Ubiquitination of alpha-synuclein

Filamentous alpha-synuclein depositions are the defining hallmarks of a subset of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. We previously reported that alpha-synuclein in those brains are extensively phosphorylated at Ser129 [Fujiwara et al. (2002) Nat. Cell Biol. 4, 160-164] and also partially ubiquitinated [Hasegawa et al. (2002) J. Biol. Chem. 277, 49071-49076]. Here, we investigate ubiquitination of alpha-synuclein in vitro and in vivo and report the ubiquitination sites and the effects of familial PD-linked mutations, phosphorylation, and fibril formation on ubiquitination. Protein-sequence analysis revealed that Lys21, Lys23, Lys32, and Lys34 within the repeats in the amino-terminal half are liable to ubiquitination in vitro. A site-directed mutagensis study confirmed that these are the major ubiquitination sites. A53T and A30P mutations had no significant effect on ubiquitination. Similarly, phosphorylation of alpha-synuclein at Ser129 did not affect ubiquitination. Notably, we show that assembled, filamentous alpha-synuclein is less ubiquitinated than the soluble form and that the major ubiquitination sites are localized to Lys6, Lys10, and Lys12 at the amino-terminal region of filamentous alpha-synuclein. Furthermore, we successfully detected ubiquitination of alpha-synuclein in 293T cells by cotransfection with alpha-synuclein and ubiquitin. The in vivo ubiquitination sites were found to be identical to those in filamentous alpha-synuclein. PD-linked mutations and phosphorylation at Ser129 had no effects on ubiquitination of alpha-synuclein in vivo. These data may have implications for the mechanisms of the formation of alpha-synuclein deposits in alpha-synucleinopathy brains.     

Proteomics analysis identifies phosphorylation-dependent alpha-synuclein protein interactions

Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.     

Ubiquitination of alpha-synuclein and autophagy in Parkinson's disease

alpha-Synuclein is mutated in Parkinson's disease (PD) and is found in cytosolic inclusions, called Lewy bodies, in sporadic forms of the disease. A fraction of alpha-synuclein purified from Lewy bodies is monoubiquitinated, but the role of this monoubiquitination has been obscure. We now review recent data indicating a role of alpha-synuclein monoubiquitination in Lewy body formation and implicating the autophagic pathway in regulating these processes. The E3 ubiquitin-ligase SIAH is present in Lewy bodies and monoubiquitinates alpha-synuclein at the same lysines that are monoubiquitinated in Lewy bodies. Monoubiquitination by SIAH promotes the aggregation of alpha-synuclein into amorphous aggregates and increases the formation of inclusions within dopaminergic cells. Such effect is observed even at low monoubiquitination levels, suggesting that monoubiquitinated alpha-synuclein may work as a seed for aggregation. Accumulation of monoubiquitinated alpha-synuclein and formation of cytosolic inclusions is promoted by autophagy inhibition and to a lesser extent by proteasomal and lysosomal inhibition. Monoubiquitinated alpha-synuclein inclusions are toxic to cells and recruit PD-related proteins, such as synphilin-1 and UCH-L1. Altogether, the new data indicate that monoubiquitination might play an important role in Lewy body formation. Decreasing alpha- synuclein monoubiquitination, by preventing SIAH function or by stimulating autophagy, constitutes a new therapeutic strategy for Parkinson's disease.     

Ubiquitination of alpha-synuclein in Lewy bodies is a pathological event not associated with impairment of proteasome function

Lewy bodies are intracellular fibrillar inclusions composed of alpha-synuclein. They constitute the pathological hallmark of Parkinson's disease, dementia with Lewy bodies, and other neurodegenerative diseases. Although the majority of Lewy bodies are stained for ubiquitin by immunohistochemistry, the substrate for this modification is poorly understood. Insoluble, urea-soluble alpha-synuclein was separated from soluble fractions and subjected to two-dimensional gel electrophoresis to further characterize pathogenic alpha-synuclein species from disease brains. By using this approach, we found that in sporadic Lewy body diseases a highly modified, disease-associated 22-24-kDa alpha-synuclein species is ubiquitinated. Conjugation of one, two, and, to a lesser extent, three ubiquitins was detected. This 22-24-kDa alpha-synuclein species represents partly phosphorylated protein. Furthermore, no generalized impairment of the proteolytic activity of the proteasome was detected in brain regions with Lewy body pathology. Because unmodified alpha-synuclein is degraded by the proteasome in a ubiquitin-independent manner, these data suggest that accumulation of modified 22-24-kDa alpha-synuclein is a disease-specific event which may overwhelm the proteolytic system, leading to aberrant ubiquitination. Accordingly, carboxyl-terminal-truncated alpha-synuclein, presumably the result of aberrant proteolysis, is found only in association with alpha-synuclein aggregates.     

Serine 129 phosphorylation of alpha-synuclein induces unfolded protein response-mediated cell death

alpha-Synuclein is a major protein component deposited in Lewy bodies and Lewy neurites that is extensively phosphorylated at Ser(129), although its role in neuronal degeneration is still elusive. In this study, several apoptotic pathways were examined in alpha-synuclein-overexpressing SH-SY5Y cells. Following the treatment with rotenone, a mitochondrial complex I inhibitor, wild type alpha-synuclein-overexpressing cells demonstrated intracellular aggregations, which shared a number of features with Lewy bodies, although cells overexpressing the S129A mutant, in which phosphorylation at Ser(129) was blocked, showed few aggregations. In wild typealpha-synuclein cells treated with rotenone, the proportion of phosphorylated alpha-synuclein was about 1.6 times higher than that of untreated cells. Moreover, induction of unfolded protein response (UPR) markers was evident several hours before the induction of mitochondrial disruption and caspase-3 activation. Eukaryotic initiation factor 2alpha, a member of the PERK pathway family, was remarkably activated at early phases. On the other hand, the S129A mutant failed to activate UPR. Casein kinase 2 inhibitor, which decreased alpha-synuclein phosphorylation, also reduced UPR activation. The alpha-synuclein aggregations were colocalized with a marker for the endoplasmic reticulum-Golgi intermediate compartment. Taken together, it seems plausible that alpha-synuclein toxicity is dependent on the phosphorylation at Ser(129) that induces the UPRs, possibly triggered by the disturbed endoplasmic reticulum-Golgi trafficking.     

Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed alpha-synuclein expression in transfected cell lines. In pulse-chase experiments alpha-synuclein appeared to be stable over long periods (t((1)/(2)) 54 h) and no endoproteolytic processing was observed. alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of alpha-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that alpha-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of alpha-synuclein is regulated by phosphorylation/dephosphorylation.     

Casein kinase II-mediated phosphorylation regulates alpha-synuclein/synphilin-1 interaction and inclusion body formation

Alpha-synuclein is a phosphoprotein that accumulates as a major component of Lewy bodies in the brains of patients with Parkinson disease. Synphilin-1, which is also present in Lewy bodies, binds with alpha-synuclein and forms cytoplasmic inclusions in transfected cells. Yet the molecular determinants of this protein-protein interaction are unknown. Here we report that casein kinase II (CKII) phosphorylates synphilin-1 and that the beta subunit of this enzyme complex binds to synphilin-1. Additionally, both CKII alpha and beta subunits are present within cytoplasmic inclusions in cells that overexpress synphilin-1. Notably, the interaction between synphilin-1 and alpha-synuclein is markedly dependent on phosphorylation. Inhibition of CKII activity by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole blocks the binding between these two proteins and significantly reduces the percentage of cells that contain eosinophilic cytoplasmic inclusions. Mutation of the major CKII phosphorylation site in alpha-synuclein (S129A) has no significant impact on the binding between alpha-synuclein and synphilin-1 or on the formation of synphilin-1/alpha-synuclein-positive inclusions. These data suggest that the CKII-mediated phosphorylation of synphilin-1 rather than that of alpha-synuclein is critical in modulating their tendency to aggregate into inclusions. These observations collectively indicate that a ubiquitous post-translational modification such as phosphorylation can regulate inclusion body formation in the context of alpha-synuclein and synphilin-1 interaction.     

Lipid droplet binding and oligomerization properties of the Parkinson's disease protein alpha-synuclein.

alpha-Synuclein is a major component of the fibrillary lesion known as Lewy bodies and Lewy neurites that are the pathologic hallmarks of Parkinson's disease (PD). In addition, point mutations in the alpha-synuclein gene imply alpha-synuclein dysfunction in the pathology of inherited forms of PD. alpha-Synuclein is a member of a family of proteins found primarily in the brain and is concentrated within presynaptic terminals. Here, we address the localization and membrane binding characteristics of wild type and PD mutants of alpha-synuclein in cultured cells. In cells treated with high concentrations of fatty acids, wild type alpha-synuclein accumulated on phospholipid monolayers surrounding triglyceride-rich lipid droplets and was able to protect stored triglycerides from hydrolysis. PD mutant synucleins showed variable distributions on lipid droplets and were less effective in regulating triglyceride turnover. Chemical cross-linking demonstrated that synuclein formed small oligomers within cells, primarily dimers and trimers, that preferentially associated with lipid droplets and cell membranes. Our results suggest that the initial phases of synuclein aggregation may occur on the surfaces of membranes and that pathological conditions that induce cross-linking of synuclein may enhance the propensity for subsequent synuclein aggregation.     

A Highlights from MBoC Selection: Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.     

Evidence for a partially folded intermediate in alpha-synuclein fibril formation

Intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) of alpha-synuclein are hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple systemic atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into such filamentous inclusions remain unknown. An intriguing aspect of this problem is that alpha-synuclein is a natively unfolded protein, with little or no ordered structure under physiological conditions. This raises the question of how an essentially disordered protein is transformed into highly organized fibrils. In the search for an answer to this question, we have investigated the effects of pH and temperature on the structural properties and fibrillation kinetics of human recombinant alpha-synuclein. Either a decrease in pH or an increase in temperature transformed alpha-synuclein into a partially folded conformation. The presence of this intermediate is strongly correlated with the enhanced formation of alpha-synuclein fibrils. We propose a model for the fibrillation of alpha-synuclein in which the first step is the conformational transformation of the natively unfolded protein into the aggregation-competent partially folded intermediate.     

Phosphorylated alpha-synuclein is ubiquitinated in alpha-synucleinopathy lesions

alpha-Synuclein is one of the major components of intracellular fibrillary aggregates in the brains of a subset of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and Hallervorden-Spatz disease, which are referred to as alpha-synucleinopathies. We have shown previously (Fujiwara, H., Hasegawa, M., Dohmae, N., Kawashima, A., Masliah, E., Goldberg, M. S., Shen, J., Takio, K., and Iwatsubo, T. (2002) Nat. Cell Biol. 4, 160-164) that alpha-synuclein deposited in synucleinopathy brains is extensively phosphorylated at Ser-129 and migrates at 15 kDa. Here we examined the biochemical characteristics of the additional, higher molecular mass species of phosphorylated alpha-synuclein-positive polypeptides that also are recovered in the Sarkosyl-insoluble fraction of synucleinopathy and migrate at about 22 and 29 kDa. These 22 and 29 kDa bands were positive for three different anti-ubiquitin antibodies and comigrated perfectly with in vitro ubiquitinated alpha-synuclein that may correspond to mono- and diubiquitinated alpha-synuclein, respectively. Furthermore, cyanogen bromide cleavage of the 22 and 29 kDa polypeptides shifted the mobility to 19 and 26 kDa, respectively, and they retained immunoreactivity for both ubiquitin and alpha-synuclein. Finally, protein sequence analysis showed that the 19 kDa band contained two amino-terminal sequences of alpha-synuclein and ubiquitin. These results strongly suggest that phosphorylated alpha-synuclein is targeted to mono- and diubiquitination in synucleinopathy brains, which may have implications for mechanisms of these diseases.     

Relationship between alpha synuclein phosphorylation, proteasomal inhibition and cell death: relevance to Parkinson's disease pathogenesis

Alpha synuclein can be phosphorylated at serine129 (P-S129), and the presence of highly phosphorylated α-synuclein in Lewy bodies suggests changes to its phosphorylation status has an important pathological role. We demonstrate that the kinase(s) responsible for α-synuclein S129 phosphorylation is constitutively active in SH-SY5Y cells and involves casein kinase 2 activity. Increased oxidative stress or proteasomal inhibition caused significant elevation of P-S129 α-synuclein levels. Under these conditions, similar increases in P-S129 α-synuclein were found in both sodium dodecyl sulphate lysates and Triton extracts indicating the phosphorylated protein was soluble and did not lead to aggregation. The rate of S129 phosphorylation was increased in response to proteasomal inhibition indicating a higher activity of the relevant kinase. Cells expressing the phosphorylation mimic, S129D α-synuclein increased cell death and enhanced sensitivity to epoxomycin exposure. Proteasomal inhibition markedly decreased S129D α-synuclein turnover suggesting proteasomal inhibition leads to the accumulation of P-S129 α-synuclein through an increase in the kinase activity and a decrease in protein turnover resulting in increased cell death. We conclude that S129 phosphorylation is toxic to dopaminergic cells and both the levels of S129 phosphorylated protein and its toxicity are increased with proteasomal inhibition emphasising the interdependence of these pathways in Parkinson's disease pathogenesis.     

Phosphorylation of α-synuclein protein at Ser-129 reduces neuronal dysfunction by lowering its membrane binding property in Caenorhabditis elegans

α-Synuclein is causative for autosomal dominant familial Parkinson disease and dementia with Lewy bodies, and the phosphorylation of α-synuclein at residue Ser-129 is a key posttranslational modification detected in Parkinson disease/dementia with Lewy bodies lesions. However, the role of Ser-129 phosphorylation on the pathogenesis of Parkinson disease/dementia with Lewy bodies remains unclear. Here we investigated the neurotoxicity of Ser-129-substituted α-synuclein in the transgenic Caenorhabditis elegans (Tg worm) model of synucleinopathy. Tg worms pan-neuronally overexpressing nonphosphorylatable (S129A) α-synuclein showed severe defects including motor dysfunction, growth retardation, and synaptic abnormalities. In contrast, Tg worms expressing phosphorylation mimic (S129D) α-synuclein exhibited nearly normal phenotypes. Biochemical fractionation revealed that the level of membrane-bound α-synuclein was significantly increased in S129A-α-synuclein Tg worms, whereas S129D- as well as A30P-α-synuclein displayed lower membrane binding properties. Furthermore, A30P/S129A double mutant α-synuclein did not cause neuronal dysfunction and displayed low membrane binding property. In human neuroblastoma SH-SY5Y cells, localization of S129A-α-synuclein to membranes was significantly increased. Finally, gene expression profiling of S129A-Tg worms revealed a dramatic up-regulation of Daf-16/FOXO pathway genes, which likely act against the dysfunction caused by S129A-α-synuclein. These results imply a role of Ser-129 phosphorylation of α-synuclein in the attenuation of α-synuclein-induced neuronal dysfunction and downstream stress response by lowering the membrane binding property.     

Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease

Parkinson disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic-ubiquitinated inclusions (Lewy bodies). Mutations in alpha-synuclein (A53T, A30P) and parkin cause familial Parkinson disease. Both these proteins are found in Lewy bodies. The absence of Lewy bodies in patients with parkin mutations suggests that parkin might be required for the formation of Lewy bodies. Here we show that parkin interacts with and ubiquitinates the alpha-synuclein-interacting protein, synphilin-1. Co-expression of alpha-synuclein, synphilin-1 and parkin result in the formation of Lewy-body-like ubiquitin-positive cytosolic inclusions. We further show that familial-linked mutations in parkin disrupt the ubiquitination of synphilin-1 and the formation of the ubiquitin-positive inclusions. These results provide a molecular basis for the ubiquitination of Lewy-body-associated proteins and link parkin and alpha-synuclein in a common pathogenic mechanism through their interaction with synphilin-1.     

Role of cytochrome c as a stimulator of alpha-synuclein aggregation in Lewy body disease.

alpha-Synuclein is a major component of aggregates forming amyloid-like fibrils in diseases with Lewy bodies and other neurodegenerative disorders, yet the mechanism by which alpha-synuclein is intracellularly aggregated during neurodegeneration is poorly understood. Recent studies suggest that oxidative stress reactions might contribute to abnormal aggregation of this molecule. In this context, the main objective of the present study was to determine the potential role of the heme protein cytochrome c in alpha-synuclein aggregation. When recombinant alpha-synuclein was coincubated with cytochrome c/hydrogen peroxide, alpha-synuclein was concomitantly induced to be aggregated. This process was blocked by antioxidant agents such as N-acetyl-L-cysteine. Hemin/hydrogen peroxide similarly induced aggregation of alpha-synuclein, and both cytochrome c/hydrogen peroxide- and hemin/hydrogen peroxide-induced aggregation of alpha-synuclein was partially inhibited by treatment with iron chelator deferoxisamine. This indicates that iron-catalyzed oxidative reaction mediated by cytochrome c/hydrogen peroxide might be critically involved in promoting alpha-synuclein aggregation. Furthermore, double labeling studies for cytochrome c/alpha-synuclein showed that they were colocalized in Lewy bodies of patients with Parkinson's disease. Taken together, these results suggest that cytochrome c, a well known electron transfer, and mediator of apoptotic cell death may be involved in the oxidative stress-induced aggregation of alpha-synuclein in Parkinson's disease and related disorders.     

Challenges and complexities of alpha-synuclein toxicity: new postulates in unfolding the mystery associated with Parkinson's disease

The discovery of two missense mutations in alpha-synuclein gene and the identification of the alpha-synuclein as the major component of Lewy bodies and Lewy neurites have imparted a new direction in understanding Parkinson's disease. Now that alpha-synuclein has been implicated in several neurodegenerative disorders makes it increasingly clear that aggregation of alpha-synuclein is a hallmark feature in neurodegeneration. Although little has been learned about its normal function, alpha-synuclein appears to be associated with membrane phospholipids and may therefore participate in a number of cell signaling pathways. Here, we review the localization, structure, and function of alpha-synuclein and provide a new hypothesis on, (a) the disruption in the membrane binding ability of synuclein which may be the major culprit leading to the alpha-synuclein aggregation and (b) the complexity associated with nuclear localization of alpha-synuclein and its possible binding property to DNA. Further, we postulated the three possible mechanisms of synuclein induced neuronal degeneration in Parkinson's disease.     

Parkinson's disease-associated alpha-synuclein is more fibrillogenic than beta- and gamma-synuclein and cannot cross-seed its homologs

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in alpha-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes beta- and gamma-synuclein. alpha-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for beta- or gamma-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between alpha- and beta-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of alpha-, beta-, and gamma- synuclein show the same natively unfolded structure. While over time alpha-synuclein forms the previously described fibrils, no fibrils could be detected for beta- and gamma-synuclein under the same conditions. Most importantly, beta- and gamma-synuclein could not be cross-seeded with alpha-synuclein fibrils. However, under conditions that drastically accelerate aggregation, gamma-synuclein can form fibrils with a lag phase roughly three times longer than alpha-synuclein. These results indicate that beta- and gamma-synuclein are intrinsically less fibrillogenic than alpha-synuclein and cannot form mixed fibrils with alpha-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to alpha-synuclein and are also abundant in brain.     

Prolyl-isomerase Pin1 accumulates in lewy bodies of parkinson disease and facilitates formation of alpha-synuclein inclusions

Parkinson disease (PD) is a relatively common neurodegenerative disorder that is characterized by the loss of dopaminergic neurons and by the formation of Lewy bodies (LBs), which are cytoplasmic inclusions containing aggregates of alpha-synuclein. Although certain post-translational modifications of alpha-synuclein and its related proteins are implicated in the genesis of LBs, the specific molecular mechanisms that both regulate these processes and initiate subsequent inclusion body formation are not yet well understood. We demonstrate in our current study, however, that the prolyl-isomerase Pin1 localizes to the LBs in PD brain tissue and thereby enhances the formation of alpha-synuclein immunoreactive inclusions. Immunohistochemical analysis of brain tissue from PD patients revealed that Pin1 localizes to 50-60% of the LBs that show an intense halo pattern resembling that of alpha-synuclein. By utilizing a cellular model of alpha-synuclein aggregation, we also demonstrate that, whereas Pin1 overexpression facilitates the formation of alpha-synuclein inclusions, dominant-negative Pin1 expression significantly suppresses this process. Consistent with these observations, Pin1 overexpression enhances the protein half-life and insolubility of alpha-synuclein. Finally, we show that Pin1 binds synphilin-1, an alpha-synuclein partner, via its Ser-211-Pro and Ser-215-Pro motifs, and enhances its interaction with alpha-synuclein, thus likely facilitating the formation of alpha-synuclein inclusions. These results indicate that Pin1-mediated prolyl-isomerization plays a pivotal role in a post-translational modification pathway for alpha-synuclein aggregation and in the resultant Lewy body formations in PD.     

Polo-like Kinase 2 (PLK2) Phosphorylates ��-Synuclein at Serine 129 in Central Nervous System

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of α-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 α-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited α-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 α-synuclein phosphorylation in central nervous system.The importance of α-synuclein to the pathogenesis of Parkinson disease (PD)⁴ and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1–3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4–6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7–9), which increase levels of α-synuclein with no alteration in sequence, also cause PD or DLB.α-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11, 12). Phosphorylation at tyrosine residues has been observed by some investigators (13, 14) but not by others (10–12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress α-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of α-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate α-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an α-synuclein kinase. The ability of PLK2 to directly phosphorylate α-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates α-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brain support a role for PLK2 as an α-synuclein kinase in vivo.