α-Synuclein serine129 phosphorylation – the physiology of pathology,Molecular Neurodegeneration

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α-Synuclein serine129 phosphorylation – the physiology of pathology
Molecular Neurodegeneration ( IF 15.1 ) Pub Date : 2023-11-13 , DOI: 10.1186/s13024-023-00680-x
Nagendran Ramalingam ₁ , Ulf Dettmer ₁

Affiliation

The study that found phospho-serine129 in the Parkinson’s-linked protein alpha-synuclein over two decades ago proposed a physiological role in regulating protein function, but this notion was neglected when alpha-synuclein serine129 phosphorylation was identified in Lewy bodies/neurites, the hallmark pathology of synucleinopathies. Recent work suggests that both are relevant: pathological phospho-serine129 in Lewy lesions and physiological phospho-serine129 that fine-tunes alpha-synuclein’s synaptic function.

α-Synuclein (αS) phospho-serine129 (pS129) is a specific marker of synucleinopathy – or is it? In the absence of robust antibodies to aggregated αS, it has become a standard to use antibodies to pS129 – in immunohistochemistry, immunocytochemistry, ELISA, or Western blot – and assume that αS aggregation and pS129 are synonyms. Indeed, pathological αS deposits in neurons - Lewy bodies (LBs) and Lewy neurites (LNs) in Parkinson’s disease and Lewy body dementia - were reported to contain about 90% of αS in the pS129 form [1, 3]. However, while there is little doubt that pS129 is present in end-stage aggregates, it has never been clear if pS129 plays any significant role in the cascade of events that lead from native to aggregated αS. On the contrary, recent work suggests pS129 is a late event in pathology, happening to large αS aggregates [4]. In this scenario, pS129 might be irrelevant to the aggregation itself, and could even represent an attempt of the neuron to dissolve the lesions. Consistent with that possibility, it has been proposed that the S129 phosphorylation by Polo-like kinase 2 (Plk2) plays a key role in the degradation of αS [8].

If the notion is true that αS aggregation predates pS129, how should one interpret the widely-documented presence of pS129 under conditions unsuspicious of aggregation: normal human brain, non-human primate brain, and rodent brain [2, 5, 6, 11]? Does αS have such a strong aggregation propensity that even under normal conditions a certain portion misfolds and precipitates? The other (and our preferred) possibility is that pS129 under normal conditions is not pathological at all. Instead, physiological pS129 has evolved to fine-tune αS function, as proposed as early as 2000 [7].

Several lines of evidence, in our view, support the occurrence of physiological pS129 (in addition to the widely documented pathological pS129 associated with αS aggregates). In a recent publication [10], we systematically studied effects of familial PD (fPD) αS missense mutations on basal pS129 in cultured αS knock-out rat neurons. The cultures were transduced with human αS WT, A30P, E46K, H50Q, G51D and A53T. All these mutations, over decades, unequivocally cause PD with classical pathology, including αS S129 hyper-phosphorylation in highly insoluble LB/LN lesions. When expressed for about 2 weeks at roughly physiological levels, however, not even trace amounts of αS were found in highly insoluble biochemical fractions and no uniform pS129 pattern arose: A30P, H50Q, and G51D were all hypo-phosphorylated, E46K αS was hyper-phosphorylated, and A53T was indistinguishable from WT. In the absence of aggregation, pS129 merely correlated with αS-membrane interaction: hypo-phosphorylated A30P, H50Q, and G51D all accumulated in the neurons’ cytosolic fractions. That, as we know, does not make these variants “protective” – in our experimental setup there may just not be enough time for aggregation. In the absence of misfolding, pS129 may be governed by a simple molecular “rule”: membrane-associated αS is a strong, cytosolic αS is a weaker substrate for the kinase (Plk2). Indeed, previous work has shown that pS129 accumulates in membrane fractions, and recombinant αS becomes dramatically more S129-phosphorylated by Plk2 if liposomes are added to the reaction [11].

In vivo support for physiological pS129 comes from “enriched-environment” experiments: animals housed under stimulating conditions exhibit both improved long-term potentiation (hippocampal slices) and increased pS129 [11]. It seems difficult to postulate that environmental enrichment causes αS to aggregate. A more likely explanation is physiological phosphorylation of S129 triggered by neuronal activity. Indeed, stimulating neuron cultures also elevates pS129, readily reversible by inhibiting neuronal activity. So, if physiological pS129 is real, what might be its function? Interestingly, synaptic transmission is impaired in pS129-deficient S129A knock-in mice, consistent with a feed-forward role of pS129. In a study accessible as a preprint, it is suggested that physiological pS129 regulates αS interactions of VAMP2 and synapsin [9], pointing at a possible molecular mechanism. This would be consistent with our finding that activity-dependent pS129 colocalizes with synapsin-containing boutons [11]. If all this is true, do we have to consider αS E46K (hyper-phosphorylated) a stimulator of synaptic transmission and A30P/H50Q/G51D (hypo-phosphorylated) a negative influence? The reality may be more complicated: compared to WT αS, the reversibility of activity-triggered pS129 is reduced for both E46K and A30P, indicating protein dyshomeostasis in both mutants. In addition, proteasomal inhibition also increases pS129, similar to neuronal activity, but with reduced reversibility [10]. More work is needed to establish and understand physiological vs. pathological pS129 (Fig. 1), and it should consider that even in the absence of endpoint aggregation, physiological and pathological pS129 may co-exist. A key question may be: is it all a matter of ratios of principally identical molecular arrangements or is there something unique about non-aggregated, pathological pS129, such as a fold that cannot be considered “normal”?

N/A.

Anderson JP, Walker DE, Goldstein JM, de Laat R, Banducci K, Caccavello RJ, Barbour R, Huang J, Kling K, Lee M, et al. Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy Body Disease. J Biol Chem. 2006;281:29739–52.
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Arlinghaus R, Iba M, Masliah E, Cookson MR, Landeck N. Specific detection of physiological S129 phosphorylated α-Synuclein in tissue using proximity ligation assay. J Parkinsons Dis. 2023;13:255–70.
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Parra-Rivas LA, Madhivanan K, Wang L, Boyer NP, Prakashchand DD, Aulston BD, Pizzo DP, Branes-Guerrero K, Tang Y, Das U et al. (2022) Serine-129 phosphorylation of α-synuclein is a trigger for physiologic protein-protein interactions and synaptic function Neuroscience.
Ramalingam N, Brontesi L, Jin S-X, Selkoe DJ, Dettmer U. (2023) Dynamic reversibility of α-synuclein serine-129 phosphorylation is impaired in synucleinopathy models. EMBO Rep: e57145.
Ramalingam N, Jin S-X, Moors TE, Fonseca-Ornelas L, Shimanaka K, Lei S, Cam HP, Watson AH, Brontesi L, Ding L, et al. Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity. NPJ Parkinsons Dis. 2023b;9:4.
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We thank the members of the Dettmer, Selkoe and Nuber labs (at BWH/HMS) for advice and scientific discussions.

Our work on αS is supported by the National Institutes of Health (grant numbers NS121826, NS099328, NS109209, NS122880, and NS133979). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors and Affiliations

Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, USA
Nagendran Ramalingam & Ulf Dettmer

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N.R. and U.D. did the literature review and wrote the manuscript.

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Correspondence to Nagendran Ramalingam or Ulf Dettmer.

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Ramalingam, N., Dettmer, U. α-Synuclein serine129 phosphorylation – the physiology of pathology. Mol Neurodegeneration 18, 84 (2023). https://doi.org/10.1186/s13024-023-00680-x

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Received: 06 November 2023
Accepted: 08 November 2023
Published: 13 November 2023
DOI: https://doi.org/10.1186/s13024-023-00680-x

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Keywords

Alpha-synuclein
Phosphorylation
Post-translational modification
Neurotransmission
Synapse
Polo-like kinase-2

中文翻译：

α-突触核蛋白丝氨酸 129 磷酸化 – 病理生理学

二十多年前在帕金森病相关蛋白 α-突触核蛋白中发现磷酸化丝氨酸 129 的研究提出了调节蛋白质功能的生理作用，但当在路易体/神经突中发现了 α-突触核蛋白丝氨酸 129 磷酸化时，这一概念被忽视了。突触核蛋白病的病理学。最近的研究表明两者都是相关的：路易损伤中的病理性磷酸丝氨酸 129 和微调 α-突触核蛋白突触功能的生理性磷酸丝氨酸 129。

α-突触核蛋白 (αS) 磷酸丝氨酸 129 (pS129) 是突触核蛋白病的特异性标志物 – 真的是这样吗？在缺乏针对聚集 αS 的强大抗体的情况下，在免疫组织化学、免疫细胞化学、ELISA 或蛋白质印迹中使用 pS129 抗体已成为标准，并假设 αS 聚集和 pS129 是同义词。事实上，据报道，神经元中的病理性 αS 沉积 - 帕金森病和路易体痴呆中的路易体 (LB) 和路易体神经突 (LN) - 含有约 90% 的 pS129 形式的 αS [1, 3]。然而，虽然毫无疑问 pS129 存在于终末期聚集体中，但尚不清楚 pS129 是否在从天然 αS 到聚集 αS 的级联事件中发挥任何重要作用。相反，最近的研究表明 pS129 是病理学中的晚期事件，发生在大型 αS 聚集体上 [4]。在这种情况下，pS129可能与聚集本身无关，甚至可能代表神经元试图溶解病变。与这种可能性一致，有人提出 Polo 样激酶 2 (Plk2) 引起的 S129 磷酸化在 αS 的降解中起着关键作用 [8]。

如果αS聚集早于pS129这一观点是正确的，那么我们应该如何解释在不怀疑聚集的条件下广泛记录的pS129的存在：正常人脑、非人灵长类动物脑和啮齿类动物脑[2,5,6,11] ？αS是否具有如此强烈的聚集倾向，以至于即使在正常条件下，也会有一部分发生错误折叠并沉淀？另一种（也是我们首选的）可能性是 pS129 在正常条件下根本不是病理性的。相反，正如早在 2000 年提出的那样，生理学 pS129 已经进化到可以微调 αS 功能 [7]。

我们认为，有几条证据支持生理性 pS129 的存在（除了广泛记录的与 αS 聚集体相关的病理性 pS129 之外）。在最近的一篇出版物中 [10]，我们系统地研究了家族性 PD (fPD) αS 错义突变对培养的 αS 敲除大鼠神经元中基础 pS129 的影响。用人 αS WT、A30P、E46K、H50Q、G51D 和 A53T 转导培养物。几十年来，所有这些突变都明确导致具有经典病理学的 PD，包括高度不溶性 LB/LN 病变中的 αS S129 过度磷酸化。然而，当在大致生理水平上表达约 2 周时，在高度不溶的生化级分中甚至没有发现微量的 αS，并且没有出现统一的 pS129 模式：A30P、H50Q 和 G51D 均低磷酸化，E46K αS 超磷酸化。磷酸化，A53T 与 WT 无法区分。在没有聚集的情况下，pS129 仅与 αS 膜相互作用相关：低磷酸化的 A30P、H50Q 和 G51D 均积聚在神经元的胞质部分中。正如我们所知，这并不会使这些变体具有“保护性”——在我们的实验设置中，可能没有足够的时间进行聚合。在没有错误折叠的情况下，pS129 可能受一个简单的分子“规则”控制：膜相关的 αS 是强的，胞质 αS 是激酶 (Plk2) 的较弱的底物。事实上，之前的研究表明，pS129 在膜组分中积累，如果将脂质体添加到反应中，重组 αS 会被 Plk2 显着增强 S129 磷酸化程度 [11]。

对生理 pS129 的体内支持来自“丰富环境”实验：在刺激条件下饲养的动物表现出长期增强作用（海马切片）的改善和 pS129 的增加[11]。似乎很难假设环境富集会导致 αS 聚集。更可能的解释是神经元活动触发 S129 的生理磷酸化。事实上，刺激神经元培养也会升高 pS129，通过抑制神经元活动很容易逆转。那么，如果生理学 pS129 是真实的，它的功能可能是什么？有趣的是，pS129 缺陷的 S129A 敲入小鼠中突触传递受损，这与 pS129 的前馈作用一致。在一项预印本研究中，表明生理学 pS129 调节 VAMP2 和突触蛋白的 αS 相互作用 [9]，指出了可能的分子机制。这与我们的发现一致，即活性依赖性 pS129 与含有突触蛋白的纽扣共定位 [11]。如果这一切都是真的，我们是否必须认为 αS E46K（高磷酸化）是突触传递的刺激剂，而 A30P/H50Q/G51D（低磷酸化）是负面影响？现实情况可能更复杂：与 WT αS 相比，E46K 和 A30P 的活性触发 pS129 的可逆性均降低，表明两种突变体中的蛋白质稳态失衡。此外，蛋白酶体抑制也会增加 pS129，与神经元活动类似，但可逆性降低 [10]。需要做更多的工作来建立和理解生理性与病理性 pS129（图 1），并且应该考虑到即使在没有终点聚集的情况下，生理性和病理性 pS129 也可能共存。一个关键问题可能是：这完全是基本相同的分子排列的比例问题，还是非聚集的病理性 pS129 存在某些独特之处，例如不能被视为“正常”的折叠？

不适用。

Anderson JP、Walker DE、Goldstein JM、de Laat R、Banducci K、Caccavello RJ、Barbour R、Huang J、Kling K、Lee M 等。Ser-129 磷酸化是家族性和散发性路易体病中 α-突触核蛋白的主要病理修饰。生物化学杂志。2006；281：29739–52。
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我们感谢 Dettmer、Selkoe 和 Nuber 实验室（BWH/HMS）的成员提供的建议和科学讨论。

我们在 αS 方面的工作得到了美国国立卫生研究院的支持（拨款号 NS121826、NS099328、NS109209、NS122880 和 NS133979）。资助者在研究设计、数据收集和分析、发表决定或手稿准备中没有任何作用。

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