Modulation of α-synuclein aggregation by dopamine in the presence of MPTP and its metabolite

The neurotransmitter dopamine has been shown to inhibit fibrillation of α-synuclein by promoting the formation of nonamyloidogenic oligomers. Fibrillation of α-synuclein is accelerated in the presence of pesticides and the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The aim of this study was to determine whether dopamine continues to have an adverse effect on the fibrillation of α-synuclein in the presence of MPTP and its metabolite 1-methyl-4-phenylpyridinum ion (MPP(+) ). We also attempted to answer the ambiguous question of whether conversion of MPTP to MPP(+) is required for the fibrillation of α-synuclein. For this, α-synuclein was incubated in the presence of MPTP and MPP(+) along with dopamine. The fibrillation of α-synuclein was monitored by Thioflavin T fluorescence and immunoblotting. The morphology of the aggregates formed was observed using scanning electron microscopy. The concentrations of the neurotoxin and its metabolite were estimated by reverse phase HPLC. We found definitive evidence that the conversion of MPTP to MPP(+) is not required for aggregation of α-synuclein. MPP(+) was found to accelerate the rate of α-synuclein aggregation even in the absence of components of mitochondrial complex I. In contrast to the effect of dopamine on the aggregation of α-synuclein alone, in the presence of MPTP or MPP(+) , the aggregates formed are Thioflavin T-positive and amyloidogenic. Thus, the effect of dopamine on the nature of aggregates formed in case of α-synuclein alone and in the presence of MPTP/MPP(+) is different.     

Assays for α-synuclein aggregation

This review describes different ways to achieve and monitor reproducible aggregation of α-synuclein, a key protein in the development of Parkinson's disease. For most globular proteins, aggregation is promoted by partially denaturing conditions which compromise the native state without destabilizing the intermolecular contacts required for accumulation of regular amyloid structure. As a natively disordered protein, α-synuclein can fibrillate under physiological conditions and this process is actually stimulated by conditions that promote structure formation, such as low pH, ions, polyamines, anionic surfactants, fluorinated alcohols and agitation. Reproducibility is a critical issue since α-synuclein shows erratic fibrillation behavior on its own. Agitation in combination with glass beads significantly reduces the variability of aggregation time curves, but the most reproducible aggregation is achieved by sub-micellar concentrations of SDS, which promote the rapid formation of small clusters of α-synuclein around shared micelles. Although the fibrils produced this way have a different appearance and secondary structure, they are rich in cross-β structure and are amenable to high-throughput screening assays. Although such assays at best provide a very simplistic recapitulation of physiological conditions, they allow the investigator to focus on well-defined molecular events and may provide the opportunity to identify, e.g. small molecule inhibitors of aggregation that affect these steps. Subsequent experiments in more complex cellular and whole-organism environments can then validate whether there is any relation between these molecular interactions and the broader biological context.     

γ-Synuclein: seeding of α-synuclein aggregation and transmission between cells

Protein misfolding and aggregation is a ubiquitous phenomenon associated with a wide range of diseases. The synuclein family comprises three small naturally unfolded proteins implicated in neurodegenerative diseases and some forms of cancer. α-Synuclein is a soluble protein that forms toxic inclusions associated with Parkinson's disease and several other synucleinopathies. However, the triggers inducing its conversion into noxious species are elusive. Here we show that another member of the family, γ-synuclein, can be easily oxidized and form annular oligomers that accumulate in cells in the form of deposits. Importantly, oxidized γ-synuclein can initiate α-synuclein aggregation. Two amino acid residues in γ-synuclein, methionine and tyrosine located in neighboring positions (Met(38) and Tyr(39)), are most easily oxidized. Their oxidation plays a key role in the ability of γ-synuclein to aggregate and seed the aggregation of α-synuclein. γ-Synuclein secreted from neuronal cells into conditioned medium in the form of exosomes can be transmitted to glial cells and cause the aggregation of intracellular proteins. Our data suggest that post-translationally modified γ-synuclein possesses prion-like properties and may induce a cascade of events leading to synucleinopathies.     

Modulation effect of filamentous phage on α-synuclein aggregation

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, including Parkinson’s disease (PD). Here we describe the modulating effect of filamentous phages on aggregation of α-synuclein (AS) in vitro and in a PD cellular model. Filamentous phages, well understood at both structural and genetic levels, have a nanotubular appearance, showing conformational similarities to amyloid fibrils. Since filamentous phages can infect only bacteria and have no tropism to mammalian cells, we utilized the f88 system to present a peptide containing a cyclic RGD (arg-gly-asp), which enabled phage internalization into the cells. Detection of intracellular AS oligomers, in differentiated SH-SY5Y cells, stably transfected with wild type AS gene, was performed using Western blot and ELISA measurements. Data presented here show reduced levels of AS soluble aggregates in phage treated cells compared to non-treated cells, suggesting new therapeutics for PD.     

Tau enhances α-synuclein aggregation and toxicity in cellular models of synucleinopathy

Background

The simultaneous accumulation of different misfolded proteins in the central nervous system is a common feature in many neurodegenerative diseases. In most cases, co-occurrence of abnormal deposited proteins is observed in different brain regions and cell populations, but, in some instances, the proteins can be found in the same cellular aggregates. Co-occurrence of tau and α-synuclein (α-syn) aggregates has been described in neurodegenerative disorders with primary deposition of α-syn, such as Parkinson''s disease and dementia with Lewy bodies. Although it is known that tau and α-syn have pathological synergistic effects on their mutual fibrillization, the underlying biological effects remain unclear.

Methodology/Principal Findings

We used different cell models of synucleinopathy to investigate the effects of tau on α-syn aggregation. Using confocal microscopy and FRET–based techniques we observed that tau colocalized and interacted with α-syn aggregates. We also found that tau overexpression changed the pattern of α-syn aggregation, reducing the size and increasing the number of aggregates. This shift was accompanied by an increase in the levels of insoluble α-syn. Furthermore, co-transfection of tau increased secreted α-syn and cytotoxicity.

Conclusions/Significance

Our data suggest that tau enhances α-syn aggregation and toxicity and disrupts α-syn inclusion formation. This pathological synergistic effect between tau and α-syn may amplify the deleterious process and spread the damage in neurodegenerative diseases that show co-occurrence of both pathologies.     

The hexapeptide PGVTAV suppresses neurotoxicity of human α-synuclein aggregates

In Parkinson’s disease patients, α-synuclein is the major component of the intracellular protein aggregates found in dopaminergic neurons. Previously, short synthetic α-synuclein-derived peptides have been shown to not only prevent α-synuclein fibrillation but also dissolve preformed α-synuclein aggregates in vitro. The hexapeptide PGVTAV was the shortest peptide that retained the ability to block α-synuclein fibrillation. For preventative or therapeutic effectiveness, a treatment must suppress the neurotoxicity of α-synuclein aggregates and remain stable in plasma. The present study shows that specific peptides can protect neuronal cells from α-synuclein aggregation-induced cell death. The β-sheet-breaking hexapeptide PGVTAV remained intact in human plasma for longer than one day, suggesting that it may be a candidate for the development of therapeutics to treat Parkinson’s disease.     

Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

Two different binding modes of α-synuclein to lipid vesicles depending on its aggregation state

Aggregation of α-synuclein is involved in the pathogenesis of Parkinson's disease (PD). Studies of in vitro aggregation of α-synuclein are rendered complex because of the formation of a heterogeneous population of oligomers. With the use of confocal single-molecule fluorescence techniques, we demonstrate that small aggregates (oligomers) of α-synuclein formed from unbound monomeric species in the presence of organic solvent (DMSO) and iron (Fe³⁺) ions have a high affinity to bind to model membranes, regardless of the lipid-composition or membrane curvature. This binding mode contrasts with the well-established membrane binding of α-synuclein monomers, which is accompanied with α-helix formation and requires membranes with high curvature, defects in the lipid packing, and/or negatively charged lipids. Additionally, we demonstrate that membrane-bound α-synuclein monomers are protected from aggregation. Finally, we identified compounds that potently dissolved vesicle-bound α-synuclein oligomers into monomers, leaving the lipid vesicles intact. As it is commonly believed that formation of oligomers is related PD progression, such compounds may provide a promising strategy for the design of novel therapeutic drugs in Parkinson's disease.     

α-synuclein interacts with SOD1 and promotes its oligomerization

Background

Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) are both neurodegenerative diseases leading to impaired execution of movement. α-Synuclein plays a central role in the pathogenesis of PD whereas Cu, Zn superoxide dismutase (SOD1) is a key player in a subset of familial ALS cases. Under pathological conditions both α-synuclein and SOD1 form oligomers and fibrils. In this study we investigated the possible molecular interaction of α-synuclein and SOD1 and its functional and pathological relevance.

Results

Using a protein-fragment complementation approach and co-IP, we found that α-synuclein and SOD1 physically interact in living cells, human erythrocytes and mouse brain tissue. Additionally, our data show that disease related mutations in α-synuclein (A30P, A53T) and SOD1 (G85R, G93A) modify the binding of α-synuclein to SOD1. Notably, α-synuclein accelerates SOD1 oligomerization independent of SOD1 activity.

Conclusion

This study provides evidence for a novel interaction of α-synuclein and SOD1 that might be relevant for neurodegenerative diseases.

     

Inhibition of α-synuclein aggregation by small heat shock proteins

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.     

PAMAM G4 dendrimers affect the aggregation of α-synuclein

α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinson's disease, and inhibition of fibril formation is a potential therapeutic strategy for these conditions. The aim of the present study was to investigate polyamidoamine (PAMAM) dendrimers (generations 4 and 3.5) as inhibitors of fibril formation in vitro by examining their interaction with ASN intrinsic tyrosine fluorescence. Furthermore, the effect of dendrimers on ASN aggregation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by a fluorescence assays using the dye thioflavin T (ThT). The PAMAM G4 dendrimer caused an increase in tyrosine residue fluorescence, and inhibited fibrillation of ASN; inhibited fibrillation was not observed with PAMAM G3.5 dendrimers.     

Supramolecular non-amyloid intermediates in the early stages of α-synuclein aggregation

The aggregation of α-synuclein is associated with progression of Parkinson's disease. We have identified submicrometer supramolecular structures that mediate the early stages of the overall mechanism. The sequence of structural transformations between metastable intermediates were captured and characterized by atomic force microscopy guided by a fluorescent probe sensitive to preamyloid species. A novel ~0.3-0.6 μm molecular assembly, denoted the acuna, nucleates, expands, and liberates fibers with distinctive segmentation and a filamentous fuzzy fringe. These fuzzy fibers serve as precursors of mature amyloid fibrils. Cryo-electron tomography resolved the acuna inner structure as a scaffold of highly condensed colloidal masses interlinked by thin beaded threads, which were perceived as fuzziness by atomic force microscopy. On the basis of the combined data, we propose a sequential mechanism comprising molecular, colloidal, and fibrillar stages linked by reactions with disparate temperature dependencies and distinct supramolecular forms. We anticipate novel diagnostic and therapeutic approaches to Parkinson's and related neurodegenerative diseases based on these new insights into the aggregation mechanism of α-synuclein and intermediates, some of which may act to cause and/or reinforce neurotoxicity.     

Haploinsufficiency of cathepsin D leads to lysosomal dysfunction and promotes cell-to-cell transmission of α-synuclein aggregates

Lysosomal dysfunction has been implicated both pathologically and genetically in neurodegenerative disorders, such as Alzheimer''s disease and Parkinson''s disease (PD). Lysosomal gene deficiencies cause lysosomal storage disorders, many of which involve neurodegeneration. Heterozygous mutations of some of these genes, such as GBA1, are associated with PD. CTSD is the gene encoding Cathepsin D (CTSD), a lysosomal protein hydrolase, and homozygous CTSD deficiency results in neuronal ceroid-lipofuscinosis, which is characterized by the early onset, progressive neurodegeneration. CTSD deficiency was also associated with deposition of α-synuclein aggregates, the hallmark of PD. However, whether partial deficiency of CTSD has a role in the late onset progressive neurodegenerative disorders, including PD, remains unknown. Here, we generated cell lines harboring heterozygous nonsense mutations in CTSD with genomic editing using the zinc finger nucleases. Heterozygous mutation in CTSD resulted in partial loss of CTSD activity, leading to reduced lysosomal activity. The CTSD mutation also resulted in increased accumulation of intracellular α-synuclein aggregates and the secretion of the aggregates. When α-synuclein was introduced in the media, internalized α-synuclein aggregates accumulated at higher levels in CTSD+/− cells than in the wild-type cells. Consistent with these results, transcellular transmission of α-synuclein aggregates was increased in CTSD+/− cells. The increased transmission of α-synuclein aggregates sustained during the successive passages of CTSD+/− cells. These results suggest that partial loss of CTSD activity is sufficient to cause a reduction in lysosomal function, which in turn leads to α-synuclein aggregation and propagation of the aggregates.Maintaining protein homeostasis (proteostasis) is crucial in not only maintenance of physiological functions of cells, but survival of cells. Proteostasis is a particularly important issue for the survival of post-mitotic cells, such as neurons, while dividing cells can dilute aged and misfolded proteins during the mitosis process.^{1, 2} For the clearance of protein burden, cells utilize two major protein degradation systems, ubiquitin proteasome system and lysosomal degradation, the latter degrades endosomal and autophagosomal cargos.^{3, 4, 5, 6} Dysregulation of ubiquitin proteasome system and lysosome has been shown to cause protein conformational diseases, including neurodegenerative disorders and metabolic disorders.^{7, 8} Genetic studies have suggested that impairment of lysosomal functions has important roles in the pathogenesis of neurodegenerative diseases. Mutations in ATP13A2, GBA1 and VPS35 have been associated with PD.^{9, 10, 11, 12} Mutations in progranulin and charged multivesicular body protein 2B (CHMP2B) have been identified as genetic causes of amyotrophic lateral sclerosis and frontotemporal dementia.^{13, 14, 15} Postmortem brain tissues of neurodegenerative diseases have exhibited deposition of endosomal and autophagic vesicles.¹⁶ Therefore, neurodegenerative proteinopathies might be attributed to lysosomal dysfunction.Pathological examinations of patient tissues have exhibited that protein aggregates, such as amyloid beta (Aβ), tau and α-synuclein aggregates, spread to larger brain regions as disease progresses.¹⁷ In animal models, intracerebrally injected α-synuclein aggregates could spread into larger brain regions both in α-synuclein transgenic and non-transgenic mice.^{18, 19, 20, 21} Inoculation of Aβ or tau aggregates into either non-transgenic or transgenic models of AD also exhibited propagation of those aggregates.^{22, 23, 24, 25, 26, 27, 28} Studies have suggested that cell-to-cell transmission of protein aggregates is the underlying mechanism of the pathological propagation.^{29, 30}Mounting evidence have suggested that lysosomal function is important for the clearance of the transferred aggregates in recipient neurons during cell-to-cell aggregate transmission.³¹ This has been extensively studied in cell culture models for α-synuclein transmission. Previous studies showed α-synculein aggregates can be internalized and transported through the endolysosomal pathway.³² Lyososomal dysfunction led to increased accumulation of the internalized α-synuclein aggregates, suggesting that the lysosomal activity in recipient cells is critical in the clearance of the transmitted α-synuclein aggregates.^{32, 33}Lysosomal storage diseases (LSDs) are caused by defects in the lysosomal degradation process. Mutations in genes encoding lysosomal catabolic enzymes and transporters manifest excessive deposition of the enzyme substrates in various organs.³⁴ Though different LSDs show different symptoms, most of LSD patients exhibit neurological symptoms such as mental retardation, motor dysfunction and progressive neurodegeneration, as well as specific pathological changes in the nervous system.^{35, 36} In addition, some of progressive neurodegenerative disorders such as AD, PD and Huntington''s disease also show similar pathological features with LSD: accumulations of endosomal and autophagosomal vesicles and undegraded macromolecules, and inflammatory responses in brain.¹⁶Gaucher''s disease (GD) is the most common LSD, which is inherited in an autosomal recessive manner. Homozygous mutations of GBA1 gene, encoding β-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase, is responsible for GD.³⁷ Evidence has suggested that GD is closely related to PD. Patients with type-1 GD, the most common form of GD, frequently develop parkinsonism.³⁸ Heterozygous carriers of GBA1 mutations are at a higher risk for PD.^{39, 40} It has been shown that about 75% of Lewy bodies, a pathological hallmark of PD, colocalized with GCase 1 in brains of PD and DLB patients with heterozygous GBA1 mutations.⁴¹ These results suggest that lysosomal enzyme deficiency is associated with the development of PD.Cathepsin D (CTSD) is a major lysosomal endopeptidase, which is critical in the degradation of long-lived proteins.⁴² Genetic and clinical studies have shown that the homozygous deficiency of CTSD results in the early onset, progressive neurodegeneration, such as congenital neuronal ceroid-lipofuscinosis.⁴³ The heterozygous missense mutations in CTSD have been known to cause the early onset motor and visual problems, brain atrophy, and progressive psychomotor symptoms.⁴⁴ However, the effects of CTSD deficiency on the late onset progressive neurodegenerative disorders, including AD and PD, remain unclear. Nevertheless, it has become clear that CTSD activity is crucial in the degradation of pathogenic protein aggregates.^{45, 46}Herein, we generated a cell line with a heterozygous nonsense mutation in CTSD and investigated the roles of the CTSD activity in lysosomal function, α-synuclein aggregation and transcellular transmission of α-synuclein aggregates.     

GM1 oligosaccharide efficacy against α-synuclein aggregation and toxicity in vitro

Fibrillary aggregated α-synuclein represents the neurologic hallmark of Parkinson's disease and is considered to play a causative role in the disease. Although the causes leading to α-synuclein aggregation are not clear, the GM1 ganglioside interaction is recognized to prevent this process. How GM1 exerts these functions is not completely clear, although a primary role of its soluble oligosaccharide (GM1-OS) is emerging. Indeed, we recently identified GM1-OS as the bioactive moiety responsible for GM1 neurotrophic and neuroprotective properties, specifically reverting the parkinsonian phenotype both in in vitro and in vivo models.Here, we report on GM1-OS efficacy against the α-synuclein aggregation and toxicity in vitro. By amyloid seeding aggregation assay and NMR spectroscopy, we demonstrated that GM1-OS was able to prevent both the spontaneous and the prion-like α-synuclein aggregation. Additionally, circular dichroism spectroscopy of recombinant monomeric α-synuclein showed that GM1-OS did not induce any change in α-synuclein secondary structure. Importantly, GM1-OS significantly increased neuronal survival and preserved neurite networks of dopaminergic neurons affected by α-synuclein oligomers, together with a reduction of microglia activation.These data further demonstrate that the ganglioside GM1 acts through its oligosaccharide also in preventing the α-synuclein pathogenic aggregation in Parkinson's disease, opening a perspective window for GM1-OS as drug candidate.     

Metalloproteomics and metal toxicology of α-synuclein

Significant evidence has been accumulated linking exposure to heavy metals and/or distortion of metal homeostasis with the development of various neurodegenerative diseases. α-Synuclein is known to be involved in pathogenesis of a subset of neurodegenerative diseases collectively known as synucleinopathies. Therefore the interplay between metals, α-synuclein and neurodegeneration has attracted significant attention of researchers. This review discusses some of the aspects of the α-synuclein metalloproteomics and represents the peculiarities and consequences of α-synuclein interaction with various metal ions. Both non-specific and specific binding of this protein to metals is considered together with the analysis of the effects of such interactions on α-synuclein structure and aggregation propensity.     

In vitro aggregation assays for the characterization of α-synuclein prion-like properties

Aggregation of α-synuclein plays a crucial role in the pathogenesis of synucleinopathies, a group of neurodegenerative diseases including Parkinson disease (PD), dementia with Lewy bodies (DLB), diffuse Lewy body disease (DLBD) and multiple system atrophy (MSA). The common feature of these diseases is a pathological deposition of protein aggregates, known as Lewy bodies (LBs) in the central nervous system. The major component of these aggregates is α-synuclein, a natively unfolded protein, which may undergo dramatic structural changes resulting in the formation of β-sheet rich assemblies. In vitro studies have shown that recombinant α-synuclein protein may polymerize into amyloidogenic fibrils resembling those found in LBs. These aggregates may be uptaken and propagated between cells in a prion-like manner. Here we present the mechanisms and kinetics of α-synuclein aggregation in vitro, as well as crucial factors affecting this process. We also describe how PD-linked α-synuclein mutations and some exogenous factors modulate in vitro aggregation. Furthermore, we present a current knowledge on the mechanisms by which extracellular aggregates may be internalized and propagated between cells, as well as the mechanisms of their toxicity.     

Gallic acid interacts with α-synuclein to prevent the structural collapse necessary for its aggregation

The accumulation of protein aggregates containing amyloid fibrils, with α-synuclein being the main component, is a pathological hallmark of Parkinson's disease (PD). Molecules which prevent the formation of amyloid fibrils or disassociate the toxic aggregates are touted as promising strategies to prevent or treat PD. In the present study, in vitro Thioflavin T fluorescence assays and transmission electron microscopy imaging results showed that gallic acid (GA) potently inhibits the formation of amyloid fibrils by α-synuclein. Ion mobility-mass spectrometry demonstrated that GA stabilises the extended, native structure of α-synuclein, whilst NMR spectroscopy revealed that GA interacts with α-synuclein transiently.     

Impact of N-terminal acetylation of α-synuclein on its random coil and lipid binding properties

N-Terminal acetylation of α-synuclein (aS), a protein implicated in the etiology of Parkinson's disease, is common in mammals. The impact of this modification on the protein's structure and dynamics in free solution and on its membrane binding properties has been evaluated by high-resolution nuclear magnetic resonance and circular dichroism (CD) spectroscopy. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein that progressively decreased with the distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone (3)J(HH) couplings are consistent with an increase in the α-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled <20% of the time. Chemical shift and (3)J(HH) data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and nonacetylated 15-residue synthetic peptides. An increase in α-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide and by weak medium-range nuclear Overhauser effect contacts indicative of α-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant differences in the fibrillation kinetics were observed between acetylated and nonacetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an "initiation-elongation" process of binding to the membrane.