Phosphorylation-dependent metal binding by α-synuclein peptide fragments

α-Synuclein (α-syn) is the major protein component of the insoluble fibrils that make up Lewy bodies, the hallmark lesions of Parkinson’s disease. Its C-terminal region contains motifs of charged amino acids that potentially bind metal ions, as well as several identified phosphorylation sites. We have investigated the metal-binding properties of synthetic model peptides and phosphopeptides that correspond to residues 119–132 of the C-terminal, polyacidic stretch of human α-syn, with the sequence Ac-Asp-Pro-Asp-Asn-Glu-Ala-Tyr-Glu-Met-Pro-Ser-Glu-Glu-Gly (α-syn119–132). The peptide pY125 replaces tyrosine with phosphotyrosine, whereas pS129 replaces serine with phosphoserine. By using Tb³⁺ as a luminescent probe of metal binding, we find a marked selectivity of pY125 for Tb³⁺ compared with pS129 and α-syn119–132, a result confirmed by isothermal titration calorimetry. Truncated or alanine-substituted peptides show that the phosphoester group on tyrosine provides a metal-binding anchor that is supplemented by carboxylic acid groups at positions 119, 121, and 126 to establish a multidentate ligand, while two glutamic acid residues at positions 130 and 131 contribute to binding additional Tb³⁺ ions. The interaction of other metal ions was investigated by electrospray ionization mass spectrometry, which confirmed that pY125 is selective for trivalent metal ions over divalent metal ions, and revealed that Fe³⁺ and Al³⁺ induce peptide dimerization through metal ion cross-links. Circular dichroism showed that Fe³⁺ can induce a partially folded structure for pY125, whereas no change was observed for pS129 or the unphosphorylated analog. The results of this study show that the type and location of a phosphorylated amino acid influence a peptide’s metal-binding specificity and affinity as well as its overall conformation. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Cross-seeding effects of amyloid β-protein and α-synuclein

J. Neurochem. (2012) 122, 883-890. ABSTRACT: Amyloid β-protein (Aβ) and α-synuclein (αS) are the primary components of amyloid plaques and Lewy bodies (LBs), respectively. Previous in vitro and in vivo studies have suggested that interactions between Aβ and αS are involved in the pathogenesis of Alzheimer's disease and LB diseases. However, the seeding effects of their aggregates on their aggregation pathways are not completely clear. To investigate the cross-seeding effects of Aβ and αS, we examined how sonicated fibrils or cross-linked oligomers of Aβ40, Aβ42, and αS affected their aggregation pathways using thioflavin T(S) assay and electron microscopy. Fibrils and oligomers of Aβ40, Aβ42, and αS acted as seeds, and affected the aggregation pathways within and among species. The seeding effects of αS fibrils were higher than those of Aβ40 and Aβ42 fibrils in the Aβ40 and Aβ42 aggregation pathways, respectively. We showed that Aβ and αS acted as seeds and affected each other's aggregation pathways in vitro, which may contribute to our understanding of the molecular mechanisms of interactions between Alzheimer's disease and LB diseases pathologies.     

Ubiquitination of α-synuclein and autophagy in Parkinson's disease

α-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 α-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 α-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 α-synuclein at the same lysines that are monoubiquitinated in Lewy bodies. Monoubiquitination by SIAH promotes the aggregation of α-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 α-synuclein may work as a seed for aggregation. Accumulation of monoubiquitinated α-synuclein and formation of cytosolic inclusions is promoted by autophagy inhibition and to a lesser extent by proteasomal and lysosomal inhibition. Monoubiquitinated α-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 α-synuclein monoubiquitination, by preventing SIAH function or by stimulating autophagy, constitutes a new therapeutic strategy for Parkinson's disease.

Addendum to: Rott R, Szargel R, Haskin J, Shani V, Shainskaya A, Manov I, Liani E, Avraham E, Engelender S. Monoubiquitination of α-synuclein by SIAH promotes its aggregation in dopaminergic cells. J Biol Chem 2007; Epub ahead of print.     

N-homocysteinylation of α-synuclein promotes its aggregation and neurotoxicity

The aggregation of α-synuclein plays a pivotal role in the pathogenesis of Parkinson's disease (PD). Epidemiological evidence indicates that high level of homocysteine (Hcy) is associated with an increased risk of PD. However, the molecular mechanisms remain elusive. Here, we report that homocysteine thiolactone (HTL), a reactive thioester of Hcy, covalently modifies α-synuclein on the K80 residue. The levels of α-synuclein K80Hcy in the brain are increased in an age-dependent manner in the TgA53T mice, correlating with elevated levels of Hcy and HTL in the brain during aging. The N-homocysteinylation of α-synuclein stimulates its aggregation and forms fibrils with enhanced seeding activity and neurotoxicity. Intrastriatal injection of homocysteinylated α-synuclein fibrils induces more severe α-synuclein pathology and motor deficits when compared with unmodified α-synuclein fibrils. Increasing the levels of Hcy aggravates α-synuclein neuropathology in a mouse model of PD. In contrast, blocking the N-homocysteinylation of α-synuclein ameliorates α-synuclein pathology and degeneration of dopaminergic neurons. These findings suggest that the covalent modification of α-synuclein by HTL promotes its aggregation. Targeting the N-homocysteinylation of α-synuclein could be a novel therapeutic strategy against PD.     

Synthesis and in vitro evaluation of α-synuclein ligands

Accumulation of misfolded α-synuclein in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson's disease (PD). To identify ligands having high binding potency toward aggregated α-synuclein, we synthesized a series of phenothiazine derivatives and assessed their binding affinity to recombinant α-synuclein fibrils using a fluorescent thioflavin T competition assay. Among 16 new analogues, the in vitro data suggest that compound 11b has high affinity to α-synuclein fibrils (K(i)=32.10 ± 1.25 nM) and compounds 11d, 16a and16b have moderate affinity to α-synuclein fibrils (K(i)≈50-100 nM). Further optimization of the structure of these analogues may yield compounds with high affinity and selectivity for aggregated α-synuclein.     

Molecular mechanisms of α-synuclein neurodegeneration

α-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 α-synuclein (SCNA) gene can cause Parkinson's disease or the related disorder dementia with Lewy bodies. α-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 α-synuclein inclusions, other modes of toxicity such as oligomers have been proposed. In this review, some of the evidence for the different mechanisms of α-synuclein toxicity is presented and discussed.     

Compact conformations of α-synuclein induced by alcohols and copper

The intrinsically disordered protein α-synuclein aggregates into amyloid fibrils, a process known to be implicated in several neurodegenerative states. Partially folded forms of the protein are thought to trigger the aggregation process. Here, α-synuclein conformers are characterized by analysis of the charge-state distributions observed in electrospray-ionization mass spectrometry under negative-ion mode. It is found that, even at neutral pH, a small fraction of the molecular population is in a compact conformation. Several distinct partially folded forms are then identified under conditions that promote α-synuclein aggregation, such as solutions of simple and fluorinated alcohols. Specific intermediates accumulate at increasing concentrations of ethanol, hexafluoro-2-propanol, and trifluoroethanol. Finally, extensive folding induced by Cu(2+) binding is revealed by titrations in the presence of Cu(2+)-glycine. The data confirm the existence of a single, high-affinity binding site for Cu(2+). Because accumulation of this partially folded form correlates with enhancement of fibrillation kinetics, it is likely to represent an amyloidogenic intermediate in α-synuclein conformational transitions.     

Improved immunodetection of endogenous α-synuclein

α-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative α-synucleinopathies such as Parkinson's disease. Despite extensive research, however, its precise function remains unclear partly because of a difficulty in immunoblotting detection of endogenous α-synuclein. This difficulty has largely restricted the progress for α-synucleinopathy research. Here, we report that α-synuclein monomers tend to easily detach from blotted membranes, resulting in no or very poor detection. To prevent this detachment, a mild fixation of blotted membranes with paraformaldehyde was applied to the immunoblotting method. Amazingly, this fixation led to clear and strong detection of endogenous α-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous α-synuclein in various human cell lines, including SH-SY5Y, HEK293, HL60, HeLa, K562, A375, and Daoy, and a mouse cell line B16 as well as in several mouse tissues such as the spleen and kidney. Moreover, it should be noted that we could clearly detect endogenous α-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous α-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous α-synuclein, thereby facilitating α-synuclein research.     

Biophysics of α-synuclein membrane interactions

Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson's disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Photophysics of diphenyl-pyrazole compounds in solutions and α-synuclein aggregates

Background

Recently diphenyl-pyrazole (DPP) compounds and especially anle138b were found to reduce the aggregation of α-synuclein or Tau protein in vitro as well as in a mouse model of neurodegenerative diseases [1,2]. Direct interaction of the DPPs with the fibrillar structure was identified by fluorescence spectroscopy. Thereby a strong dependence of the fluorescence on the surroundings could be identified [3].

Direct observation of the interconversion of normal and toxic forms of α-synuclein

Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.     

Synergistic influence of phosphorylation and metal ions on tau oligomer formation and coaggregation with α-synuclein at the single molecule level

Background

Abnormal proteostasis due to alterations in protein turnover has been postulated to play a central role in several neurodegenerative diseases. Therefore, the development of techniques to quantify protein turnover in the brain is critical for understanding the pathogenic mechanisms of these diseases. We have developed a bolus stable isotope-labeling kinetics (SILK) technique coupled with multiple reaction monitoring mass spectrometry to measure the clearance of proteins in the mouse brain.

Results

Cohorts of mice were pulse labeled with ¹³?C₆-leucine and the brains were isolated after pre-determined time points. The extent of label incorporation was measured over time using mass spectrometry to measure the ratio of labeled to unlabeled apolipoprotein E (apoE) and amyloid ?? (A??). The fractional clearance rate (FCR) was then calculated by analyzing the time course of disappearance for the labeled protein species. To validate the technique, apoE clearance was measured in mice that overexpress the low-density lipoprotein receptor (LDLR). The FCR in these mice was 2.7-fold faster than wild-type mice. To demonstrate the potential of this technique for understanding the pathogenesis of neurodegenerative disease, we applied our SILK technique to determine the effect of ATP binding cassette A1 (ABCA1) on both apoE and A?? clearance. ABCA1 had previously been shown to regulate both the amount of apoE in the brain, along with the extent of A?? deposition, and represents a potential molecular target for lowering brain amyloid levels in Alzheimer's disease patients. The FCR of apoE was increased by 1.9- and 1.5-fold in mice that either lacked or overexpressed ABCA1, respectively. However, ABCA1 had no effect on the FCR of A??, suggesting that ABCA1 does not regulate A?? metabolism in the brain.

Conclusions

Our SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain. We expect that this technique will be applicable to the study of protein dynamics in the pathogenesis of several neurodegenerative diseases, and could aid in the evaluation of novel therapeutic agents.     

Inhibition and disaggregation of α-synuclein oligomers by natural polyphenolic compounds

Aggregation of alpha-synuclein (αS) into oligomers is critically involved in the pathogenesis of Parkinson's disease (PD). Using confocal single-molecule fluorescence spectroscopy, we have studied the effects of 14 naturally-occurring polyphenolic compounds and black tea extract on αS oligomer formation. We found that a selected group of polyphenols exhibited potent dose-dependent inhibitory activity on αS aggregation. Moreover, they were also capable of robustly disaggregating pre-formed αS oligomers. Based upon structure-activity analysis, we propose that the key molecular scaffold most effective in inhibiting and destabilizing self-assembly by αS requires: (i) aromatic elements for binding to the αS monomer/oligomer and (ii) vicinal hydroxyl groups present on a single phenyl ring. These findings may guide the design of novel therapeutic drugs in PD.     

Aggregation-defective α-synuclein mutants inhibit the fibrillation of Parkinson’s disease-linked α-synuclein variants

α-Synuclein comprises the fibrillar core of Lewy bodies, which is one of the histologically defining lesions of Parkinson’s disease. Previously, we screened for α-synuclein substitution mutants that do not form fibrils. For preventative or therapeutic uses, it is essential to suppress the oligomerization/fibrillation of the wild-type and PD-linked α-synuclein proteins. Here we have examined the effects of fibrillation-retarded α-synuclein mutants on fibril formation by wild-type and PD-linked α-synuclein molecules. Six self-aggregation-defective α-synuclein mutants completely inhibit the fibrillation of both wild-type and Parkinson’s disease-linked α-synuclein variants. These results suggest future applications for gene therapy: the transplantation of a fibrillation-blocking mutant α-synuclein gene into individuals who carry an early-onset PD-associated α-synuclein allele. Short synthetic peptides derived from these mutant sequences may also serve as a lead compound for the development of therapeutics for 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.     

Assessment of α-synuclein secretion in mouse and human brain parenchyma

Genetic, biochemical, and animal model studies strongly suggest a central role for α-synuclein in the pathogenesis of Parkinson's disease. α-synuclein lacks a signal peptide sequence and has thus been considered a cytosolic protein. Recent data has suggested that the protein may be released from cells via a non-classical secretory pathway and may therefore exert paracrine effects in the extracellular environment. However, proof that α-synuclein is actually secreted into the brain extracellular space in vivo has not been obtained. We developed a novel highly sensitive ELISA in conjugation with an in vivo microdialysis technique to measure α-synuclein in brain interstitial fluid. We show for the first time that α-synuclein is readily detected in the interstitial fluid of both α-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that α-synuclein is physiologically secreted by neurons in vivo. This interstitial fluid pool of the protein may have a role in the propagation of synuclein pathology and progression of 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.     

Solid-state NMR sequential assignments of α-synuclein

Parkinson’s disease is amongst the most frequent and most devastating neurodegenerative diseases. It is tightly associated with the assembly of proteins into high-molecular weight protein species, which propagate between neurons in the central nervous system. The principal protein involved in this process is α-synuclein which is a structural component of the Lewy bodies observed in diseased brain. We here present the solid-state NMR sequential assignments of a new fibrillar form of this protein, the first one with a well-ordered and rigid N-terminal part.