Physiological roles of α-synuclein serine-129 phosphorylation — not an oxymoron

Abstract

α-Synuclein (αS) is an abundant presynaptic protein that regulates neurotransmission. It is also a key protein implicated in a broad class of neurodegenerative disorders termed “synucleinopathies” including Parkinson’s disease and dementia with Lewy bodies. Pathological αS deposits in these diseases, Lewy bodies/neurites, contain about 90% of αS in its phospho-serine129 (pS129) form. Therefore, pS129 is widely used as a surrogate marker of pathology. However, recent findings demonstrate that pS129 is also physiologically triggered by neuronal activity to positively regulate synaptic transmission. In this opinion article, we contrast the literature on pathological and physiological pS129, with a special focus on the latter. We emphasize that pS129 is ambiguous and knowledge about the context is necessary to correctly interpret changes in pS129.

α-Synuclein pathology entails phospho-serine129 – and vice versa?

α-synuclein (αS) is a 140-amino acid neuronal protein. It has a strong propensity to interact with synaptic vesicles, even though the binding is dynamic and only transient (reviewed in [1]). αS accumulates at synapses in neurons [2] and a variety of functions related to synaptic vesicle biology have been proposed, including synaptic vesicle exocytosis [3], endocytosis [4], synaptic vesicle clustering [5], controlling the size of presynaptic fusion pore [6], regulating SNARE assembly [7], stabilization of synaptic vesicle membranes [8], and facilitation as well as depression of dopamine release [9]. However, αS is most widely recognized for being linked to Parkinson’s disease (PD) and Lewy body dementia (LBD). These neurodegenerative diseases are characterized by the aggregation of αS into large cytoplasmic inclusions termed Lewy bodies (LBs; present in the cell body) and Lewy neurites (LNs; present in neuronal processes).

Several other lines of evidence point at a central role of αS in PD pathogenesis. Both αS missense mutations and gene duplication/triplication are associated with rare familial forms of PD (reviewed in [10]). Importantly, familial PD (fPD) is largely indistinguishable in its clinical manifestation from idiopathic PD, the latter of which is caused by a combination of genetic risk factors and environmental influences. Clinically, both familial and idiopathic PD patients suffer from similar impairment of movement (including tremor and stiffness). Histologically, LBs/LNs are found in nearly all forms of PD (prominent exceptions are Parkin- and certain LRRK2-linked forms [11]). While there is a current debate about the exact nature of LBs – are they more proteinaceous or more lipid-rich? [12,13] – there is no doubt that αS is abundant in these lesions. Notably, up to 90% of LB/LN αS is present in its phospho-serine-129 (pS129) form [14,15]. Other potential phospho-sites in the αS C-terminus such as tyrosine (Y)125 seem to be observed inconsistently and less frequently [16]. In other words: Lewy-like aggregation is invariably associated with pS129.

It is tempting to assume that, conversely, pS129 is invariably associated with pathology. In line with this notion, antibodies to pS129 are widely used in histology and immunoblot techniques as a proxy for αS aggregation. While in PD brains and models of late-stage pathology, this may well be appropriate, there is growing evidence that in the absence of pathology, a physiological form of pS129 serves the purpose of fine-tuning αS’s synaptic function [17–19]. In fact, the study that first described (to our knowledge) that αS can be phosphorylated at S129 speculated that pS129 may serve a role in αS regulation [20]. Thus, in the absence of gross pathology, pS129 should be cautiously interpreted.

The proposal of physiological roles for pS129 is not intended to challenge the well established presence of pS129 in pathological aggregates, which we highlight in the following section. We also touch upon open questions regarding pathological pS129, most importantly whether phosphorylation occurs early or late in the aggregation process and whether there may be a neuroprotective role of pS129 in αS aggregates. We then showcase evidence that pS129 occurs in the absence of any obvious synucleinopathy-relevant conditions, e.g., in the brains of healthy humans, primates, and mice, as well as in unperturbed cultured neurons and immortalized cell lines. In further support to the notion that pS129 is not unequivocally associated with pathology, we discuss the diverging effects that fPD-linked αS point mutations have on pS129 levels; specifically, these effects have been interchangeably reported to be greater, lesser, and unchanged levels of pS129 in comparison to wildtype (WT) αS. Most importantly, we then highlight recent insights into the function of physiological pS129 at the synapse, starting from the notion that neuronal activity drives synaptic pS129, and ending with speculations on how this post-translational modification may fine-tune αS’s synaptic role. Finally, we share our views on the relationship between physiological and pathological αS, and bring attention to open questions to be addressed in future work. Given the relative novelty of the concept, the number of publications on physiological pS129 is still small. As far as pathological pS129 is concerned, we had to limit ourselves to a number of representative studies, with emphasis on early publications reporting the discovery of pS129 in LBs, as well as advances from recent years.

Pathological pS129

pS129 is found in pathology

About a decade after its initial discovery in Torpedo [21], αS was identified as the main component of LBs [22]. Fast forward 5 years, it was revealed that 90% of αS in these lesions is phosphorylated at S129 [14]. The authors used mass-spectrometry and subsequently substantiated their findings using well-characterized antibodies to total αS and pS129. Their rigorous results were later confirmed by many groups (e.g., [15]). Consequently, both total and pS129 αS levels as well as pS129/total αS ratios have been suggested to be elevated in PD patient cerebrospinal fluid [23], plasma [24–26], and even erythrocyte [27,28] samples. However, pS129 distribution in diseased brain and body fluids seems to depend on details of the diagnosed synucleinopathy [29,30]. Optimized detection methods for pS129 in body fluids are still being developed (e.g., [31]), and the choice of the pS129-detecting antibodies is certainly critical [32,33]. Independent of whether pS129 can be harnessed as a biomarker or not, its abundance in Lewy pathology is firmly established. The certainty, however, ends with the question of whether pS129 plays any causal role in aggregation. Related to that, there is lack of consensus on whether S129 undergoes phosphorylation in early or late stages of αS aggregation and whether S129-phosphorylation of aggregated αS is detrimental or protective, as discussed in the following two subsections.

pS129 in pathology: early or late?

Biochemical analyses in cingulate and temporal cortex of DLB patients at different disease stages revealed a progressive accumulation of pS129 immunoreactivity in diseased brains, and a positive correlation between pS129 and the severity of disease symptoms [34]. Importantly, the accumulation of pS129-positive insoluble species became detectable only at the late stages of the disease, i.e., stage IV and V, according to the Unified staging system for Lewy body disorders [35]. A similar study, using brain samples from PD patients, also reported a dramatic accumulation of pS129-positive inclusions in different brain regions at the late stages of the disease [36]. Recent work verified that the αS S129 residue is more efficiently phosphorylated when the protein is aggregated [37]. Based on these observations, it is tempting to propose that abnormal accumulation of insoluble pS129 αS characterizes the advanced stages of synucleinopathies (Fig. 1A). However, pS129 was also suggested to be the earliest αS post-translational modification in LB formation, followed by Y39 nitrosylation and lower amounts of phospho-serine87 later in disease progression [38].

Figure 1. Early vs. late αS phospho-serine 129 (pS129) in pathology.

A, Early pS129: pathway from native αS to Lewy pathology. Oligomeric aggregates, or even monomers in disequilibrium, are markedly pS129-modified, and so are Lewy lesions. B, Late pS129: large Lewy-like deposits are heavily pS129-modified, but not their precursors.

In addition, there is evidence for the existence of pathological pS129 without obvious aggregation (Fig. 1B). For example, abnormal elevation of pS129 upon proteasomal inhibition was documented exclusively in RIPA-soluble fractions in cultured neurons [39]. Similarly, methamphetamine or iron overload may cause rapid formation of detergent-soluble pS129 in cultured cells and rodents in vivo [40,41]. Thus, pathological S129 may well be observed before the formation of insoluble aggregates as a result of conformational changes that render αS a better substrate for S129 kinases such as polo-like kinase 2 (Plk2) and/or a poorer substrate for S129 phosphatases such as PP2A [39]. Notably, a recent preprint article implies that dephosphorylation of pS129 in detergent-insoluble aggregates is impaired, which might account for the pronounced pS129 in LB/LN (Fig. 1A) [42]. Moreover, astroglial accumulations of αS were shown to be pS129-negative, but positive for phosphorylated and nitrated forms of αS at Y39 [43], indicating that at least in glial cells αS deposition does not depend on pS129. However, in another study, both glia and neurons in DLB cases were shown to have increased nuclear pS129 compared to controls [44], so the presence of pS129 in glial αS deposits requires further investigation.

All in all, it seems premature to make a definite statement on whether pS129 occurs late (Fig. 1A) or early (Fig. 1B) in pathology. We speculate, however, that as soon as αS deviates from one of its native conformations, altered interactions with kinases and phosphatases will increase pS129. Proposing experiments to definitely resolve the question of pS129 timing seems challenging. A study in post-mortem brains may not provide the necessary level of clarity, even if a large number of non-synucleinopathy controls, incidental LB disease and various stages of PD are included. A well-controlled study in rodents that combines preformed-fibril (PFF) injection, monitoring aggregate formation by seeded amplification assay [45], assessing PLA positivity [46], and studying pS129 kinase or phosphatase sensitivity [39,42], may be more suitable. We also speculate that critical protein-protein interactions [19] will be altered during the course of aggregate formation.

pS129 in pathology: detrimental or protective?

A better understanding of when and how pS129 occurs in disease might also help answer another important question, i.e., what the significance of pS129 modification under pathological conditions might be. The possibilities range from pS129 aggravating pathology to pS129 preventing further aggregation and pS129 being a tag for improved turnover by protein degradation systems. Some reports suggest that pS129 accelerates neuronal cell death in cellulo [47,48] as well as in vivo [49] and could potentially inhibit Plk2 from properly regulating stress signaling [50]. However, others suggest that pS129 does not confer toxicity in vivo [51–53]. Studies using diverse aggregation assays involving recombinant αS (phospho or non-phospho S129) and brain homogenates from PD and DLB cases suggest that pS129 αS may actually inhibit αS fibril formation and seeded aggregation [54,37]. This thought-provoking notion needs to be thoroughly validated in other systems. Interestingly, in a yeast αS overexpression model, pS129 indeed increased αS turnover, which was interpreted as relevant for pathological αS [55]. Furthermore, it was demonstrated in HEK cells that selective autophagic degradation of ectopically expressed αS may be governed by Plk2-mediated S129 phosphorylation [56]. In the same study, Plk2 overexpression in a rat model of PD suppressed αS-induced toxicity, possibly without affecting αS levels. Additionally, proteasome inhibition increases endogenous pS129 levels without altering total αS protein levels in cultured neurons [57,58]. These results suggest a complex relationship between pS129 and αS turnover. In summary, it is tempting to speculate that pS129 overall plays a protective role in disease, preventing aggregation and/or promoting the degradation of misfolded αS, but a firm conclusion seems premature. Notwithstanding, pathological pS129 could still serve as a therapeutic target or diagnostic marker even if it is not actively participating in pathology progression, but the distinction from physiological pS129 may be critical.

Physiological pS129

pS129 is detected under non-pathological conditions

The first study (to our knowledge) to describe pS129 suggested it to be a physiological event [20], studying HEK 293 and rat pheochromocytoma PC12 cells. The authors further speculated that αS function is regulated by phosphorylation/dephosphorylation. Subsequently, pS129 was detected in a wide range of other sources unsuspicious of synucleinopathy context, including normal human brain, non-human primate brain, rodent brain, and cultured rodent neurons [14,18, 19,39,46,59–61]. A frequently cited number is 4% of total αS being pS129 in normal rodent brains [14]. A recent study, employing a tyramide signal amplification technique in non-diseased WT mice, detected relatively enriched pS129 in the olfactory bulb and several brain regions across the neuroaxis [61]. pS129 enrichment was also found in olfactory bulb mitral cells of rats, non-human primates, and humans in the absence of synucleinopathy. Also recently, a proximity-ligation assay was developed that specifically detects physiological and soluble pS129 αS in cell culture, mouse brain sections, and human brain tissue, while not picking up aggregated αS in LBs [46]. The most plausible interpretation of these various “sightings” of pS129 in the absence of disease, in our opinion, is the existence of physiological pS129 that plays a role in regulating αS biology. The alternative hypothesis, that αS has such a strong aggregation propensity that even under normal conditions a relevant portion always misfolds, precipitates, and becomes pathologically phosphorylated, seems less likely. Human αS is the result of more than 300 million years of evolution since the first appearance of synucleins in early vertebrates [62]. A constant threat to proteostasis imposed by readily aggregating αS and the need to continuously remove aggregates should not stand the test of selective pressure. Interestingly, S129 appeared relatively late in evolution: based on existing evidence, it seems that only placental mammals possess it, and it is highly conserved among them (Fig. 2).

Figure 2: S129 evolution.

Sequence alignment of αS amino-acids 123-140 (human nomenclature) across vertebrates; only vertebrates possess αS. Residues that potentially undergo phosphorylation are marked with downward arrows. S129 may have evolved in placental animals as a way to fine-tune αS function. Protein sequences from www.ncbi.nlm.nih.gov/protein. Silhouettes from www.phylopic.org under Public Domain Mark 1.0 or CC0 1.0 Universal Public Domain Dedication licenses.

In light of an increased need to fine-tune proteins involved in synaptic function in complex brain structures [63], the tightly regulated post-translational modification of the synaptic protein αS [17–19,61] may serve this purpose. Experimental evidence suggests that pS129 levels can go up three-fold during neuronal activity[18], sufficient to markedly affect, e.g., pS129-dependent protein-protein interactions [19]. Furthermore, observed differences in pS129 intensity in specific brain regions (e.g., prominent levels in superficial cortex, hippocampus, olfactory bulb, and midbrain) suggest a region- or cell type-specific regulation of synaptic functions by pS129 [19].

Familial PD variants have diverging effects on pS129 in the absence of aggregation

A recent publication systematically addressed how fPD-linked αS missense mutations affect basal pS129 in cultured neurons [39]. αS knock-out rat neuron cultures were transduced with human αS WT, A30P, E46K, H50Q, G51D and A53T for about two weeks to achieve expression at roughly endogenous levels. All these mutations cause human PD with classical pathology, including αS S129 hyper-phosphorylation in LB/LN lesions. When cells were subjected to sequential extraction, all αS variants were recovered in both cytosolic and membrane fractions. The highly insoluble fraction was devoid of αS, ruling out the presence of aggregates. Moreover, no uniform pS129 pattern was found: A30P, H50Q, and G51D were all hypo-phosphorylated, E46K αS was hyper-phosphorylated, and A53T was indistinguishable from WT. These findings confirmed previous work on the effects of A30P [64], and G51D [65,66] on membrane interaction in the absence of aggregation. The observations from these studies are overall consistent with a model in which cytosolic αS is a weaker substrate for the cognate kinase Plk2 (Fig. 3A i). Mechanistically, it was demonstrated that recombinant pS129 increases dramatically if liposomes are added to the reaction [18]. And, consistent with that, pS129 accumulates in membrane fractions: while total WT αS distributes 50:50 between cytosol and membrane, it is 30:70 for pS129 [18,67]. Conversely, certain engineered αS variants that accumulate at membranes have been shown to be rich in pS129 [68–70]. However, elevated pS129 is not necessarily accompanied by increased membrane association: neuronal expression of αS E46K or proteasomal inhibition seem to enhance pS129 without markedly affecting the solubility of αS [39] (Fig. 3A ii and iii) and increased synaptic activity also elevates pS129 without changing its membrane interaction [18]. Thus, pS129 accumulation does not necessarily reflect increased membrane binding. In summary, αS fPD variants have diverging effects on pS129 in the absence of aggregation, with an overall tendency to decrease it. In other words, under steady state conditions, both reduction and increase in pS129 can reflect PD-relevant αS dyshomeostasis. Under conditions of Lewy-like aggregation, however, pS129 may very well serve as a proxy for the presence of aggregates because the extent of pS129 on aggregates well exceeds that of any residual physiological pS129 (Fig. 3A, iv and v).

Figure 3. αS pS129 is present in pathological and in physiological, functional states.

A, Lewy bodies and Lewy neurites accumulate pathological pS129 [14] (top). The presynaptic terminal inset is zoomed in at the bottom. Without obvious insoluble aggregates, familial PD-linked αS mutations A30P, H50Q and G51D accumulate less pS129 (less membrane association; i) but E46K accumulates more (unknown mechanism; ii). Proteasome inhibition also increases pS129 (iii). The relationship between excess pS129 of native αS species (iv) and excess pS129 in aggregates is unclear (v). This model is mainly based on [39]; please see main text for more details. B, In response to synaptic activity, Plk2 is activated by Ca²⁺-regulated calcineurin (CaN). CaN* refers to the active form. The exact mechanism is unknown (i-iii). Increased Plk2 activity (Plk2* refers to the active form) then counteracts constitutive PP2A action (iv) to increase pS129. Thus, in a feed-forward mechanism, pS129 promotes neurotransmission (v). This model is mainly based on [18,19].

Neuronal activity drives pS129 under physiological conditions

Animals housed under stimulating conditions with toys and changes in their daily routines (“enriched environment”) have improved cognitive abilities, reflected by increased long-term potentiation measured in hippocampal brain slices [71,72]. Interestingly, the stimulating environment also leads to increases in pS129 when whole brain lysates are subjected to Western blot [18], consistent with a physiological event in vivo. In vitro, the stimulation of rodent neuron cultures with picrotoxin (an inhibitor to inhibitory GABAergic neurons), increases pS129steadily over ~8 h before it plateaus at around 200-300% of basal levels. Activity-dependent pS129 elevation in cultured neurons was subsequently confirmed upon electrical stimulation or stimulation via 4-aminopyridine; the latter also augmented pS129 in mouse brains upon peritoneal injection [19]. Consistent with a dynamic, physiological event, activity-dependent pS129 is readily reversible by inhibiting neuronal activity, e.g., when picrotoxin treatment is followed by tetrodotoxin (a sodium channel blocker), or by inhibitingPlk2 with the small molecule BI2536 [18]. 2h BI2536 treatment reduces both basal and activity-dependent pS129 to virtually zero, indicating that Plk2 is the key kinase regulating physiological pS129. In vitro kinase reactions followed by nuclear magnetic resonance analysis indeed suggested that αS is a direct substrate of Plk2 with a specificity for S129 (no other phospho-sites were altered). The counteracting phosphatase was identified as PP2A. In contrast, inhibition of the calcium-sensitive phosphatase PP2B/calcineurin prevented activity-dependent pS129. In line with this notion, the activation of PP2B/calcineurin by the cytosolic calcium wave that happens during synaptic activity, could be the first step in the incompletely understood cascade that governs activity-dependent pS129 (Fig. 3B).

pS129 fine-tunes the function of αS at the synapse

S129A knock-in mice (S129A^KI) are deficient for pS129. Electrophysiology in hippocampal slices of S129A^KI mice revealed impaired neurotransmission vs. WT by several measures [18]. This finding is consistent with a feed-forward mechanism, i.e., neuronal activity stimulates Plk2-dependent pS129 (Fig. 3B i–iv), which in turn increases synaptic transmission (Fig. 3B v). In the proposed mechanism (largely based on findings from [18]), neurotransmission increases Ca²⁺ (Fig. 3B i), Calcineurin gets activated (Fig. 3B ii), and Plk2 activity increases by an unknown mechanism (Fig. 3B iii). Plk2 is counteracted by PP2A (Fig. 3B iv). The mechanism by which pS129 then stimulates neurotransmission remains to be elucidated (Fig. 3B v). It was ruled out that pS129 affects αS-membrane interactions [18]. Thus, a simple model in which pS129 keeps αS off the vesicle membrane is currently not supported by data. Nonetheless, injection of pS129 into lamprey axons was reported to induce defects in synaptic vesicle trafficking and declustering, indicating that – while not affecting vesicle binding - S129 phosphorylation may still modulate αS effects on vesicles [73]. It was also shown that pS129 strongly increases αS interactions with VAMP2 and synapsin [19]. Consistent with that, activity-dependent pS129 colocalizes with synapsin-containing boutons [18]. In a feedforward scenario, the pS129-triggered αS-VAMP2 and αS-synapsin interactions could stimulate synaptic vesicle transport and/or the kinetics of neurotransmitter release, thereby amplifying synaptic transmission, but other scenarios are possible. For example, an S129 phospho-mimetic version of αS (S129D) more efficiently attenuated exocytosis compared to WT, and an interaction between VAMP2/Synapsin and αS may be crucial to this novel role described for pS129 [19]. The data on physiological pS129 are so far not consistent with enhanced αS degradation: stimulated and control neurons have indistinguishable αS total levels, and an obvious upregulation of αS in S129 knock-in animals was not observed either [18]. All in all, activity-dependent pS129 at the synapse seems to affect αS-protein interactions rather than αS-membrane interactions or αS half-life [19,74,75].

The relationship between physiological and pathological αS

The most parsimonious scenario – whether satisfying from the researchers’ standpoint or not – may be that physiological and pathological pS129 are not related at all [76]. Physiological pS129 occurs on monomeric αS at synapses and is readily reversible. Pathological pS129 is prominently found in LBs and LNs that are not readily degradable or dissolvable. As stated elsewhere, the only fPD-linked αS variant that increases basal pS129 in the absence of insoluble αS aggregates seems to be E46K. A30P, H50Q, G51D reduce pS129, and A53T leaves it unchanged, which is seemingly at odds with the notion that all variants are associated with pS129-rich Lewy lesions in PD patients. A possible resolution to this conundrum comes from a recent study that suggests alterations of pS129 in fPD-linked αS variants in the absence of aggregation could be an indication of impaired αS homeostasis. It was observed that the two divergent αS variants A30P (strongly mis-localized to the cytosol) and E46K (similar to WT) had in common an impaired pS129 reversibility when neuronal stimulation was blocked [39]. In addition, proteasomal inhibition was also shown to increase pS129 in cultured neurons (Fig. 3A iii), similar to neuronal activity, but the reversibility was again impaired. Accordingly, reduced proteasomal activity in neurons could explain the increased pS129 associated with normal aging, as reported for instance in non-human primate brains [77]. The pathway, if any, from excess pS129 in non-aggregated conformations to excess pS129 in aggregates (Fig. 3A iv, v) needs to be elucidated, and currently we consider it more likely that there is no direct connection between the two.

All in all, a parsimonious working hypothesis for the relationship between different cellular forms of pS129 is as follows. The kinases and phosphatases involved in pS129 – in health and disease – have different affinities for the different αS folding states, governing the extent of pS129 for each species. Both helical membrane-associated αS (Fig. 4B) and β-sheet aggregated αS (Fig. 4C, D) seem to be relatively better substrates for Plk2 than cytosolic αS (Fig. 4A), but that does not imply a straight pathway from membrane-associated pS129 to pS129 in aggregates (see previous paragraph). Moreover, it is possible that different S129 kinases are involved in health vs. disease. One study for example suggested that Plk2 may be predominantly involved in S129-phosphorylation of the soluble physiological fraction of αS because Plk2 inhibition did not reduce S129 phosphorylation or inter-neuronal spreading of aggregated αS in organotypic slices, in vivo mouse models or human dopaminergic neurons, [78]. Other studies, however, involved Plk2 also in the phosphorylation of S129 under pathological conditions [79].

Figure 4. αS conformation determines the extent of pS129.

A, Soluble native αS is a poor substrate for Plk2 and PP2A phosphatase activity prevails. As a result, pS129 is low [18]. B, Native membrane-associated helical αS is a preferred substrate of Plk2. As a result, pS129 increases relative to cytosolic αS [18]. C, Oligomeric αS and other precursors of Lewy-like aggregates may be better substrates to Plk2 and poorer substrates to PP2A than native αS (the extent of pS129 is still lower than in advanced aggregates) [42]. D, Pathological αS in Lewy-like aggregates is heavily phosphorylated, presumably as a result of increased Plk2 and decreased PP2A interaction [42].

Concluding remarks and future perspectives

There is now accumulating evidence that the serine-129 phosphorylation naturally occurs in brains in the absence of pathology. A role of physiological pS129 in fine-tuning synaptic activity is emerging that needs further detailed analysis. However, the presence of pS129 in αS aggregates is also well-documented. Thus, upon detection of pS129 a second criterium is necessary to distinguish between physiological and pathological pS129. This additional criterium could be αS folding state (e.g., using seeded amplification assay [45] or proteinase K digestion [68]), αS solubility (e.g., using biochemical fractionation [39]), pS129 detection by the PLA method [46], or pS129 reversibility (upon inhibition of activity in live neurons [39]; by kinase/phosphatase treatment of cells and brain tissue [39,42]). A strong abundance of pS129 may indicate pathology [14], but recent work suggests that in certain regions, brains can also be of high pS129 abundance naturally [61,19]. Outside the brain, the presence of high pS129 in skin biopsies seems to correlate well with synucleinopathy [80]. Future work to understand physiological vs. pathological pS129 will also need to consider that under certain conditions, physiological and pathological pS129 can co-exist. A key question in this context is whether anything about pathological pS129 is uniquely different from physiological pS129. One intriguing possibility is that a loss of pS129 dynamics in pathological states is a starting point of worsening pathology. Two broad areas that remain to be addressed in future mechanistic work on physiological pS129 are: (1) What pathways lead from synaptic activity to physiological pS129? (2) How does pS129 alter the molecular interactions of αS as well as its synaptic and possibly non-synaptic functions? (see Outstanding Questions).

Outstanding questions.

• Activity-dependent pS129 was observed to stimulate neuronal activity, consistent with a feed-forward mechanism. However, pS129 was also shown to impair synaptic vesicle exocytosis. How can these two seemingly contradictory results be reconciled, ideally in vivo?

• Are the altered protein interactions of pS129 key to understanding its mechanisms? Beyond the protein interactions that have been identified, are other interactions, such as with lipids or other biomolecules, affected by pS129?

• Plk2 catalyzes physiological pS129 downstream of calcineurin, counteracted by PP2A. How exactly is this pathway regulated and what other players might be involved?

• In healthy brains, why is pS129 found to be concentrated in the olfactory bulb? Are there region- or neuron-type-specific functions for pS129 in the brain?

• Are physiological and pathological pS129 truly unrelated or is there a pathway from excess activity-dependent pS129 to αS aggregation? Does pathological pS129 play a causal role in the aggregation or is it merely a bystander, occurring on aggregated material?

• Does the notion of physiological pS129 affect PD biomarker considerations? And what are the therapeutic implications of physiological pS129?

Lastly, important physiological roles for αS have been described in the literature, ranging from synaptic vesicle recycling, synaptic vesicle clustering, neurotransmitter release, and SNARE assembly [81] to the formation of synaptic protein condensates [82] and DNA repair [83]. Some of these functions have not been characterized in vivo in animals lacking αS. It will be interesting to systematically study the processes first in αS-depleted mice and then to examine the potential contribution of pS129 in each process leveraging S129A^KI (and possibly S129D^KI) mice. As pS129, the most prominent post-translational modification of αS, seems to be linked to the protein’s synaptic function [18,19], it seems prudent to study both aspects together.

Highlights.

• There is growing evidence that in the absence of pathology, serine-129 phosphorylation of α-synuclein (αS) serves the purpose of fine-tuning αS’s synaptic function.

• We posit that pS129 has a dual role in health and disease: a physiological role in regulating αS biology, and a pathological role related to the deposition of insoluble αS into Lewy bodies.

• We further propose that there is no direct conversion of physiological to pathological pS129. Instead, physiological pS129 is driven by a pathway that involves neuronal activity, calcium, calcineurin, Plk2, and PP2A, and is readily reversible. Pathological pS129 likely accumulates because aggregated αS is easier for Plk2 to phosphorylate and/or more difficult for PP2A to dephosphorylate than native αS.