Mechanisms of Parkinson's disease linked to pathological alpha-synuclein: new targets for drug discovery

Classic Parkinson's disease (PD) is characterized by fibrillar alpha-synuclein inclusions known as Lewy bodies in the substantia nigra, which are associated with nigrostriatal degeneration. However, alpha-synuclein pathologies accumulate throughout the CNS in areas that also undergo progressive neurodegeneration, leading to dementia and other behavioral impairments in addition to parkinsonism. Although mutations in the alpha-synuclein gene only cause Lewy body PD in rare families, and although there are multiple other, albeit rare, genetic causes of familial parkinsonism, sporadic Lewy body PD is the most common movement disorder, and insights into mechanisms underlying alpha-synuclein-mediated neurodegeneration provide novel targets for the discovery of disease-modifying therapies for PD and related neurodegenerative alpha-synucleinopathies.     

Molecular mechanisms of alpha-synuclein neurodegeneration

alpha-Synuclein is an abundant highly charged protein that is normally predominantly localized around synaptic vesicles in presynaptic terminals. Although the function of this protein is still ill-defined, genetic studies have demonstrated that point mutations or genetic alteration (duplications or triplications) that increase the number of copies of the alpha-synuclein (SCNA) gene can cause Parkinson's disease or the related disorder dementia with Lewy bodies. alpha-Synuclein can aberrantly polymerize into fibrils with typical amyloid properties, and these fibrils are the major component of many types of pathological inclusions, including Lewy bodies, which are associated with neurodegenerative diseases, such as Parkinson's disease. Although there is substantial evidence supporting the toxic nature of alpha-synuclein inclusions, other modes of toxicity such as oligomers have been proposed. In this review, some of the evidence for the different mechanisms of alpha-synuclein toxicity is presented and discussed.     

Filamentous nerve cell inclusions in neurodegenerative diseases: tauopathies and alpha-synucleinopathies.

Alzheimer's disease and Parkinson's disease are the most common neurodegenerative diseases. They are characterized by the degeneration of selected populations of nerve cells that develop filamentous inclusions before degeneration. The neuronal inclusions of Alzheimer's disease are made of the microtubule-associated protein tau, in a hyperphosphorylated state. Recent work has shown that the filamentous inclusions of Parkinson's disease are made of the protein alpha-synuclein and that rare, familial forms of Parkinson's disease are caused by missense mutations in the alpha-synuclein gene. Besides Parkinson's disease, the filamentous inclusions of two additional neurodegenerative diseases, namely dementia with Lewy bodies and multiple system atrophy, have also been found to be made of alpha-synuclein. Abundant filamentous tau inclusions are not limited to Alzheimer's disease. They are the defining neuropathological characteristic of frontotemporal dementias such as Pick's disease, and of progressive supranuclear palsy and corticobasal degeneration. The recent discovery of mutations in the tau gene in familial forms of frontotemporal dementia has provided a direct link between tau dysfunction and dementing disease. The new work has established that tauopathies and alpha-synucleinopathies account for most late-onset neurodegenerative diseases in man. The formation of intracellular filamentous inclusions might be the gain of toxic function that leads to the demise of affected brain cells.     

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.     

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.     

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.     

Induction of neuronal cell death by Rab5A-dependent endocytosis of alpha-synuclein

The presynaptic alpha-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinson's disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimer's disease. Here we examined the pathogenic mechanism of neuronal cell death induced by alpha-synuclein. The exogenous addition of alpha-synuclein caused a marked decrease of cell viability in primary and immortalized neuronal cells. The neuronal cell death appeared to be correlated with the Rab5A-specific endocytosis of alpha-synuclein that subsequently caused the formation of Lewy body-like intracytoplasmic inclusions. This was further supported by the fact that the expression of GTPase-deficient Rab5A resulted in a significant decrease of its cytotoxicity as a result of incomplete endocytosis of alpha-synuclein.     

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.     

Association of the cytoskeletal GTP-binding protein Sept4/H5 with cytoplasmic inclusions found in Parkinson's disease and other synucleinopathies

alpha-Synuclein-positive cytoplasmic inclusions are a pathological hallmark of several neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Here we report that Sept4, a member of the septin protein family, is consistently found in these inclusions, whereas five other septins (Sept2, Sept5, Sept6, Sept7, and Sept8) are not found in these inclusions. Sept4 and alpha-synuclein can also be co-immunoprecipitated from normal human brain lysates. When co-expressed in cultured cells, FLAG-tagged Sept4 and Myc-tagged alpha-synuclein formed detergent-insoluble complex, and upon treatment with a proteasome inhibitor, they formed Lewy body-like cytoplasmic inclusions. The tagged Sept4 and alpha-synuclein synergistically accelerated cell death induced by the proteasome inhibitor, and this effect was further enhanced by expression of another Lewy body-associated protein, synphilin-1, tagged with the V5 epitope. Moreover, co-expression of the three proteins (tagged Sept4, alpha-synuclein, and synphilin-1) was sufficient to induce cell death. These data raise the possibility that Sept4 is involved in the formation of cytoplasmic inclusions as well as induction of cell death in alpha-synuclein-associated neurodegenerative disorders.     

Microtubule-associated protein 1B is a component of cortical Lewy bodies and binds alpha-synuclein filaments

Lewy bodies, neuropathological hallmarks of Parkinson's disease and dementia with Lewy bodies, comprise alpha-synuclein filaments and other less defined proteins. Characterization of Lewy body proteins that interact with alpha-synuclein may provide insight into the mechanism of Lewy body formation. Double immunofluorescence labeling and confocal microscopy revealed approximately 80% of cortical Lewy bodies contained microtubule-associated protein 1B (MAP-1B) that overlapped with alpha-synuclein. Lewy bodies were isolated using an immunomagnetic technique from brain tissue of patients dying with dementia with Lewy bodies. Lewy body proteins were resolved by polyacrylamide gel electrophoresis. Immunoblotting confirmed the presence of MAP-1B and alpha-synuclein in purified Lewy bodies. Direct binding studies revealed a high affinity interaction (IC(50) approximately 20 nm) between MAP-1B and alpha-synuclein. The MAP-1B-binding sites were mapped to the last 45 amino acids of the alpha-synuclein C terminus. MAP-1B also bound in vitro assembled alpha-synuclein fibrils. Thus, MAP-1B may be involved in the pathogenesis of Lewy bodies via its interaction with monomeric and fibrillar alpha-synuclein.     

Stabilization of partially folded conformation during alpha-synuclein oligomerization in both purified and cytosolic preparations

Aggregation of alpha-synuclein is tightly associated with many neurodegenerative diseases, such as Parkinson's disease, dementia with Lewy body, Lewy body variant of Alzheimer's disease, multiple system atrophy, and Hallervorden-Spatz disease, implicating a crucial role of aggregated forms of alpha-synuclein in the pathogenesis. Here, we examined the effect of elevated temperature on the oligomerization and structural changes of alpha-synuclein in the early stage of aggregation and show that self-assembly is crucial for the stabilization of a partially folded conformation. The efficiency of alpha-synuclein oligomerization increased proportional to the temperature increase, both in purified form and in crude cytosolic preparation. This oligomerization coincided with a small but reproducible change in the circular dichroism spectrum and an increase in the 1-anilinonaphthalene-8-sulfonic acid binding. The hydrodynamic dimensions of the dimer measured by size exclusion chromatography suggest a pre-molten globule-like structure. These data suggest that partially folded alpha-synuclein, which is unstable in the monomeric form, is stabilized by self-assembly and that these oligomers may evolve into the fibril nucleus.     

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.     

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.     

Structural Evidence for α-Synuclein Fibrils Using in Situ Atomic Force Microscopy

Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson‘s disease, dementia with Lewy bodies and Lewy body variant of Alzheimer‘s disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentous topography for over 1h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how 13-sheets assemble into α-synuclein fibrils on a nanometer scale.     

Conformational behavior and aggregation of alpha-synuclein in organic solvents: modeling the effects of membranes

Intracellular proteinaceous inclusions (Lewy bodies and Lewy neurites) of alpha-synuclein are pathological hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies (DLB), and multiple systemic atrophy. The molecular mechanisms underlying the aggregation of alpha-synuclein into such filamentous inclusions remain unknown, although many factors have been implicated, including interactions with lipid membranes. To model the effects of membrane fields on alpha-synuclein, we analyzed the structural and fibrillation properties of this protein in mixtures of water with simple and fluorinated alcohols. All solvents that were studied induced folding of alpha-synuclein, with the common first stage being formation of a partially folded intermediate with an enhanced propensity to fibrillate. Protein fibrillation was completely inhibited due to formation of beta-structure-enriched oligomers with high concentrations of methanol, ethanol, and propanol and moderate concentrations of trifluoroethanol (TFE), or because of the appearance of a highly alpha-helical conformation at high TFE and hexafluoro-2-propanol concentrations. At least to some extent, these conformational effects mimic those observed in the presence of phospholipid vesicles, and can explain some of the observed effects of membranes on alpha-synuclein fibrillation.     

TorsinA and heat shock proteins act as molecular chaperones: suppression of alpha-synuclein aggregation

TorsinA, a protein with homology to yeast heat shock protein104, has previously been demonstrated to colocalize with alpha-synuclein in Lewy bodies, the pathological hallmark of Parkinson's disease. Heat shock proteins are a family of chaperones that are both constitutively expressed and induced by stressors, and that serve essential functions for protein refolding and/or degradation. Here, we demonstrate that, like torsinA, specific molecular chaperone heat shock proteins colocalize with alpha-synuclein in Lewy bodies. In addition, using a cellular model of alpha-synuclein aggregation, we demonstrate that torsinA and specific heat shock protein molecular chaperones colocalize with alpha-synuclein immunopositive inclusions. Further, overexpression of torsinA and specific heat shock proteins suppress alpha-synuclein aggregation in this cellular model, whereas mutant torsinA has no effect. These data suggest that torsinA has chaperone-like activity and that the disease-associated GAG deletion mutant has a loss-of-function phenotype. Moreover, these data support a role for chaperone proteins, including torsinA and heat shock proteins, in cellular responses to neurodegenerative inclusions.     

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.     

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.     

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.