-syn pathology; (2) E46 and E83 arginyla- tion targets a different percentage of α-syn in different Declarations patients, ranging from a few percent to nearly 50% of the Ethics approval and consent to participate total α-syn pool; (3) α-syn arginylation on these two sites The research was performed with IRB approval according to the ethics guide- strongly decreases with age, and negatively impacts α-syn lines using de-identified human patient samples. pathological intracellular aggregation; and (4) α-syn argi- Consent for publication nylation counteracts S129 phosphorylation. All involved parties consent to publication. Competing interests Abbreviations The authors declare that they have no competing interests. α-syn: α-Synuclein; PD: Parkinson’s disease; LB: Lewy bodies; PTM: Posttransla- tional modifications; ATE1: Arginyl transfer enzyme 1. Author details Department of Biomedical Sciences, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA. Depar t- Supplementary Information ment of Chemistry, University of Pennsylvania School of Arts and Sciences, The online version contains supplementary material available at https:// doi. Philadelphia, Pennsylvania 19104, USA. Center for Neurodegenerative Disease org/ 10. 1186/ s40035- 022- 00295-0. Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA. Additional file 1. Table S1: Clinical profiles of donors with and without PD. Table S2: Primary antibodies used for western blot. Fig. S1: Characteri- Received: 18 November 2021 Accepted: 23 March 2022 zation of E46- and E83-arginylated α-syn antibodies. Fig. S2: Both bands in the doublet recognized with α-syn antibodies contain α-syn. Fig. S3: S129 phosphorylation and E46 and E83 arginylation shows negative correlation with total α-syn levels. Fig. S4: E46 and E83 arginylation in the human Zhao  et al. Translational Neurodegeneration (2022) 11:20 Page 13 of 13 References 24. Luk KC, Covell DJ, Kehm VM, Zhang B, Song IY, Byrne MD, et al. Molecular 1. Bennett MC. The role of alpha-synuclein in neurodegenerative diseases. and biological compatibility with host alpha-synuclein influences fibril Pharmacol Ther. 2005;105(3):311–31. pathogenicity. Cell Rep. 2016;16(12):3373–87. 2. Kim WS, Kagedal K, Halliday GM. Alpha-synuclein biology in Lewy body 25. Fredenburg RA, Rospigliosi C, Meray RK, Kessler JC, Lashuel HA, Eliezer diseases. Alzheimers Res Ther. 2014;6(5):73. D, et al. 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Journal

Translational Neurodegeneration – Springer Journals

Published: Apr 8, 2022

Keywords: Arginylation; Neurodegeneration; Aging; α-Synuclein

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