Disorders characterized by α-synuclein (α-syn) accumulation, Lewy body formation and parkinsonism (and in some cases dementia) are collectively known as Lewy body diseases. The molecular mechanism(s) through which α-syn abnormally accumulates and contributes to neurodegeneration in these disorders remains unknown. Here, we provide an overview of current knowledge and prevailing hypotheses regarding the conformational, oligomerization and aggregation states of α-syn and their role in regulating α-syn function in health and disease. Understanding the nature of the various α-syn structures, how they are formed, and their relative contributions to α-syn-mediated toxicity may inform future studies aiming to develop therapeutic prevention and intervention.

Increasing evidence suggests that phosphorylation may play an important role in the oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity of ␣-synuclein (␣-syn) in Parkinson disease. Herein we demonstrate that ␣-syn is phosphorylated at S87 in vivo and within LBs. The levels of S87-P are increased in brains of transgenic (TG) models of synucleinopathies and human brains from Alzheimer disease (AD), LB disease (LBD), and multiple system atrophy (MSA) patients. Using antibodies against phosphorylated ␣-syn (S129-P and S87-P), a significant amount of immunoreactivity was detected in the membrane in the LBD, MSA, and AD cases but not in normal controls. In brain homogenates from diseased human brains and TG animals, the majority of S87-P ␣-syn was detected in the membrane fractions. A battery of biophysical methods were used to dissect the effect of S87 phosphorylation on the structure, aggregation, and membranebinding properties of monomeric ␣-syn. These studies demonstrated that phosphorylation at S87 expands the structure of ␣-syn, increases its conformational flexibility, and blocks its fibrillization in vitro. Furthermore, phosphorylation at S87, but not S129, results in significant reduction of ␣-syn binding to membranes. Together, our findings provide novel mechanistic insight into the role of phosphorylation at S87 and S129 in the pathogenesis of synucleinopathies and potential roles of phosphorylation in ␣-syn normal biology.

A better understanding of the molecular and cellular determinants that influence the pathology of Parkinson's disease (PD) is essential for developing effective diagnostic, preventative and therapeutic strategies to treat this devastating disease. A number of post-translational modifications to a-syn are present within the Lewy bodies in the brains of affected patients and transgenic models of PD and related disorders. However, whether disease-associated a-syn post-translational modifications promote or inhibit a-syn aggregation and neurotoxicity in vivo remains unknown. Herein, we summarize and discuss the major disease-associated post-translational modifications (phosphorylation, truncation and ubiquitination) and present our current understanding of the effect of these modifications on a-syn aggregation and toxicity. Elucidating the molecular mechanisms underlying post-translation modifications of a-syn and the consequences of such modifications on the biochemical, structural, aggregation and toxic properties of the protein is essential for unravelling the molecular basis of its function(s) in health and disease. Furthermore, the identification of the natural enzymes involved in regulating the post-translational modifications of a-synuclein will yield novel and more tractable therapeutic targets to treat PD and related synucleinopathies.

Phosphorylation of ␣-synuclein (␣-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating ␣-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate ␣-syn and ␤-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate ␣-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate ␤-syn at Ser-118, whereas no phosphorylation of ␥-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103-140) of ␣-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of ␣-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) ␣-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in ␣-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of ␣-syn and ␤-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies.Increasing evidence suggests that phosphorylation may play an important role in the oligomerization and fibrillogenesis (1), Lewy body formation (1, 2) and neurotoxicity of ␣-synuclein (␣-syn) 5 in vivo (3). The majority of ␣-syn within Lewy bodies (LBs) in diseased human brains and animal models of Parkinson disease (PD) and related synucleinopathies is phosphorylated at Ser-129 (Ser(P)-129) (1, 2, 4 -7). Although recent studies support the notion that phosphorylation at Ser-129 is related to pathology and blocks ␣-syn fibrillization in vitro (8, 9), the exact mechanisms by which phosphorylation at Ser-129 modulates ␣-syn aggregation and toxicity in vivo remain elusive. Unraveling the role of phosphorylation in modulating the physiological and pathogenic activities of ␣-syn requires identification of the kinases and phosphatases involved in regulating its phosphorylation in vivo.Several kinases that phosphorylate ␣-syn at serine and tyrosine residues, primarily in its C-terminal region, have been identified using in vitro kinase assays and co-transfection studies. Casein kinase I and II, G-protein-coupled receptor kinases (GRK1, GRK2, GRK5, and GRK6), and calmodulin-dependent kinase II (10 -12) phosphorylate ␣-syn at Ser-129. Ser-87 is the only residue outside the C-terminal region report...

Abnormal accumulation of proteinaceous intraneuronal inclusions called Lewy bodies (LBs) is the neurpathological hallmark of Parkinson’s disease (PD) and related synucleinopathies. These inclusions are mainly constituted of a presynaptic protein, α-synuclein (α-syn). Over the past decade, growing amounts of studies reported an aberrant accumulation of phosphorylated α-syn at the residue S129 (pS129) in the brain of patients suffering from PD, as well as in transgenic animal models of synucleinopathies. Whereas only a small fraction of α-syn (<4%) is phosphorylated in healthy brains, a dramatic accumulation of pS129 (>90%) has been observed within LBs, suggesting that this post-translational modification may play an important role in the regulation of α-syn aggregation, LBs formation and neuronal degeneration. However, whether phosphorylation at S129 suppresses or enhances α-syn aggregation and toxicity in vivo remains a subject of active debate. The answer to this question has important implications for understanding the role of phosphorylation in the pathogenesis of synucleinopathies and determining if targeting kinases or phosphatases could be a viable therapeutic strategy for the treatment of these devastating neurological disorders. In the present review, we explore recent findings from in vitro, cell-based assays and in vivo studies describing the potential implications of pS129 in the regulation of α-syn physiological functions, as well as its implication in synucleinopathies pathogenesis and diagnosis.

An increase in α-synuclein levels due to gene duplications/triplications or impaired degradation is sufficient to trigger its aggregation and cause familial Parkinson disease (PD). Therefore, lowering α-synuclein levels represents a viable therapeutic strategy for the treatment of PD and related synucleinopathies. Here, we report that Polo-like kinase 2 (PLK2), an enzyme up-regulated in synucleinopathy-diseased brains, interacts with, phosphorylates and enhances α-synuclein autophagic degradation in a kinase activity-dependent manner. PLK2-mediated degradation of α-synuclein requires both phosphorylation at S129 and PLK2/α-synuclein complex formation. In a rat genetic model of PD, PLK2 overexpression reduces intraneuronal human α-synuclein accumulation, suppresses dopaminergic neurodegeneration, and reverses hemiparkinsonian motor impairments induced by α-synuclein overexpression. This PLK2-mediated neuroprotective effect is also dependent on PLK2 activity and α-synuclein phosphorylation. Collectively, our findings demonstrate that PLK2 is a previously undescribed regulator of α-synuclein turnover and that modulating its kinase activity could be a viable target for the treatment of synucleinopathies.adeno-associated virus | animal model | serum inducible kinase P arkinson disease (PD) is a neurodegenerative disorder characterized by the progressive neuronal loss in different brain regions, including the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc) (1, 2). Pathologically, PD is characterized by the presence, in surviving DA neurons, of intracellular inclusions called Lewy Bodies (LBs) and Lewy Neurites (LNs) (3). These fibrillar aggregates are mainly composed of the presynaptic protein α-synuclein (α-syn) (4). Converging evidence from pathologic, genetic, biochemical, and biophysical studies supports the hypothesis that α-syn accumulation and misfolding play a central role in the pathogenesis of PD and related disorders, which are altogether referred to as synucleinopathies (5).Although α-syn is constitutively phosphorylated at low levels (<4%) at S129 (pS129) in normal brains (6-8), a clear accumulation of pS129 (>90%) is found in LBs (8, 9) and in the brain of animal models of synucleinopathies (7,(10)(11)(12). The roles of this major posttranslational modification in regulating α-syn physiology and LB formation and/or neurodegeneration in PD remain elusive.Recently, our group and others (13-15) demonstrated that Pololike kinase 2 (PLK2), a serine/threonine kinase playing a central role in cell division, oncogenesis, and synaptic regulation in the adult brain (16-19), efficiently phosphorylates α-syn, with an exclusive preference for the S129 site. Unlike other kinases that were reported to partially phosphorylate α-syn at S129, PLK2 phosphorylates α-syn with quantitative conversion in vitro (>95%) and in mammalian cell lines (13-15).Interestingly, PLK2 expression levels are significantly up-regulated in the midbrain of aged monkeys and correlate with increased pS129 levels in dopaminer...

Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates α-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that α-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with α-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 α-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces α-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of α-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating α-syn clearance and contribute to the pathogenesis of PD.

Background:A new SNCA mutation, H50Q, has been linked to familial Parkinson disease (PD). Results: The H50Q mutation does not affect the structure, membrane binding, or subcellular localization of ␣-Syn but alters its pathogenic properties. Conclusion: The H50Q mutation increases ␣-Syn aggregation, secretion, and extracellular toxicity. Significance: ␣-Syn mutations contribute to the pathogenesis of PD via multiple mechanisms.