Phosphorylated α-synuclein in Parkinson's disease.

Phosphorylated α-synuclein (PS-129), a protein implicated in the pathogenesis of Parkinson's disease (PD), was identified by mass spectrometry in human cerebrospinal fluid (CSF). A highly sensitive and specific assay was established and used to measure PS-129 together with total α-synuclein in the CSF of patients with PD, other parkinsonian disorders such as multiple system atrophy (MSA) and progressive supranuclear palsy (PSP), and healthy individuals (a total of ~600 samples). PS-129 CSF concentrations correlated weakly with PD severity and, when combined with total α-synuclein concentrations in CSF, contributed to distinguishing PD from MSA and PSP. Further rigorous validation in independent cohorts of patients, especially those where samples have been collected longitudinally, will determine whether the concentration of PS-129 in CSF will be useful for diagnosing PD and for monitoring PD severity and progression.

Characterization of inhibitor-bound alpha-synuclein dimer: role of alpha-synuclein N-terminal region in dimerization and inhibitor binding.

alpha-Synuclein is a major component of filamentous inclusions that are histological hallmarks of Parkinson's disease and other alpha-synucleinopathies. Previous analyses have revealed that several polyphenols inhibit alpha-synuclein assembly with low micromolar IC(50) values, and that SDS-stable, noncytotoxic soluble alpha-synuclein oligomers are formed in their presence. Structural elucidation of inhibitor-bound alpha-synuclein oligomers is obviously required for the better understanding of the inhibitory mechanism. In order to characterize inhibitor-bound alpha-synucleins in detail, we have prepared alpha-synuclein dimers in the presence of polyphenol inhibitors, exifone, gossypetin, and dopamine, and purified the products. Peptide mapping and mass spectrometric analysis revealed that exifone-treated alpha-synuclein monomer and dimer were oxidized at all four methionine residues of alpha-synuclein. Immunoblot analysis and redox-cycling staining of endoproteinase Asp-N-digested products showed that the N-terminal region (1-60) is involved in the dimerization and exifone binding of alpha-synuclein. Ultra-high-field NMR analysis of inhibitor-bound alpha-synuclein dimers showed that the signals derived from the N-terminal region of alpha-synuclein exhibited line broadening, confirming that the N-terminal region is involved in inhibitor-induced dimerization. The C-terminal portion still predominantly exhibited the random-coil character observed in monomeric alpha-synuclein. We propose that the N-terminal region of alpha-synuclein plays a key role in the formation of alpha-synuclein assemblies.

Inhibition of alpha-synuclein fibril assembly by small molecules: analysis using epitope-specific antibodies.

The conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases. Existing treatments are at best symptomatic. Accordingly, small molecule inhibitors of amyloid fibril formation and their mechanisms are of great interest. Here we report that the conformational changes undergone by alpha -synuclein as it assembles into amyloid fibrils can be detected by epitope-specific antibodies. We show that the conformations of polyphenol-bound alpha-synuclein monomers and dimers differ from those of unbound monomers and resemble amyloid fibrils. This strongly suggests that small molecule inhibitors bind and stabilize intermediates of amyloid fibril formation, consistent with the view that inhibitor-bound molecular species are on-pathway intermediates.

Conversion of wild-type alpha-synuclein into mutant-type fibrils and its propagation in the presence of A30P mutant.

Fibrillization or conformational change of alpha-synuclein is central in the pathogenesis of alpha-synucleinopathies, such as Parkinson disease. We found that the A30P mutant accelerates nucleation-dependent fibrillization of wild type (WT) alpha-synuclein. Electron microscopy observation and ultracentrifugation experiments revealed that shedding of fragments occurs from A30P fibrils and that these fragments accelerate fibrillization by serving as seeds. Immunochemical analysis using epitope-specific antibodies and biochemical analyses of protease-resistant cores demonstrated that A30P fibrils have a distinct conformation. Interestingly, WT fibrils formed with A30P seeds exhibited the same character as A30P fibrils, as did A30P fibrils formed with WT seeds, indicating that the A30P mutation affects the conformation and fibrillization of both WT and A30P. These effects of A30P mutation may explain the apparent conflict between the association of A30P with Parkinson disease and the slow fibrillization of A30P itself and therefore provide new insight into the molecular mechanisms of alpha-synucleinopathies.

Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of alpha-synuclein.

Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of alpha-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies. Based on NMR spectral data collected at a 920 MHz proton frequency, we performed epitope mapping of an anti-alpha-synuclein monoclonal antibody, and furthermore, characterized conformational effects of phosphorylation at Ser129 of alpha-synuclein.

Small molecule inhibitors of alpha-synuclein filament assembly.

Alpha-synuclein is the major component of the filamentous inclusions that constitute defining characteristics of Parkinson's disease and other alpha-synucleinopathies. Here we have tested 79 compounds belonging to 12 different chemical classes for their ability to inhibit the assembly of alpha-synuclein into filaments in vitro. Several polyphenols, phenothiazines, porphyrins, polyene macrolides, and Congo red and its derivatives, BSB and FSB, inhibited alpha-synuclein filament assembly with IC(50) values in the low micromolar range. Many compounds that inhibited alpha-synuclein assembly were also found to inhibit the formation of Abeta and tau filaments. Biochemical analysis revealed the formation of soluble oligomeric alpha-synuclein in the presence of inhibitory compounds, suggesting that this may be the mechanism by which filament formation is inhibited. Unlike alpha-synuclein filaments and protofibrils, these soluble oligomeric species did not reduce the viability of SH-SY5Y cells. These findings suggest that the soluble oligomers formed in the presence of inhibitory compounds may not be toxic to nerve cells and that these compounds may therefore have therapeutic potential for alpha-synucleinopathies and other brain amyloidoses.

Cysteine misincorporation in bacterially expressed human alpha-synuclein.

Bacterially expressed human alpha-synuclein (alpha-syn) has been widely used in structural and functional studies. Here we show that approximately 20% of human alpha-syn expressed in Escherichia coli is mistranslated and that a Cys residue is incorporated at position 136 instead of a Tyr. Site-directed mutagenesis of codon 136 (TAC to TAT) resulted in the expression of alpha-syn lacking Cys. Although wild-type (Y136-TAC and Y136-TAT) and mutant (C136-TGC) alpha-syn had similar propensities to assemble into filaments, the levels of dimeric alpha-syn were increased by misincorporation. To avoid potential artefacts, we recommend use of the Y136-TAT construct for the expression of human alpha-syn.

Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins.

Tau protein is the major component of the intraneuronal filamentous inclusions that constitute defining neuropathological characteristics of Alzheimer's disease and other tauopathies. The discovery of tau gene mutations in familial forms of frontotemporal dementia has established that dysfunction of the tau protein is sufficient to cause neurodegeneration and dementia. Here we have tested 42 compounds belonging to nine different chemical classes for their ability to inhibit heparin-induced assembly of tau into filaments in vitro. Several phenothiazines (methylene blue, azure A, azure B, and quinacrine mustard), polyphenols (myricetin, epicatechin 5-gallate, gossypetin, and 2,3,4,2',4'-pentahydroxybenzophenone), and the porphyrin ferric dehydroporphyrin IX inhibited tau filament formation with IC(50) values in the low micromolar range as assessed by thioflavin S fluorescence, electron microscopy, and Sarkosyl insolubility. Disassembly of tau filaments was observed in the presence of the porphyrin phthalocyanine. Compounds that inhibited tau filament assembly were also found to inhibit the formation of Abeta fibrils. Biochemical analysis revealed the formation of soluble oligomeric tau in the presence of the inhibitory compounds, suggesting that this may be the mechanism by which tau filament formation is inhibited. The compounds investigated did not affect the ability of tau to interact with microtubules. Identification of small molecule inhibitors of heparin-induced assembly of tau will form a starting point for the development of mechanism-based therapies for the tauopathies.