Identification of human alpha-synuclein specific single chain antibodies

Parkinson's disease (PD) is a common neurodegenerative disease of unknown etiology. Evidence suggests a role for protein misfolding in disease pathogenesis. One pathologic feature observed in dopaminergic neurons is the intracytoplasmic eosinophilic inclusions known as Lewy bodies. One component of Lewy bodies, the presynaptic protein, alpha-synuclein forms oligomers and higher order aggregates and is proposed to be involved in dopaminergic neuronal death. In an effort to discriminate between alpha-synuclein conformational forms as well as design potential disruptors of pathogenic misfolding we panned a human phage antibody library for anti-synuclein single chain antibodies (scFvs). We identified six scFvs which recognize different conformers of alpha-synuclein in both an ELISA and Western blot analysis. These scFvs may further our understanding of alpha-synuclein's role in PD.     

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.     

Transgenic mice overexpressing tyrosine-to-cysteine mutant human alpha-synuclein: a progressive neurodegenerative model of diffuse Lewy body disease

Abnormal aggregation of human alpha-synuclein in Lewy bodies and Lewy neurites is a pathological hallmark of Parkinson disease and dementia with Lewy bodies. Studies have shown that oxidation and nitration of alpha-synuclein lead to the formation of stable dimers and oligomers through dityrosine cross-linking. Previously we have reported that tyrosine-to-cysteine mutations, particularly at the tyrosine 39 residue (Y39C), significantly enhanced alpha-synuclein fibril formation and neurotoxicity. In the current study, we have generated transgenic mice expressing the Y39C mutant human alpha-synuclein gene controlled by the mouse Thy1 promoter. Mutant human alpha-synuclein was widely expressed in transgenic mouse brain, resulting in 150% overexpression relative to endogenous mouse alpha-synuclein. At age 9-12 months, transgenic mice began to display motor dysfunction in rotarod testing. Older animals aged 15-18 months showed progressive accumulation of human alpha-synuclein oligomers, associated with worse motor function and cognitive impairment in the Morris water maze. By age 21-24 months, alpha-synuclein aggregates were further increased, accompanied by severe behavioral deficits. At this age, transgenic mice developed neuropathology, such as Lewy body-like alpha-synuclein and ubiquitin-positive inclusions, phosphorylation at Ser(129) of human alpha-synuclein, and increased apoptotic cell death. In summary, Y39C human alpha-synuclein transgenic mice show age-dependent, progressive neuronal degeneration with motor and cognitive deficits similar to diffuse Lewy body disease. The time course of alpha-synuclein oligomer accumulation coincided with behavioral and pathological changes, indicating that these oligomers may initiate protein aggregation, disrupt cellular function, and eventually lead to neuronal death.     

alpha-Synuclein implicated in Parkinson's disease catalyses the formation of hydrogen peroxide in vitro

Some rare inherited forms of Parkinson's disease (PD) are due to mutations in the gene encoding a 140-amino acid presynaptic protein called alpha-synuclein. In PD, and some other related disorders such as dementia with Lewy bodies, alpha-synuclein accumulates in the brain in the form of fibrillar aggregates, which are found inside the neuronal cytoplasmic inclusions known as Lewy bodies. By means of an electron spin resonance (ESR) spin trapping method, we show here that solutions of full-length alpha-synuclein, and a synthetic peptide fragment of alpha-synuclein corresponding to residues 61-95 (the so-called non-Abeta component or NAC), both liberate hydroxyl radicals upon incubation in vitro followed by the addition of Fe(II). We did not observe this property for the related beta- and gamma-synucleins, which are not found in Lewy bodies, and are not linked genetically to any neurodegenerative disorder. There is abundant evidence for the involvement of free radicals and oxidative stress in the pathogenesis of nigral damage in PD. Our new data suggest that the fundamental molecular mechanism underlying this pathological process could be the production of hydrogen peroxide by alpha-synuclein.     

Is malfunction of the ubiquitin proteasome system the primary cause of alpha-synucleinopathies and other chronic human neurodegenerative disease?

Neuropathological investigations have identified major hallmarks of chronic neurodegenerative disease. These include protein aggregates called Lewy bodies in dementia with Lewy bodies and Parkinson's disease. Mutations in the alpha-synuclein gene have been found in familial disease and this has led to intense focused research in vitro and in transgenic animals to mimic and understand Parkinson's disease. A decade of transgenesis has lead to overexpression of wild type and mutated alpha-synuclein, but without faithful reproduction of human neuropathology and movement disorder. In particular, widespread regional neuronal cell death in the substantia nigra associated with human disease has not been described. The intraneuronal protein aggregates (inclusions) in all of the human chronic neurodegenerative diseases contain ubiquitylated proteins. There could be several reasons for the accumulation of ubiquitylated proteins, including malfunction of the ubiquitin proteasome system (UPS). This hypothesis has been genetically tested in mice by conditional deletion of a proteasomal regulatory ATPase gene. The consequences of gene ablation in the forebrain include extensive neuronal death and the production of Lewy-like bodies containing ubiquitylated proteins as in dementia with Lewy bodies. Gene deletion in catecholaminergic neurons, including in the substantia nigra, recapitulates the neuropathology of Parkinson's disease.     

Biochemical characterization of the core structure of alpha-synuclein filaments

Intracellular filamentous aggregates comprised of alpha-synuclein such as Lewy bodies and glial cytoplasmic inclusions are the defining hallmarks of a subset of neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. We have analyzed biochemical and structural properties of alpha-synuclein filaments assembled in vitro or extracted from brains of patients with multiple system atrophy and found that both types of filaments are insoluble to detergents and partially resistant to proteinase K digestion. Immunoelectron microscopy and immunoblot analysis showed that both amino and carboxyl termini of alpha-synuclein in in vitro assembled filaments were degraded by proteinase K treatment, whereas the central portion of alpha-synuclein is resistant to proteinase K and retains filamentous structures. Protein sequencing and mass spectrometric analyses of the proteinase K-resistant, minimal fragment of 7 kDa revealed that amino acid residues 31-109 of alpha-synuclein constitute the core unit of the filaments. These observations suggest that the central half of the alpha-synuclein polypeptide, containing five tandem repeats as well as a part of the carboxyl-terminal acidic region, forms the core structure of alpha-synuclein filaments, which is coated by the amino- and carboxyl-terminal portions at the periphery.     

Monoubiquitylation of alpha-synuclein by seven in absentia homolog (SIAH) promotes its aggregation in dopaminergic cells

alpha-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of alpha-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. alpha-Synuclein is ubiquitylated in Lewy bodies, but the role of alpha-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates alpha-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates alpha-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished alpha-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates alpha-synuclein. Moreover, SIAH co-immunoprecipitated with alpha-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of alpha-synuclein by SIAH. Monoubiquitylation increased the aggregation of alpha-synuclein in vitro. At the electron microscopy level, monoubiquitylated alpha-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased alpha-synuclein aggregation in vivo as observed by increased formation of alpha-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for alpha-synuclein aggregation and Lewy body formation.     

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.     

Interaction between parkin and alpha-synuclein

A dramatic paradigm shift in understanding Parkinson's disease (PD) has emerged with implications for Alzheimer's disease (AD) because: (1) Mutations in the alpha-synuclein (AS) gene cause familial PD, (2) Antibodies to AS detect Lewy bodies (LBs) and dystrophic Lewy neurites in PD, dementia with LBs (DLB), sporadic AD and the LB variant of AD (LBVAD), (3) Insoluble AS filaments are recovered from DLB brains and purified LBs, (4) Recombinant AS assembles into LB-like filaments and residues 71–82 are essential for filament assembly, (5) AS transgenic mice and flies develop a PD-like phenotype, (6) Cortical LBs detected with antibodies to AS correlate with dementia in PD, DLB and LBVAD, (7) Antibodies to AS detect LBs in 50% of familial AD, sporadic AD and Down's syndrome brains, (8) AS forms glial cytoplasmic inclusions (GCIs) in multiple system atrophy, (9) Epitopes throughout AS in LBs and GCIs, (10) Filamentous AS aggregates in LBs, GCIs and related lesions contain nitrated tyrosines, (11) Cells transfected with AS and treated with nitric oxide generators develop LB-like AS inclusions, (12) Bigenic mice overexpressing mutant human APP and AS show an augmentation in AS inclusions. Thus, neurodegenerative diseases characterized by AS pathologies are synucleinopathies, and the filamentous AS lesions in these disorders may result in part from oxidative/nitrative damage to AS. Abnormal interactions of brain proteins may underlie synucleinopathies and other neurodegenerative disorders. Acknowledgements:
Supported by NIA/NIH and Alzheimer's Association.     

Dityrosine cross-linking promotes formation of stable alpha -synuclein polymers. Implication of nitrative and oxidative stress in the pathogenesis of neurodegenerative synucleinopathies

Intracellular proteinaceous aggregates are hallmarks of many common neurodegenerative disorders, and recent studies have shown that alpha-synuclein is a major component of several pathological intracellular inclusions, including Lewy bodies in Parkinson's disease (PD) and glial cell inclusions in multiple system atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into filamentous inclusions remain unknown. Since oxidative and nitrative stresses are potential pathogenic mediators of PD and other neurodegenerative diseases, we asked if oxidative and/or nitrative events alter alpha-synuclein and induce it to aggregate. Here we show that exposure of human recombinant alpha-synuclein to nitrating agents (peroxynitrite/CO(2) or myeloperoxidase/H(2)O(2)/nitrite) induces formation of nitrated alpha-synuclein oligomers that are highly stabilized due to covalent cross-linking via the oxidation of tyrosine to form o,o'-dityrosine. We also demonstrate that oxidation and nitration of pre-assembled alpha-synuclein filaments stabilize these filaments to withstand denaturing conditions and enhance formation of SDS-insoluble, heat-stable high molecular mass aggregates. Thus, these data suggest that oxidative and nitrative stresses are involved in mechanisms underlying the pathogenesis of Lewy bodies and glial cell inclusions in PD and multiple system atrophy, respectively, as well as alpha-synuclein pathologies in other synucleinopathies.     

Amyloid fibril formation of alpha-synuclein is accelerated by preformed amyloid seeds of other proteins: implications for the mechanism of transmissible conformational diseases

Alpha-synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only alpha-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of alpha-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human alpha-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of alpha-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of alpha-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of alpha-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.     

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.     

Tubulin seeds alpha-synuclein fibril formation.

Increasing evidence suggests that alpha-synuclein is a common pathogenic molecule in several neurodegenerative diseases, particularly in Parkinson's disease. To understand alpha-synuclein pathology, we investigated molecules that interact with alpha-synuclein in human and rat brains and identified tubulin as an alpha-synuclein binding/associated protein. Tubulin co-localized with alpha-synuclein in Lewy bodies and other alpha-synuclein-positive pathological structures. Tubulin initiated and promoted alpha-synuclein fibril formation under physiological conditions in vitro. These findings suggest that an interaction between tubulin and alpha-synuclein might accelerate alpha-synuclein aggregation in diseased brains, leading to the formation of Lewy bodies.     

Formation and removal of alpha-synuclein aggregates in cells exposed to mitochondrial inhibitors.

Mitochondrial dysfunction has been associated with Parkinson's disease. However, the role of mitochondrial defects in the formation of Lewy bodies, a pathological hallmark of Parkinson's disease has not been addressed directly. In this report, we investigated the effects of inhibitors of the mitochondrial electron-transport chain on the aggregation of alpha-synuclein, a major protein component of Lewy bodies. Treatment with rotenone, an inhibitor of complex I, resulted in an increase of detergent-resistant alpha-synuclein aggregates and a reduction in ATP level. Another inhibitor of the electron-transport chain, oligomycin, also showed temporal correlation between the formation of aggregates and ATP reduction. Microscopic analyses showed a progressive evolution of small aggregates of alpha-synuclein to a large perinuclear inclusion body. The inclusions were co-stained with ubiquitin, 20 S proteasome, gamma-tubulin, and vimentin. The perinuclear inclusion bodies, but not the small cytoplasmic aggregates, were thioflavin S-positive, suggesting the amyloid-like conformation. Interestingly, the aggregates disappeared when the cells were replenished with inhibitor-free medium. Disappearance of aggregates coincided with the recovery of mitochondrial metabolism and was partially inhibited by proteasome inhibitors. These results suggest that the formation of alpha-synuclein inclusions could be initiated by an impaired mitochondrial function and be reversed by restoring normal mitochondrial metabolism.     

Parkin accumulation in aggresomes due to proteasome impairment

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and by the presence of ubiquitinated cytoplasmic inclusions known as Lewy bodies. Alpha-synuclein and Parkin are two of the proteins associated with inherited forms of PD and are found in Lewy bodies. Whereas numerous reports indicate the tendency of alpha-synuclein to aggregate both in vitro and in vivo, no information is available about similar physical properties for Parkin. Here we show that overexpression of Parkin in the presence of proteasome inhibitors leads to the formation of aggresome-like perinuclear inclusions. These eosinophilic inclusions share many characteristics with Lewy bodies, including a core and halo organization, immunoreactivity to ubiquitin, alpha-synuclein, synphilin-1, Parkin, molecular chaperones, and proteasome subunit as well as staining of some with thioflavin S. We propose that the process of Lewy body formation may be akin to that of aggresome-like structures. The tendency of wild-type Parkin to aggregate and form inclusions may have implications for the pathogenesis of sporadic PD.     

The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson's disease

Accumulation of misfolded proteins as insoluble aggregates occurs in several neurodegenerative diseases. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), alpha-synuclein (alpha S) accumulates in insoluble inclusions. To identify soluble alpha S oligomers that precede insoluble aggregates, we probed the cytosols of mesencephalic neuronal (MES) cells, normal and alpha S-transgenic mouse brains, and normal, PD, and DLB human brains. All contained highly soluble oligomers of alpha S whose detection was enhanced by delipidation. Exposure of living MES neurons to polyunsaturated fatty acids (PUFAs) increased alpha S oligomer levels, whereas saturated FAs decreased them. PUFAs directly promoted oligomerization of recombinant alphaS. Transgenic mice accumulated soluble oligomers with age. PD and DLB brains had elevated amounts of the soluble, lipid-dependent oligomers. We conclude that alpha S interacts with PUFAs in vivo to promote the formation of highly soluble oligomers that precede the insoluble alpha S aggregates associated with neurodegeneration.     

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.     

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.     

Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells

Mice treated with the psychostimulant methamphetamine (MA) showed the appearance of intracellular inclusions in the nucleus of medium sized striatal neurones and cytoplasm of neurones of the substantia nigra pars compacta but not in the frontal cortex. All inclusions contained ubiquitin, the ubiquitin activating enzyme (E1), the ubiquitin protein ligase (E3-like, parkin), low and high molecular weight heat shock proteins (HSP 40 and HSP 70). Inclusions found in nigral neurones stained for alpha-synuclein, a proteic hallmark of Lewy bodies that are frequently observed in Parkinson's disease and other degenerative disorders. However, differing from classic Lewy bodies, MA-induced neuronal inclusions appeared as multilamellar bodies resembling autophagic granules. Methamphetamine reproduced this effect in cultured PC12 cells, which offered the advantage of a simple cellular model for the study of the molecular determinants of neuronal inclusions. PC12 inclusions, similar to those observed in nigral neurones, were exclusively localized in the cytoplasm and stained for alpha-synuclein. Time-dependent experiments showed that inclusions underwent a progressive fusion of the external membranes and developed an electrodense core. Inhibition of dopamine synthesis by alpha-methyl-p-tyrosine (alphaMpT), or administering the antioxidant S-apomorphine largely attenuated the formation of inclusions in PC12 cells exposed to MA. Inclusions were again observed when alphaMpT-treated cells were loaded with l-DOPA, which restored intracellular dopamine levels.     

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.