alpha-Synuclein forms a complex with transcription factor Elk-1

alpha-Synuclein has been identified as a component of Lewy bodies in Parkinson's disease and diffuse Lewy body disease, and glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA). To explore the role of alpha-synuclein in the pathogenesis, we searched for molecules interacting with alpha-synuclein and discovered that GCIs are stained by anti-Elk-1 antibody. To seek the role of Elk-1 in synucleinopathies, we cotransfected alpha-synuclein and Elk-1 to cultured cells, and found small granular structure complexes where the two molecules colocalized. Moreover, alpha-synuclein and Elk-1 were co-immunoprecipitated from the cell lysates. For formation of the complex, the presence of both ETS and B-box domains of Elk-1 was required. Although there was no evidence of direct binding between alpha-synuclein and Elk-1, we discovered that alpha-synuclein and Elk-1 both bind to ERK-2, a MAP kinase. The effect of alpha-synuclein on the MAP kinase pathway was assessed using the Pathdetect system, which showed prominent attenuation of Elk-1 phosphorylation with alpha-synuclein, and especially A53T mutant. Our results suggest that alpha-synuclein reacts with the MAP kinase pathway, which might cause dysfunction of neurons and oligodendrocytes and lead to neurodegeneration in Parkinson's disease and MSA.     

Lipid interaction of alpha-synuclein during the metal-catalyzed oxidation in the presence of Cu2+ and H2O2

Alpha-synuclein co-exists with lipids in the Lewy bodies, a pathological hallmark of Parkinson's disease. Molecular interaction between alpha-synuclein and lipids has been examined by observing lipid-induced protein self-oligomerization in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. Lipids such as phosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and even arachidonic acid induced the self-oligomerization whereas phosphatidylcholine did not affect the protein. Because the oligomerizations occurred from critical micelle concentrations of the lipids, the self interaction of alpha-synuclein was shown to be a lipid-surface dependent phenomenon with head group specificity. By employing beta-synuclein and a C-terminally truncated alpha-synuclein (alpha-syn97), the head-group dependent self-oligomerization was demonstrated to occur preferentially at the N-terminal region while the fatty acid interaction leading to the protein self-association required the presence of the acidic C-terminus of alpha-synuclein. In the presence of Cu2+ and H2O2, phosphatidylinositol (PI), along with other acidic lipids, actually enhanced the metal-catalyzed oxidative self-oligomerization of alpha-synuclein. The dityrosine crosslink formation responsible for the PI-enhanced covalent self-oligomerization was more sensitive to variation of copper concentrations than that of H2O2 during the metal-catalyzed oxidation. The enhancement by PI was shown to be due to facilitation of copper localization to the protein because actual binding affinity between copper and alpha-synuclein increased from Kd of 44.7 microm to 5.9 microm in the presence of the lipid. Taken together, PI not only affects alpha-synuclein to be more self-interactive by providing the lipid surface, but also enhances the metal-catalyzed oxidative protein self-oligomerization by facilitating copper localization to the protein when the metal and H2O2 are provided. This observation therefore could be implicated in the formation of Lewy bodies as lipids and metal-catalyzed oxidative stress have been considered to be a part of pathological causes leading to the neurodegeneration.     

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.     

DJ-1 is present in a large molecular complex in human brain tissue and interacts with alpha-synuclein

DJ-1 is a ubiquitously expressed protein involved in various cellular processes including cell proliferation, RNA-binding, and oxidative stress. Mutations that result in loss of DJ-1 function lead to early onset parkinsonism in humans, and DJ-1 protein is present in pathological lesions of several tauopathies and synucleinopathies. In order to further investigate the role of DJ-1 in human neurodegenerative disease, we have generated novel polyclonal and monoclonal antibodies to human DJ-1 protein. We have characterized these antibodies and confirmed the pathological co-localization of DJ-1 with other neurodegenerative disease-associated proteins, as well as the decrease in DJ-1 solubility in disease tissue. In addition, we report the presence of DJ-1 in a large molecular complex (> 2000 kDa), and provide evidence for an interaction between endogenous DJ-1 and alpha-synuclein in normal and diseased tissue. These findings provide new avenues towards the study of DJ-1 function and how loss of its activity may lead to parkinsonism. Furthermore, our results provide further evidence for the interplay between neurodegenerative disease-associated proteins.     

α - synuclein and Parkinson's disease: the first roadblock

α-synuclein gene mutations are major underlying genetic defects known in familial juvenile onset Parkinson's disease (PD), and α-synuclein is a major constituent of Lewy Bodies, the pathological hallmark of PD. The normal cellular function of α-synuclein has been elusive, and its exact etiological mechanism in causing dopaminergic neuronal death in PD is also not clearly understood. Very recent reports now indicate that mutant or simply over-expressed α-synuclein could cause damage by interfering with particular steps of neuronal membrane traffic. α-synuclein selectively blocks endoplamic reticulum-to-Golgi transport, thus causing ER stress. A screen in a yeast revealed that α-synuclein toxicity could be suppressed by over-expression of the small GTPase Ypt1/Rab1, and that over-expression of the latter rescues neuron loss in invertebrate and mammalian models of α-synuclein-induced neurodegeneration. α-synuclein may also serve a chaperone function for the proper folding of synaptic SNAREs that are important for neurotransmitter release. We discuss these recent results and the emerging pathophysiological interaction of α-synuclein with components of neuronal membrane traffic.     

Effect of the overexpression of wild-type or mutant alpha-synuclein on cell susceptibility to insult

Mutations in alpha-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of alpha-synuclein overexpression on the response of cells to various insults. Wild-type alpha-synuclein and alpha-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type alpha-synuclein delayed cell death induced by serum withdrawal or H(2)O(2), but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP(+)). By contrast, wild-type alpha-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-trans-nonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type alpha-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant alpha-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant alpha-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal alpha-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.     

Amino acid sequence motifs and mechanistic features of the membrane translocation of alpha-synuclein

Many lines of evidence suggest that alpha-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of alpha-synuclein, and the mechanistic features of the phenomenon. We first showed that not only alpha-synuclein but also beta- and gamma-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of alpha-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. alpha-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of alpha-synuclein at 4 degrees C was comparable with the import observed at 37 degrees C. Furthermore, membrane translocation of alpha-synuclein was not significantly affected by treatment with inhibitors of endocytosis. These results suggest that the internalization of alpha-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis.     

Identification of the region of non-Aβ component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity

The non-beta-amyloid (Abeta) component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and alpha-synuclein both form beta-sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3-18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY-5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Circular dichroism indicates that none of the peptides displays beta-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce beta sheet, fragments NAC(8-18) and NAC(8-16) both form beta-sheet structure. Only NAC(8-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8-16 of NAC, equivalent to residues 68-76 in alpha-synuclein, comprise the region crucial for toxicity.     

Overexpression of alpha-synuclein in rat substantia nigra results in loss of dopaminergic neurons, phosphorylation of alpha-synuclein and activation of caspase-9: resemblance to pathogenetic changes in Parkinson's disease

To elucidate the role of alpha-synuclein in the pathogenesis of Parkinson's disease, both human alpha-synuclein transgenic mice and targeted overexpression of human alpha-synuclein in rat substantia nigra using viral vector-based methods have been studied, however, little is known about the pathogenetic changes of dopaminergic neuron loss. Therefore, it is necessary to address whether the pathogenetic changes in brains with Parkinson's disease are recapitulated in these models. Here, we used the recombinant adeno-associated viral (rAAV) vector system for human alpha-synuclein gene transfer to rat substantia nigra and observed approximately 50% loss of dopaminergic neurons at 13 weeks after infection, which was comparably slower than the progression of neurodegeneration reported in other studies. In the slower progression of neurodegeneration, we identified several important features in common with the pathogenesis of Parkinson's disease, such as phosphorylation of alpha-synuclein at Ser129 and activation of caspase-9. Both findings were also evident in cortical tissues overexpressing alpha-synuclein via rAAV. Our results indicate that overexpression of alpha-synuclein via rAAV apparently recapitulates several important features of brains with Parkinson's disease and dementia with Lewy bodies, and thus alpha-synucleinopathy described here is likely to be an ideal model for the study of the pathogenesis of Parkinson's disease and dementia with Lewy bodies.     

alpha-Synuclein, oxidative stress and apoptosis from the perspective of a yeast model of Parkinson's disease

The neuronal protein α-synuclein (α-syn) has been suggested to be one of the factors linked to Parkinson's disease (PD). Several organisms, including the rat, mouse, worm, and fruit fly, are being used to study α-syn pathobiology. A new model organism was recently added to this armamentarium: the budding yeast Saccharomyces cerevisiae . The yeast system recapitulates many of the findings made with higher eukaryotes. For example, yeast cells expressing α-syn accumulate lipid droplets, have vacuolar/lysosomal defects, and exhibit markers of apoptosis, including the externalization of phosphatidylserine, the release of cytochrome c , and the accumulation of reactive oxygen species. This MiniReview focuses on the mechanisms by which α-syn induces oxidative stress and the mechanisms by which yeast cells respond to this stress. Three classes of therapeutics are discussed.     

The ubiquitin ligase E6-AP promotes degradation of α-synuclein

Parkinson's disease (PD) is a common neurodegenerative disorder caused mainly because of the loss of dopaminergic neurons in the substantia nigra. Protein inclusions called Lewy bodies are the most common pathological hallmark of PD and other synucleinopathies. Because the main component of these inclusions is α-synuclein, aggregation of this protein is thought to be a key pathogenic event in this disease. In the present investigation we report that E6 associated protein (E6-AP), a HECT (homologous to E6-AP C-terminus) domain ubiquitin ligase is a component of Lewy bodies in post-mortem PD brain. In the cell culture model, we demonstrate that endogenous E6-AP colocalizes with α-synuclein in juxtanuclear aggregates. E6-AP is also recruited to the centrosome upon inhibition of the proteasome function suggesting its involvement in the degradation of misfolded proteins. Over-expression of E6-AP enhances the degradation of wild type as well as the mutant forms of α-synuclein in a proteasome-dependent manner. E6-AP also promotes the degradation of the more toxic oligomeric forms of α-synuclein. Our data suggests that E6-AP is involved in the clearance of α-synuclein.     

Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro

Parkinson's disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha-synuclein (alpha-syn), the protein mutated in some patients with familial PD. The mechanisms of alpha-syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave alpha-syn in vitro. Calpain I cleaved wild-type alpha-syn predominantly after amino acid 57 and within the non-amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized alpha-syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild-type alpha-syn after amino acid 57, this did not occur in mutant A53T alpha-syn. This paucity of proteolysis could increase the stability of A53T alpha-syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild-type alpha-syn. However, once alpha-syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of alpha-syn by cleaving at aberrant sites within the C-terminal region. Elucidating the role of calpain I in the proteolytic processing of alpha-syn in normal and diseased brains may clarify mechanisms of neurodegenerative alpha-synucleinopathies.     

Alpha-synuclein and polyunsaturated fatty acids promote clathrin-mediated endocytosis and synaptic vesicle recycling

α-Synuclein (αS) is an abundant neuronal cytoplasmic protein implicated in Parkinson's disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, αS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in αS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity. We now report that αS acts with PUFAs to enhance the internalization of the membrane-binding dye, FM 1-43. Specifically, αS expression coupled with exposure to physiological levels of certain PUFAs enhanced clathrin-mediated endocytosis in neuronal and non-neuronal cultured cells. Moreover, αS expression and PUFA-enhanced basal and -evoked synaptic vesicle (SV) endocytosis in primary hippocampal cultures of wild type (wt) and genetically depleted αS mouse brains. We suggest that αS and PUFAs normally function in endocytic mechanisms and are specifically involved in SV recycling upon neuronal stimulation.     

Regulated interactions of the norepineprhine transporter by the actin and microtubule cytoskeletons

One role of the actin cytoskeleton is to maintain the structural morphology and activity of the pre-synaptic terminal. We sought to determine if the actin cytoskeleton plays a role in regulating interactions between the norepinephrine transporter (NET) and alpha-Synuclein (α-Syn), two proteins expressed in the pre-synaptic terminal. In cells transfected with either 0.5 μg/mL or 3 μg/mL of α-Syn and 1 μg/mL of NET DNA, treatment with cytochalasin D, an actin depolymerizing agent, caused a dose-dependent decrease and increase, respectively, in [³H]-NE uptake. Protein interactions between NET, β-actin, and α-Syn were modified, along with levels of surface transporters. Treatment of primary brainstem neurons and frontal cortex synaptosomes with cytochalasin D caused a 115% and 28% increase, respectively, in NET activity. Depolymerization of both actin and microtubules did not alter NET activity in cells with 0.5 μg/mL α-Syn, but caused an increase in [³H]-NE uptake in cells transfected with 3 μg/mL of α-Syn and primary neurons. This is the first direct demonstration of NET activity being regulated via actin and modulated by interactions with α-Syn.     

Inducible over-expression of wild type α-synuclein in human neuronal cells leads to caspase-dependent non-apoptotic death

Alpha-synuclein (ASYN) is central in Parkinson's disease pathogenesis. Converging pieces of evidence suggest that the levels of ASYN expression play a critical role in both familial and sporadic Parkinson's disease. To elucidate the mechanism underlying wild type (WT) ASYN-mediated neurotoxicity, we have generated a novel Tet-Off SHSY-5Y cell line, conditionally expressing WT ASYN. Induction of human WT ASYN in retinoic acid-differentiated SHSY-5Y cells leads to accumulation of soluble ASYN oligomers, in the absence of inclusions, and to gradual cellular degeneration. Morphologically, the death observed is non-apoptotic. Caspases other than caspase 3, including caspase 9, are activated and caspase inhibition diminishes death by acting at a point upstream of cytochrome c release. Application of Scyllo-inositol, an oligomer-stabilizing compound, prevents neuronal death in this model. These findings are consistent with a model in which oligomeric ASYN triggers the initial activation of the apoptotic pathway, which is however blocked downstream of the mitochondrial checkpoint, thus leading to a death combining in a unique fashion both apoptotic and non-apoptotic features. This novel inducible cell model system may prove valuable in the deciphering of WT ASYN-induced pathogenic effects and in the assessment and screening of potential therapeutic strategies.     

Seeding induced by α-synuclein oligomers provides evidence for spreading of α-synuclein pathology

Lewy bodies, α-synuclein (α-syn) immunopositive intracellular deposits, are the pathological hallmark of Parkinson's disease (PD). Interestingly, Lewybody-like structures have been identified in fetal tissue grafts about one decade after transplantation into the striatum of PD patients. One possible explanation for the accelerated deposition of α-syn in the graft is that the aggregation of α-syn from the host tissue to the graft is spread by a prion disease-like mechanism. We discuss here an in vitro model which might recapitulate some aspects of disease propagation in PD. We found here that in vitro -generated α-syn oligomers induce transmembrane seeding of α-syn aggregation in a dose- and time-dependent manner. This effect was observed in primary neuronal cultures as well as in neuronal cell lines. The seeding oligomers were characterized by a distinctive lithium dodecyl sulfate-stable oligomer pattern and could be generated in a dynamic process out of pore-forming oligomers. We propose that α-syn oligomers form as a dynamic mixture of oligomer types with different properties and that α-syn oligomers can be converted into different types depending on the brain milieu conditions. Our data indicate that extracellular α-syn oligomers can induce intracellular α-syn aggregation, therefore we hypothesize that a similar mechanism might lead to α-syn pathology propagation.     

The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease

Intracellular inclusions containing alpha-synuclein (alpha SN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). In order to identify disease-associated changes of alpha SN, this study compared the levels, solubility and molecular weight species of alpha SN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent-soluble and detergent-insoluble alpha SN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of alpha SN in the detergent-soluble fraction of brain samples from pons and white matter but detergent-insoluble alpha SN was not detected. There was an inverse correlation between buffered saline-soluble and detergent-soluble levels of alpha SN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of alpha SN between grey and white matter in disease may result from different processing of alpha SN in neurons compared with oligodendrocytes. Highly insoluble alpha SN is not involved in the pathogenesis of MSA. It is therefore possible that buffered saline-soluble or detergent-soluble forms of alpha SN are involved in the pathogenesis of other alpha SN-related diseases.