Ramalingam N, Jin SX, Moors TE, Fonseca-Ornelas L, Shimanaka K, Lei S, Cam HP, Watson AH, Brontesi L, Ding L, Hacibaloglu DY, Jiang H, Choi SJ, Kanter E, Liu L, Bartels T, Nuber S, Sulzer D, Mosharov EV, Chen WV, Li S, Selkoe DJ, Dettmer U. Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity. NPJ Parkinsons Dis. 2023 Jan 16;9(1):4. PubMed.

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  1. These two studies provide new and intriguing information about a physiological role for α-synuclein phosphorylation at S129. pS129 α-synuclein is best known as a marker for Lewy pathology in Parkinson’s disease. However, a small portion of α-synuclein is phosphorylated under physiological conditions and the two teams cited here sought to determine what the role of that phosphorylated α-synuclein may be. In some ways, the two stories are remarkably similar. Both teams found that pS129 α-synuclein is present at low levels in the rodent brain and in primary neuron cultures, and that stimulation of neuronal activity increases the pool of pS129 α-synuclein. Phosphorylated α-synuclein seems to have an increased localization to the presynaptic terminal, where it has an effect on synaptic function. The two stories diverge on what this function is.

    Parra-Rivas et al. use fluorescent live imaging to track synaptic vesicle dynamics and find that α-synuclein attenuates activity-dependent synaptic vesicle cycling, and that phosphorylation of α-synuclein exacerbates this effect. Ramalingam et al. use patch clamp electrophysiology and find that α-synuclein phosphorylation seems to promote neuronal activity through a feed-forward mechanism. It should be noted that both sets of experiments are largely supported by overexpression of phospho-mimetic or phospho-null mutant α-synuclein. It seems that future experiments will be necessary to determine the impact of pS129 α-synuclein on synaptic physiology.

    How does activity stimulate α-synuclein phosphorylation? Ramalingam et al. provide convincing evidence that neuronal activity can stimulate PlK2 phosphorylation of α-synuclein through a calcium-calcineurin-dependent pathway. Parra-Rivas show via immunohistochemistry, that select neuronal populations appear to express elevated pS129 α-synuclein, while others express non-phosphorylated α-synuclein. It will be interesting to investigate what those neuronal populations are that express the highest level of physiological pS129 α-synuclein. Are they highly active neurons? Are they more susceptible to developing Lewy pathology?

    These studies are also an important reminder to interpret antibody-dependent data carefully. For example, pS129 staining alone is insufficient to establish the presence of Lewy pathology. Non-pathological controls must stain negatively for positive staining to be interpreted as pathological. This control is especially important in transgenic mice that overexpress α-synuclein because the pool of soluble phosphorylated α-synuclein is likely much higher in these mice. The two studies presented here used a variety of pS129 α-synuclein antibodies to establish the noted phenotypes, and the congruent findings by both groups support the reproducibility of the findings. However, phospho-selective antibodies often have nonspecific staining, and this topic was revisited for α-synuclein antibodies in a recent publication (Lashuel et al., 2022). 

    The function of α-synuclein has been difficult to pin down over the years. Is this because its function is dynamically regulated via phosphorylation? Is it the same or a different pathway that regulates phosphorylation of pathological α-synuclein? Another aggregation-prone protein, tau, is physiologically phosphorylated, and there is some evidence that this phosphorylation could precipitate further pathological phosphorylation and misfolding. It will be interesting to see if α-synuclein phosphorylation fits a similar mold.

    References:

    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. Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity. NPJ Parkinsons Dis. 2022 Oct 20;8(1):136. PubMed.

    View all comments by Michael Henderson

  2. The novel findings presented in the two papers, which suggest that normal α-synuclein has functions under physiological conditions, are quite interesting and unexpected. We had reported in 2002 that aggregated α-synuclein, but not the normal soluble species, is exclusively and highly phosphorylated at Ser129 (Fujiwara et al., 2002). Our estimation was that only ~4 percent of soluble α-synuclein was Ser129-phosphorylated, prompting us to speculate that the effect of α-synuclein phosphorylation in the normal state might be negligible. Since then, a number of studies using the Ser129Ala “unphosphorylatable” mutant have been conducted in variable contexts (e.g., Kuwahara et al., 2012), although the studies were mostly focused on toxicity, membrane binding, or aggregation properties of S129A mutant α-synuclein under an overexpression paradigm aiming at mimicking PD in model organisms.

    The data presented in the two papers show that Ser129 phosphorylation of α-synuclein at physiological level is indeed increased in a neuronal activity-dependent manner. Furthermore, detailed analyses of the S129A mutant implicated the role of this phosphorylation in neuronal plasticity and/or synaptic vesicle transport in neurons. It is notable that the loss of Ser129 phosphorylation, which normally occurs in a few percent of molecules, elicited these effects, which may indicate that the phosphorylation leads to an acquisition of function, not an inactivation of constitutive activities. Of course, the results using the S129A mutant are always not free from the concern that the Ala substitution might have artifactually caused misconformation of α-synuclein, not a simple loss of phosphorylation.

    The results of these studies further arouse our interest in the following points. First, is the regulatory mechanism(s) of α-synuclein phosphorylation in soluble state distinct from that of insoluble α-synuclein in synucleinopathy brains? Second, given our previous finding that soluble α-synuclein is extracellularly released in a neuronal activity-dependent manner in vitro and in vivo (Yamada and Iwatsubo, 2018), does the phosphorylation at Ser129 also affect the release of soluble α-synuclein? These questions may be of further interest in terms of the relevance of α-synuclein phosphorylation in disease and synaptic functions.

    References:

    Fujiwara H, Hasegawa M, Dohmae N, Kawashima A, Masliah E, Goldberg MS, Shen J, Takio K, Iwatsubo T. alpha-Synuclein is phosphorylated in synucleinopathy lesions. Nat Cell Biol. 2002 Feb;4(2):160-4. PubMed.

    Kuwahara T, Tonegawa R, Ito G, Mitani S, Iwatsubo T. Phosphorylation of α-Synuclein Protein at Ser-129 Reduces Neuronal Dysfunction by Lowering Its Membrane Binding Property in Caenorhabditis elegans. J Biol Chem. 2012 Mar 2;287(10):7098-109. PubMed.

    Yamada K, Iwatsubo T. Extracellular α-synuclein levels are regulated by neuronal activity. Mol Neurodegener. 2018 Feb 22;13(1):9. PubMed.

    View all comments by Tomoki Kuwahara

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