Ghanem SS, Majbour NK, Vaikath NN, Ardah MT, Erskine D, Jensen NM, Fayyad M, Sudhakaran IP, Vasili E, Melachroinou K, Abdi IY, Poggiolini I, Santos P, Dorn A, Carloni P, Vekrellis K, Attems J, McKeith I, Outeiro TF, Jensen PH, El-Agnaf OM. α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity. Proc Natl Acad Sci U S A. 2022 Apr 12;119(15):e2109617119. Epub 2022 Mar 30 PubMed.

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  1. A crucial tilting point in the field of synucleinopathies was the discovery that α-synuclein within Lewy bodies is aberrantly phosphorylated at the residue ser 129 (pS129). Since then, there has been a big controversy surrounding the exact role of this post-translational modification (PTM) in α-synuclein aggregation and toxicity. Answering this question is crucial, as many therapeutic approaches targeting this PTM have been initiated.

    El Agnaf and colleagues sought to address this question in a complete and well-executed study, combining in vitro, cell culture, in vivo, and human postmortem analysis. The authors concluded that phosphorylation at Ser 129 (pS129) is not required for the initiation of α-synuclein clustering, and that it negatively regulates α-synuclein aggregation process.

    The in vitro and cell culture findings fit with previous studies. They show that phosphorylation at ser 129 is not required for the initiation of α-synuclein clustering and it rather inhibits the aggregation process (Oueslati 2016). Interestingly, the authors showed in a very clever way that the proportion of phosphorylated protein in the solution is critical for modeling α-syn aggregation, with a complete inhibition of inclusion formation when half of the protein is phosphorylated. In vivo, the authors used a battery of anti-α-syn and anti-pS129 antibodies generated in-house to corroborate the cell-culture data, and to confirm that phosphorylation might occur after inclusion formation.

    Finally, postmortem analysis from PD-diseased brains did not bring conclusive data regarding the question of when is α-syn phosphorylated during the disease progression, as no link between pS129 levels and the disease duration has been observed. The lack of conclusive data could be related to the fact that the structure and the composition of Lewy bodies in diseased brains could be different compared to α-synuclein inclusions in cell culture and in rodent brain.

    Moreover, another degree of complexity should be taken into consideration; it relates to the use of pS129 antibodies, as affinity of these antibodies could be affected by other PTMs surrounding the Ser 129 (Lashuel et al., 2022). 

    In conclusion, the present study adds more argument supporting the hypothesis that phosphorylation is not required for α-syn aggregation and could be a late event in the aggregation process. Together with previous work (Oueslati et al., 2013), the present study also suggests a potential neuroprotective role of this post-translational modification.

    References:

    Oueslati A. Implication of Alpha-Synuclein Phosphorylation at S129 in Synucleinopathies: What Have We Learned in the Last Decade?. J Parkinsons Dis. 2016;6(1):39-51. PubMed.

    Lashuel HA, Mahul-Mellier AL, Novello S, Hegde RN, Jasiqi Y, Altay MF, Donzelli S, DeGuire SM, Burai R, Magalhães P, Chiki A, Ricci J, Boussouf M, Sadek A, Stoops E, Iseli C, Guex N. Neighbouring modifications interfere with the detection of phosphorylated alpha-synuclein at Serine 129: Revisiting the specificity of pS129 antibodies. bioRxiv, March 31, 2022 bioRxiv

    Oueslati A, Schneider BL, Aebischer P, Lashuel HA. Polo-like kinase 2 regulates selective autophagic α-synuclein clearance and suppresses its toxicity in vivo. Proc Natl Acad Sci U S A. 2013 Oct 8;110(41):E3945-54. PubMed.

    View all comments by Abid Oueslati

  2. This study is interesting because it tackles an outstanding issue in the field. Over the last years, there has been some controversy about whether S129 α-synuclein phosphorylation is pathological or not, and whether it takes place before or after α-synuclein deposition. Granem et al. show, using a variety of techniques, that pS129 seems to occur after an initial step of α-syn aggregation, and that this phosphorylation reduced its aggregation propensity, attenuating cytotoxicity. This is consistent with some previous findings that reported that S129 α-syn phosphorylation reduced cytotoxicity compared to mutant forms of α-syn that prevent phosphorylation (Tenreiro et al., 2014).

    While more studies are needed, the findings by Granem et al. are important for the field, since it relies heavily on using pS129 as a readout of pathological α-syn. It is therefore important to better understand the relevance of this modification in the pathogenesis of PD, DLB, and other synucleinopathies, and the implications for the diagnosis and therapeutic approaches of these disorders.

    References:

    Tenreiro S, Reimão-Pinto MM, Antas P, Rino J, Wawrzycka D, Macedo D, Rosado-Ramos R, Amen T, Waiss M, Magalhães F, Gomes A, Santos CN, Kaganovich D, Outeiro TF. Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease. PLoS Genet. 2014 May;10(5):e1004302. Epub 2014 May 8 PubMed.

    View all comments by Alberto Lleó

  3. This interesting study confirms observations reported by our group and others, namely that 1) phosphorylation at S129 inhibits α-Syn aggregation (Paleologou et al., 2008; Stephens et al., 2020); 2) phosphorylation at S129 represents a late event that occurs post α-Syn aggregation (Paleologou et al., 2008; Mahul-Mellier et al., 2020); 3) phosphorylation at S129 is not required for α-Syn PFF's seeding activity and aggregation in vitro and in vivo (Luk et al., 2012). 

    The two main new findings in this report are 1) the development of the 4B1 antibody, which is specific for non-phosphorylated α-Syn, and 2) new data suggesting that phosphorylated fibrils do not efficiently seed the aggregation of monomeric α-Syn in vitro or endogenous α-Syn in cells. These findings should be interpreted with caution for the following reasons: 1) Neither the antigen nor the epitope of the 4B1 antibody was disclosed, making it difficult to interpret some of the data, particularly labeling of aggregates in human tissues; 2) The reported seeding activity for pS129 α-Syn fibrils contradicts previous findings by the same group, where they reported increased seeding and exacerbated α-Syn pathology and toxicity by phosphorylated fibrils compared to unmodified fibrils in neurons and in vivo, and findings by other groups.

    Phosphorylation at S129 inhibits α-Syn fibril formation.
    As the authors indicate, the inhibitory effect of pS129 on α-Syn aggregation they report supports findings from our group, which we published in 2008 (Paleologou et al., 2008) and were independently validated by the Kaminiski Schierle group (Strohäker et al., 2019). 

    Phosphorylated fibrils at S129 have reduced ability to seed the aggregation of α-Syn
    The authors investigated the seeding activity of pS129 α-Syn-PFFs in stable HEK cell lines expressing α-Syn fused to EGFP, and reported increased seeding activity of WT α-Syn fibrils compared to pS129 fibrils. Interestingly, these findings differ from those reported in a previous study by Ma et al. using an N2a stable cell-line also expressing GFP-tagged α-Syn, where they reported similar levels of seeding activity for WT and pS129 α-Syn fibrils, although they showed reduced seeding activity for pS129 fibrils in a cell-free aggregation assay (Ma et al., 2016). 

    In addition, a previous study by several members of the same team (Karampetsou et al., 2017) reported that phosphorylated α-Syn fibrils at S129 “triggered the formation of more α-Synuclein inclusions in the Substantia Nigra pars compacta (SNpc), exacerbated pathology in the cortex and caused dopaminergic neuronal loss and fine motor impairment as early as 60 days post-injection." They also reported that pS129 α-Syn fibrils were more efficiently taken up by neurons and led to accelerated seeding and accumulation of the endogenous α-Synuclein, compared to the unmodified WT fibrils. Thus, they concluded that “targeting phosphorylated α-Synuclein assemblies might be important for delaying inclusion formation.”

    Although the authors cite this paper (Karampetsou et al., 2017) when referring to conflicting reports in the literature, they did not explain why they obtained opposite results compared to their previous study, especially given that the same team produced and characterized the fibrils preparations used in both studies. The two studies used different models of α-Syn seeding. Ghanem et al. used HEK cells stably expressing α-Syn fused to GFP and which has not been shown to form LB-like inclusions. The studies by Karampetsou were carried out in primary neurons and mouse models of α-Syn seeding and pathology spreading. Also, differences in seeding activity could arise from differences in the level of uptake of the unmodified vs. modified fibrils. In such experiments, it is essential first to determine that differences in uptake or amounts of fibrils used do not contribute to the observed differences in seeding activity. It is not clear from the text if this was assessed and how.

    Several other experimental observations argue against the claim that phosphorylated fibrils exhibit a dramatically reduced seeding activity than unmodified α-Syn fibrils.

    1. A previous study from our group demonstrated that insoluble native α-Syn aggregates isolated from our neuronal model efficiently seed α-Syn aggregation and induce the formation of LB-like inclusions in neurons. These native aggregates are heavily ubiquitinated and phosphorylated at S129 (Mahul-Mellier et al., 2020). Similarly, several studies have consistently shown that brain-derived preparations of aggregated α-Syn fibrils, which are heavily phosphorylated at S129, induce efficient seeding of α-Syn aggregation in primary neurons and in vivo. (Marotta et al., 2021; Masuda-Suzukake et al., 2013; Woerman et al., 2018; Morgan et al., 2020). 
    2. The authors' own data (Figure 2) also show that brain-derived aggregates from PD and DLB brain efficiently seed the aggregation of monomeric α-Syn in vitro.
    3. In 2018, we demonstrated that most α-Syn PFFs undergo rapid cleavage within six to 20 hours post internalization into neurons in culture and in vivo, resulting in the removal of the C-terminal domain containing S129. α-Syn seeding in neurons occurs only after a few days post-treatment with PFFs. This means that pS129 is no longer present on α-Syn fibrils and thus should not inhibit α-Syn seeding in neurons. Consistent with these observations, several studies have shown that the C-terminal domain is not required for α-Syn PFFs seeding activity.
    4. Finally, α-Syn seeding studies in primary neurons have consistently shown the appearance of pS129 aggregates at very early stages during the process of α-Syn fibrillization. Suppose phosphorylation of fibrils at S129 inhibits α-Syn fibrils seeding activity. In that case, it is surprising why we continue to see seeding and increased aggregation and accumulation of pS129 fibrils over time during α-Syn fibrillization and LB formation.

    Phosphorylation at S129 occurs after α-Syn aggregation.
    Several studies from our group have previously shown that phosphorylation at S129, a late event after PFF, induced seeding and fibrillization of endogenous α-Syn in primary neurons.

    1. In our 2008 JBC paper, we concluded that our data on the in vitro aggregation inhibitory effects of phosphorylation at S129 “combined with the fact that the C-terminal region containing Ser-129 remains exposed in the aggregated forms (protofibrils and fibrils) of α-syn support the hypothesis that phosphorylation of α-syn could also occur within LBs and is not a prerequisite for α-syn fibrillation and LB formation in PD.”
    2. In a manuscript uploaded to BioRxiv in 2018, titled “The making of a Lewy body: the role of α-synuclein post-fibrillization modifications in regulating the formation and the maturation of pathological inclusions,” we systematically dissected the occurrence of post-aggregation PTMs during early and late events of α-Syn seeding and LB formation in neurons. We reported that “we did not observe the accumulation of pS129 α-syn immunoreactive species in the insoluble fractions during the first 4 days post-seeding, which suggests that phosphorylation at S129 is not required in the initial aggregation events.” We then investigated the temporal relationship between α-Syn aggregation and PTMs, namely phosphorylation at S129, ubiquitination, and C-terminal cleavages. These observations were depicted in our models of the mechanisms of LB formation, which we presented first in BioRxiv (Mahul-Mellier et al., 2018) and in 2020 in PNAS (Mahul-Mellier et al., 2020) Figure 8. This model shows the following sequence of events, i) internalized fibrils are completely cleaved within 20 hours, resulting in complete removal of the C-terminal domain containing pS129, ii) the truncated PFFs seed the aggregation of endogenous α-Syn fibrils, which then become phosphorylated and continue to accumulate over the next two weeks. This model also shows that the phosphorylated aggregates are seeding competent.

    Development and validation of specific antibody against non-phosphorylated α-Syn at serine129.
    The specificity of the 4B1 antibody toward WT aggregated α-Syn is puzzling. The authors’ data clearly suggest that the epitope comprises residue S129, as evidenced by the fact that it does not recognize pS129 or S129A α-Syn. Previous studies have consistently shown that the C-terminal domain containing S129 is highly dynamic and remains accessible in the monomeric, oligomeric, and fibrillar states of α-Syn. It is also well-established that antibodies against pS129 can readily detect different aggregated forms of α-Syn phosphorylated at S129. Although the monomer is known to adopt conformation where the C- and N-terminal proteins participate in intramolecular interactions, such interactions are transient and represent the conformation of only one population of α-Syn conformations, in addition to extended conformations. Concerning the performance of this antibody in tissues from transgenic mice and human brains, it would be nice to see the data (full WBs) on soluble and insoluble fractions from these samples. Finally, it is important to emphasize that this antibody most likely will not detect C-terminally truncated forms of α-Syn which are abundant in LBs and in the insoluble α-Syn aggregates from human brains. Surprisingly, the authors do not elaborate on the sequence of structural basis of the specificity of this antibody. It is crucial that all information about the antibodies used be disclosed to allow better understanding of the data and comparison to studies using different antibodies.

    Finally, a technical comment. To generate the phosphorylated α-Syn monomers and fibrils, the authors performed in-vitro phosphorylation of α-Syn using PLK2, and the phosphorylated proteins were used without further purification or removal of the phosphorylation buffer. The efficiency of phosphorylation before use was not assessed.

    References:

    Paleologou KE, Schmid AW, Rospigliosi CC, Kim HY, Lamberto GR, Fredenburg RA, Lansbury PT, Fernandez CO, Eliezer D, Zweckstetter M, Lashuel HA. Phosphorylation at Ser-129 but not the phosphomimics S129E/D inhibits the fibrillation of alpha-synuclein. J Biol Chem. 2008 Jun 13;283(24):16895-905. PubMed.

    Stephens AD, Zacharopoulou M, Moons R, Fusco G, Seetaloo N, Chiki A, Woodhams PJ, Mela I, Lashuel HA, Phillips JJ, De Simone A, Sobott F, Schierle GS. Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity. Nat Commun. 2020 Jun 4;11(1):2820. PubMed.

    Mahul-Mellier AL, Burtscher J, Maharjan N, Weerens L, Croisier M, Kuttler F, Leleu M, Knott GW, Lashuel HA. The process of Lewy body formation, rather than simply α-synuclein fibrillization, is one of the major drivers of neurodegeneration. Proc Natl Acad Sci U S A. 2020 Mar 3;117(9):4971-4982. Epub 2020 Feb 19 PubMed.

    Luk KC, Kehm VM, Zhang B, O'Brien P, Trojanowski JQ, Lee VM. Intracerebral inoculation of pathological α-synuclein initiates a rapidly progressive neurodegenerative α-synucleinopathy in mice. J Exp Med. 2012 May 7;209(5):975-86. PubMed.

    Strohäker T, Jung BC, Liou SH, Fernandez CO, Riedel D, Becker S, Halliday GM, Bennati M, Kim WS, Lee SJ, Zweckstetter M. Structural heterogeneity of α-synuclein fibrils amplified from patient brain extracts. Nat Commun. 2019 Dec 4;10(1):5535. PubMed.

    Strohäker T, Jung BC, Liou SH, Fernandez CO, Riedel D, Becker S, Halliday GM, Bennati M, Kim WS, Lee SJ, Zweckstetter M. Structural heterogeneity of α-synuclein fibrils amplified from patient brain extracts. Nat Commun. 2019 Dec 4;10(1):5535. PubMed.

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    Shillcock JC, Hastings J, Riguet N, Lashuel HA. Non-monotonic fibril surface occlusion by GFP tags from coarse-grained molecular simulations. Comput Struct Biotechnol J. 2022;20:309-321. Epub 2021 Dec 15 PubMed.

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    Woerman AL, Kazmi SA, Patel S, Freyman Y, Oehler A, Aoyagi A, Mordes DA, Halliday GM, Middleton LT, Gentleman SM, Olson SH, Prusiner SB. MSA prions exhibit remarkable stability and resistance to inactivation. Acta Neuropathol. 2018 Jan;135(1):49-63. Epub 2017 Aug 28 PubMed.

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    View all comments by Hilal Lashuel

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