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Polo-like kinase 2 inhibition reduces serine-129 phosphorylation of physiological nuclear alpha-synuclein but not of the aggregated alpha-synuclein.

ABSTRACT: Accumulation of aggregated alpha-synuclein (α-syn) is believed to play a pivotal role in the pathophysiology of Parkinson's disease (PD) and other synucleinopathies. As a key constituent of Lewy pathology, more than 90% of α-syn in Lewy bodies is phosphorylated at serine-129 (pS129) and hence, it is used extensively as a marker for α-syn pathology. However, the exact role of pS129 remains controversial and the kinase(s) responsible for the phosphorylation have yet to be determined. In this study, we investigated the effect of Polo-like kinase 2 (PLK2) inhibition on formation of pS129 using an ex vivo organotypic brain slice model of synucleinopathy. Our data demonstrated that PLK2 inhibition has no effect on α-syn aggregation, pS129 or inter-neuronal spreading of the aggregated α-syn seen in the organotypic slices. Instead, PLK2 inhibition reduced the soluble pS129 level in the nuclei. The same finding was replicated in an in vivo mouse model of templated α-syn aggregation and in human dopaminergic neurons, suggesting that PLK2 is more likely to be involved in S129-phosphorylation of the soluble physiological fraction of α-syn. We also demonstrated that reduction of nuclear pS129 following PLK2 inhibition for a short time before sample collection improves the signal-to-noise ratio when quantifying pS129 aggregate pathology.

SUBMITTER: Elfarrash S

PROVIDER: S-EPMC8494365 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Pramipexole reduces phosphorylation of α-synuclein at serine-129.

Project description:α-Synuclein is a central component of the pathogenesis of Parkinson's disease (PD). Phosphorylation at serine-129 represents an important post-translational modification and constitutes the major form of the protein in Lewy bodies. Several kinases have been implicated in the phosphorylation of α-synuclein. The targeting of kinase pathways as a potential to influence the pathogenesis of PD is an important focus of attention, given that mutations of specific kinases (LRRK2 and PINK1) are causes of familial PD. Pramipexole (PPX) is a dopamine agonist developed for the symptomatic relief of PD. Several in vitro and in vivo laboratory studies have demonstrated that PPX exerts neuroprotective properties in model systems of relevance to PD. The present study demonstrates that PPX inhibits the phosphorylation of α-synuclein and that this is independent of dopamine receptor activation. PPX blocks the increase in phosphorylated α-synuclein induced by inhibition of the ubiquitin proteasomal system. The phosphorylation of α-synuclein occurs in part at least through casein kinase 2, and PPX in turn reduces the phosphorylation of this enzyme, thereby inhibiting its activity. Thus, PPX decreases the phosphorylation of α-synuclein, and this mechanism may contribute to its protective properties in PD models.

| S-EPMC3779594 | biostudies-literature

CK1BP Reduces α-Synuclein Oligomerization and Aggregation Independent of Serine 129 Phosphorylation.

Project description:The pathological accumulation of α-Synuclein (α-Syn) is the hallmark of neurodegenerative α-synucleinopathies, including Parkinsons's disease (PD). In contrast to the mostly non-phosphorylated soluble α-Syn, aggregated α-Syn is usually phosphorylated at serine 129 (S129). Therefore, S129-phosphorylation is suspected to interfere with α-Syn aggregation. Among other kinases, protein kinase CK1 (CK1) is known to phosphorylate α-Syn at S129. We overexpressed CK1 binding protein (CK1BP) to inhibit CK1 kinase activity. Using Bimolecular Fluorescence Complementation (BiFC) in combination with biochemical methods, we monitored the S129 phosphorylation and oligomerization of α-Syn in HEK293T cells. We found that CK1BP reduced the overall protein levels of α-Syn. Moreover, CK1BP concomitantly reduced S129 phosphorylation, oligomerization and the amount of insoluble α-Syn. Analyzing different α-Syn variants including S129 mutations, we show that the effects of CK1BP on α-Syn accumulation were independent of S129 phosphorylation. Further analysis of an aggregating polyglutamine (polyQ) protein confirmed a phosphorylation-independent decrease in aggregation. Our results imply that the inhibition of CK1 activity by CK1BP might exert beneficial effects on NDDs in general. Accordingly, CK1BP represents a promising target for the rational design of therapeutic approaches to cease or at least delay the progression of α-synucleinopathies.

| S-EPMC8616157 | biostudies-literature

Genetic deletion of Polo-like kinase 2 reduces alpha-synuclein serine-129 phosphorylation in presynaptic terminals but not Lewy bodies.

Project description:Phosphorylation of alpha-synuclein at serine-129 is an important marker of pathologically relevant, aggregated forms of the protein in several important human diseases, including Parkinson's disease, Dementia with Lewy bodies, and Multiple system atrophy. Although several kinases have been shown to be capable of phosphorylating alpha-synuclein in various model systems, the identity of the kinase that phosphorylates alpha-synuclein in the Lewy body remains unknown. One member of the Polo-like kinase family, PLK2, is a strong candidate for being the Lewy body kinase. To examine this possibility, we have used a combination of approaches, including biochemical, immunohistochemical, and in vivo multiphoton imaging techniques to study the consequences of PLK2 genetic deletion on alpha-synuclein phosphorylation in both the presynaptic terminal and preformed fibril-induced Lewy body pathology in mouse cortex. We find that PLK2 deletion reduces presynaptic terminal alpha-synuclein serine-129 phosphorylation, but has no effect on Lewy body phosphorylation levels. Serine-129 mutation to the phosphomimetic alanine or the unphosphorylatable analog aspartate does not change the rate of cell death of Lewy inclusion-bearing neurons in our in vivo multiphoton imaging paradigm, but PLK2 deletion does slow the rate of neuronal death. Our data indicate that inhibition of PLK2 represents a promising avenue for developing new therapeutics, but that the mechanism of neuroprotection by PLK2 inhibition is not likely due to reducing alpha-synuclein serine-129 phosphorylation and that the true Lewy body kinase still awaits discovery.

| S-EPMC7948797 | biostudies-literature

Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system.

Project description:Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of alpha-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to alpha-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates alpha-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited alpha-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in alpha-synuclein phosphorylation in central nervous system.

| S-EPMC2631975 | biostudies-literature

Dynamic reversibility of α-synuclein serine-129 phosphorylation is impaired in synucleinopathy models

Project description:α-synuclein phosphorylation at serine-129 (pS129) is a widely used surrogate marker of pathology in Parkinson's disease and other synucleinopathies. However, we recently demonstrated that phosphorylation of S129 is also a physiological activator of synaptic transmission. In a feed-forward fashion, neuronal activity triggers reversible pS129. Here, we show that Parkinson's disease-linked missense mutations in SNCA impact activity-dependent pS129. Under basal conditions, cytosol-enriched A30P, H50Q, and G51D mutant forms of α-synuclein exhibit reduced pS129 levels in rat primary cortical neurons. A53T pS129 levels are similar to wild-type, and E46K pS129 levels are higher. A30P and E46K mutants show impaired reversibility of pS129 after stimulation. For the engineered profoundly membrane-associated α-synuclein mutant '3K' (E35K+E46K+E61K), de-phosphorylation was virtually absent after blocking stimulation, implying that reversible pS129 is severely compromised. Importantly, pS129 excess resulting from proteasome inhibition is also associated with reduced reversibility - by neuronal inhibition, kinase inhibition, or phosphatase activation. Our findings suggest that perturbed pS129 dynamics are probably a shared characteristic of pathology-associated α-synuclein, with possible implications for synucleinopathy treatment and diagnosis.

| S-SCDT-10_15252-EMBR_202357145 | biostudies-other

Serine 129 phosphorylation reduces the ability of alpha-synuclein to regulate tyrosine hydroxylase and protein phosphatase 2A in vitro and in vivo.

Project description:Alpha-synuclein (a-Syn), a protein implicated in Parkinson disease, contributes significantly to dopamine metabolism. a-Syn binding inhibits the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Phosphorylation of TH stimulates its activity, an effect that is reversed by protein phosphatase 2A (PP2A). In cells, a-Syn overexpression activates PP2A. Here we demonstrate that a-Syn significantly inhibited TH activity in vitro and in vivo and that phosphorylation of a-Syn serine 129 (Ser-129) modulated this effect. In MN9D cells, a-Syn overexpression reduced TH serine 19 phosphorylation (Ser(P)-19). In dopaminergic tissues from mice overexpressing human a-Syn in catecholamine neurons only, TH-Ser-19 and TH-Ser-40 phosphorylation and activity were also reduced, whereas PP2A was more active. Cerebellum, which lacks excess a-Syn, had PP2A activity identical to controls. Conversely, a-Syn knock-out mice had elevated TH-Ser-19 phosphorylation and activity and less active PP2A in dopaminergic tissues. Using an a-Syn Ser-129 dephosphorylation mimic, with serine mutated to alanine, TH was more inhibited, whereas PP2A was more active in vitro and in vivo. Phosphorylation of a-Syn Ser-129 by Polo-like-kinase 2 in vitro reduced the ability of a-Syn to inhibit TH or activate PP2A, identifying a novel regulatory role for Ser-129 on a-Syn. These findings extend our understanding of normal a-Syn biology and have implications for the dopamine dysfunction of Parkinson disease.

| S-EPMC2878529 | biostudies-literature

Behavioral Deficits and Brain α-Synuclein and Phosphorylated Serine-129 α-Synuclein in Male and Female Mice Overexpressing Human α-Synuclein.

Project description:BackgroundAlpha-synuclein (α-syn) is involved in pathology of Parkinson's disease, and 90% of α-syn in Lewy bodies is phosphorylated at serine 129 (pS129 α-syn).ObjectiveTo assess behavior impairments and brain levels of α-syn and pS129 α-syn in mice overexpressing human α-syn under Thy1 promoter (Thy1-α-syn) and wild type (wt) littermates.MethodsMotor and non-motor behaviors were monitored, brain human α-syn levels measured by ELISA, and α-syn and pS129 α-syn mapped by immunohistochemistry.ResultsMale and female wt littermates did not show differences in the behavioral tests. Male Thy1-α-syn mice displayed more severe impairments than female counterparts in cotton nesting, pole tests, adhesive removal, finding buried food, and marble burying. Concentrations of human α-syn in the olfactory regions, cortex, nigrostriatal system, and dorsal medulla were significantly increased in Thy1-α-syn mice, higher in males than females. Immunoreactivity of α-syn was not simply increased in Thy1-α-syn mice but had altered localization in somas and fibers in a few brain areas. Abundant pS129 α-syn existed in many brain areas of Thy1-α-syn mice, while there was none or only a small amount in a few brain regions of wt mice. The substantia nigra, olfactory regions, amygdala, lateral parabrachial nucleus, and dorsal vagal complex displayed different distribution patterns between wt and transgenic mice, but not between sexes.ConclusionThe severer abnormal behaviors in male than female Thy1-α-syn mice may be related to higher brain levels of human α-syn, in the absence of sex differences in the altered brain immunoreactivity patterns of α-syn and pS129 α-syn.

| S-EPMC8577576 | biostudies-literature

Tyrosine 136 phosphorylation of α-synuclein aggregates in the Lewy body dementia brain: involvement of serine 129 phosphorylation by casein kinase 2.

Project description:Serine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replacement of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was significantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target.

| S-EPMC8590312 | biostudies-literature

Alpha-synuclein aggregation and Ser-129 phosphorylation-dependent cell death in oligodendroglial cells.

Project description:Multiple system atrophy is a neurodegenerative disorder characterized by accumulation of aggregated Ser-129-phosphorylated alpha-synuclein in oligodendrocytes. p25alpha is an oligodendroglial protein that potently stimulates alpha-synuclein aggregation in vitro. To model multiple system atrophy, we coexpressed human p25alpha and alpha-synuclein in the rat oligodendroglial cell line OLN-93 and observed a cellular response characterized by a fast retraction of microtubules from the cellular processes to the perinuclear region followed by a protracted development of apoptosis. This response was dependent on phosphorylation at Ser-129 in alpha-synuclein as demonstrated by site-directed mutagenesis. Treatment of the cells with the kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole that targets kinases like casein kinase 2, and polo-like kinases abrogated the toxicity. The polo-like kinase inhibitor BI 2536 caused apoptosis in the model. Ser-129 phosphorylation was linked to the formation of phosphorylated oligomers detectable by immunoblotting, and their formation was inhibited by 2-dimethylamino-4,5,6,7-tetrabromo-1H benzimidazole. The process of microtubule retraction was also dependent on aggregation as demonstrated by the protective effect of treating the cells with the specific peptide inhibitor of alpha-synuclein aggregation ASI1D and the non-selective inhibitors Congo Red and baicalein. The fast microtubule retraction was followed by the development of the apoptotic markers: activated caspase-3, phosphatidylserine externalization, nuclear condensation, and fragmentation. These markers could all be blocked by the inhibitors of phosphorylation, aggregation, and caspase-3. Hence, the model predicts that both Ser-129 phosphorylation and aggregation control the toxic alpha-syn pathway in oligodendroglial cells and may represent therapeutic intervention points in multiple system atrophy.

| S-EPMC2665075 | biostudies-literature

Effect of Ser-129 phosphorylation on interaction of α-synuclein with synaptic and cellular membranes.

Project description:In the healthy brain, less than 5% of α-synuclein (α-syn) is phosphorylated at serine 129 (Ser(P)-129). However, within Parkinson disease (PD) Lewy bodies, 89% of α-syn is Ser(P)-129. The effects of Ser(P)-129 modification on α-syn distribution and solubility are poorly understood. As α-syn normally exists in both membrane-bound and cytosolic compartments, we examined the binding and dissociation of Ser(P)-129 α-syn and analyzed the effects of manipulating Ser(P)-129 levels on α-syn membrane interactions using synaptosomal membranes and neural precursor cells from α-syn-deficient mice or transgenic mice expressing human α-syn. We first evaluated the recovery of the Ser(P)-129 epitope following either α-syn membrane binding or dissociation. We demonstrate a rapid turnover of Ser(P)-129 during both binding to and dissociation from synaptic membranes. Although the membrane binding of WT α-syn was insensitive to modulation of Ser(P)-129 levels by multiple strategies (the use of phosphomimic S129D and nonphosphorylated S129A α-syn mutants; by enzymatic dephosphorylation of Ser(P)-129 or proteasome inhibitor-induced elevation in Ser(P)-129; or by inhibition or stable overexpression of PLK2), PD mutant Ser(P)-129 α-syn showed a preferential membrane association compared with WT Ser(P)-129 α-syn. Collectively, these data suggest that phosphorylation at Ser-129 is dynamic and that the subcellular distribution of α-syn bearing PD-linked mutations, A30P or A53T, is influenced by the phosphorylation state of Ser-129.

| S-EPMC3195582 | biostudies-literature

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