Sequence Determinants for Amyloid Fibrillogenesis of Human alpha-Synuclein

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by the presence of filamentous inclusions in nerve cells. These filaments are amyloid fibrils that are made of the protein α-synuclein, which is genetically linked to rare cases of PD and DLB. β-Synuclein, which shares 60% identity with α-synuclein, is not found in the inclusions. Furthermore, while recombinant α-synuclein readily assembles into amyloid fibrils, β-synuclein fails to do so. It has been suggested that this may be due to the lack in β-synuclein of a hydrophobic region that spans residues 73-83 of α-synuclein. Here, fibril assembly of recombinant human α-synuclein, α-synuclein deletion mutants, β-synuclein and β/α-synuclein chimeras was assayed quantitatively by thioflavin T fluorescence and semi-quantitatively by transmission electron microscopy. Deletion of residues 73-83 from α-synuclein did not abolish filament formation. Furthermore, a chimera of β-synuclein with α-synuclein(73-83) inserted was significantly less fibrillogenic than wild-type α-synuclein. These findings, together with results obtained using a number of recombinant synucleins, showed a correlation between fibrillogenesis and mean β-strand propensity, hydrophilicity and charge of the amino acid sequences. The combination of these simple physicochemical properties with a previously described calculation of β-strand contiguity allowed us to design mutations that changed the fibrillogenic propensity of α-synuclein in predictable ways.     

Neuroprotective effects of extract of Acanthopanax senticosus harms on SH-SY5Y cells overexpressing wild-type or A53T mutant α-synuclein

Extract of Acanthopanax senticosus harms (EAS) has been shown to have neuroprotective effects on dopaminergic neurons in Parkinson's disease (PD) mice model. α-Synuclein is a key player in the pathogenesis of PD, the elevated level of which is deleterious to dopaminergic neurons, and enhancing its clearance might be a promising strategy for treating PD. To assess the potential of EAS in this regard, we investigated its effect on the SH-SY5Y cells overexpressing wild-type α-synuclein (WT-α-Syn) or A53T mutant α-synuclein (A53T-α-Syn), and the implicated pathway it might mediate. After treatment with EAS, the changes of α-synuclein, caspase-3, parkin, phospho-protein kinase B (Akt), phospho-glycogen synthase kinase 3 beta (GSK3β), and phospho-microtubule-associated protein tau (Tau) in WT-α-Syn or A53T-α-Syn transgenic cells were reverted back to near normal levels, demonstrated by the western blotting and quantitative real-time PCR outcomes. The neuroprotective effects of EAS may be able to protect WT-α-Syn or A53T-α-Syn transgenic SH-SY5Y cells from α-synuclein overexpression and toxicity. Therefore, we speculate that EAS might be a promising candidate for prevention or treatment of α-synuclein-related neurodegenerative disorders such as PD.     

Inhibition of formation of α-synuclein inclusions by mannosylglycerate in a yeast model of Parkinson's disease

Background

Protein aggregation in the brain is a central hallmark in many neurodegenerative diseases. In Parkinson's disease, α-synuclein (α-Syn) is the major component of the intraneuronal inclusions found in the brains of patients. Current therapeutics is merely symptomatic, and there is a pressing need for developing novel therapies. Previously we showed that mannosylglycerate (MG), a compatible solute typical of marine microorganisms thriving in hot environments, is highly effective in protecting a variety of model proteins against thermal denaturation and aggregation in vitro.

Methods

Saccharomyces cerevisiae cells expressing eGFP-tagged α-Syn, were further engineered to synthesize MG. The number of cells with fluorescent foci was assessed by fluorescence microscopy. Fluorescence spectroscopy and transmission electron microscopy were used to monitor fibril formation in vitro.

Results

We observed a 3.3-fold reduction in the number of cells with α-Syn foci and mild attenuation of α-Syn-induced toxicity. Accordingly, sucrose gradient analysis confirmed a clear reduction in the size-range of α-Syn species in the cells. MG did not affect the expression levels of α-Syn or its degradation rate. Moreover, MG did not induce molecular chaperones (Hsp104, Hsp70 and Hsp40), suggesting the implication of other mechanisms for α-Syn stabilization. MG also inhibited α-Syn fibrillation in vitro.

Conclusions

MG acts as a chemical chaperone and the stabilization mechanism involves direct solute/protein interactions.

General significance

This is the first demonstration of the anti-aggregating ability of MG in the intracellular milieu. The work shows that MG is a good candidate to inspire the development of new drugs for protein-misfolding diseases.     

Neuroprotective and nootropic drug noopept rescues α-synuclein amyloid cytotoxicity

Parkinson's disease is a common neurodegenerative disorder characterized by α-synuclein (α-Syn)-containing Lewy body formation and selective loss of dopaminergic neurons in the substantia nigra. We have demonstrated the modulating effect of noopept, a novel proline-containing dipeptide drug with nootropic and neuroprotective properties, on α-Syn oligomerization and fibrillation by using thioflavin T fluorescence, far-UV CD, and atomic force microscopy techniques. Noopept does not bind to a sterically specific site in the α-Syn molecule as revealed by heteronuclear two-dimensional NMR analysis, but due to hydrophobic interactions with toxic amyloid oligomers, it prompts their rapid sequestration into larger fibrillar amyloid aggregates. Consequently, this process rescues the cytotoxic effect of amyloid oligomers on neuroblastoma SH-SY5Y cells as demonstrated by using cell viability assays and fluorescent staining of apoptotic and necrotic cells and by assessing the level of intracellular oxidative stress. The mitigating effect of noopept against amyloid oligomeric cytotoxicity may offer additional benefits to the already well-established therapeutic functions of this new pharmaceutical.     

Alpha-synuclein overexpression increases phospho-protein phosphatase 2A levels via formation of calmodulin/Src complex

Alpha-synuclein (α-Syn) is the principal protein component of Lewy bodies, a pathological hallmark of Parkinson’s disease (PD). This protein may regulate protein phosphatase 2A (PP2A) activity, although the molecular mechanisms for α-Syn-mediated regulation of PP2A and the potential neuroprotective actions of PP2A against PD-associated pathology remain largely unexplored. We found that α-Syn gene overexpression in SK-N-SH cells and primary neurons led to PP2A/C phosphorylation at Y307, a known target of Src kinase, and consequent phosphatase inhibition. In addition, phospho-activated Src (p-Y416 Src, pSrc) was higher in SK-N-SH cells and primary neurons overexpressing α-Syn. Thus, α-Syn may promote Src activation and PP2A inactivation, leading to hyperphosphorylation of proteins. Immunoprecipitation revealed higher calmodulin/Src complex formation in α-Syn-overexpressing cells and α-Syn transgenic mice. A TUNEL apoptosis assay and an MTT cell viability assay demonstrated that the PP2A activator C₂-ceramide protected neurons against α-Syn-induced cell injury. Buffering the Ca²⁺ elevations induced by α-Syn overexpression ameliorated the cytotoxicity of α-Syn. Our findings define a potential molecular mechanism for α-Syn-mediated regulation of PP2A through formation of the calmodulin/Src complex, activation of Src, and Src-mediated phospho-inhibition of PP2A. Overexpression of α-Syn may lead to neurodegeneration in PD in part by suppressing the endogenous neuroprotective activity of PP2A.     

At Low Concentrations, 3,4-Dihydroxyphenylacetic Acid (DOPAC) Binds Non-Covalently to α-Synuclein and Prevents Its Fibrillation

Several studies have shown that catecholamines can inhibit the fibrillation of α-synuclein (α-Syn), a small presynaptic protein whose aggregation is believed to be a critical step in the etiology of Parkinson's disease and several other neurodegenerative disorders. However, the mechanism of this inhibition is uncertain. We show here that substoichiometric concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), a normal product of the metabolism of dopamine, can inhibit the fibrillation of α-Syn, due to non-covalent binding of DOPAC to α-Syn monomer. Intriguingly, the presence of α-Syn accelerates the spontaneous oxidation of DOPAC, and the oxidized form of DOPAC (the quinone) is responsible for the fibrillation inhibition. In addition, the presence of DOPAC leads to the oxidation of the methionine residues of α-Syn, probably due to the H₂O₂ production as a by-product of DOPAC oxidation. The lack of fibrillation results from the formation of stable oligomers, which are very similar to those observed transiently at early stages of the α-Syn fibrillation. A possible explanation for this phenomenon is that DOPAC stabilizes the normally transient oligomers and prevents them from subsequent fibril formation. The analysis of the α-Syn Y39W variant suggests that DOPAC binds non-covalently to the same N-terminal region of α-Syn as lipid vesicles, probably in the vicinity of residue 39. In contrast to the compounds with 1,2-dihydroxyphenyl groups (DOPAC and catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone and homogentisic acid) are able to modify α-Syn covalently, probably due to the less steric hindrance in the Michael addition.     

C-terminal truncation exacerbates the aggregation and cytotoxicity of α-Synuclein: A vicious cycle in Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disease which usually associates with neuroinflammation. The main pathological characteristics of PD are dopaminergic neurons death and the presence of Lewy bodies which are composed of aggregated α-synuclein (α-Syn). Truncated forms of α-Syn are found in the brain of PD patients, and account for 10–30% of total synuclein in Lewy bodies. Caspase-1, which plays an important role in neuroinflammation, cleaves full-length α-Syn (α-Syn FL) to generate a C-terminus 19-residues truncated α-Syn (α-Syn121). However, the role of truncated α-Syn in the onset and/or pathogenesis of PD is unclear. Here, we used α-Syn121 as a model to explore its aggregation, membrane disruption and cytotoxicity properties. Compared with α-Syn FL, α-Syn121 aggregated at an accelerated rate, and formed amorphous aggregates rich in random coil structures rather than β-sheet-rich linear fibrils formed by α-Syn FL. Importantly, higher cytotoxicity with lower membrane disruption capacity was found for α-Syn121 aggregates. Furthermore, α-Syn121 aggregates could activate the apoptosis signaling pathway and stimulate the caspase-1-mediated cleavage of α-Syn FL to generate α-Syn121, which as a result leading to increased levels of endogenous α-Syn121 and intracellular S129 phosphorylated α-Syn inclusions. Together, our data suggests a hidden vicious cycle in PD that α-Syn121 rapidly forms amorphous aggregates, which activate caspase-1 to cleave α-Syn FL and generate more α-Syn121, and this cycle may contribute to the onset and/or pathogenesis of PD.     

Oligomerization of adenosine A2A and dopamine D2 receptors in living cells

We investigated whether oligomerization of adenosine A(2A) receptor (A(2A)R) and dopamine D(2) receptor (D(2)R) exists in living cells using modified bioluminescence resonance energy transfer (BRET(2)) technology. Fusion of these receptors to a donor, Renilla luciferase (Rluc), and to an acceptor, modified green fluorescent protein (GFP(2)), did not affect the ligand binding affinity, subcellular distribution, and coimmunoprecipitation of the receptors. BRET was detected not only between Myc-D(2)R-Rluc and A(2A)R-GFP(2) but also between HA-tagged A(2A)R-Rluc and A(2A)R-GFP(2). These results indicate A(2A)R, either homomeric or heteromeric with D(2)R, exists as an oligomer in living cells.     

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular β-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular β-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.     

Receptor crosstalk: haloperidol treatment enhances A2A adenosine receptor functioning in a transfected cell model

A(2A) adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A(2A) adenosine receptors are regulated by D(2) dopamine receptor ligands. These two receptors are co-expressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A(2A) adenosine receptor functional responses caused by the chronic blockade/activation of D(2) dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A(2A) adenosine receptors induced by antipsychotic drugs, commonly acting as D(2) dopamine receptor antagonists, in a cellular model co-expressing both A(2A) and D(2) receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A(2A) receptor and also affected the degree of A(2A)-D(2) receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A(2A) adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosine-dopamine receptor interaction. Modifications to A(2A) adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A(2A) adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-010-9201-z) contains supplementary material, which is available to authorized users.     

α-Synuclein蛋白聚集状态与帕金森病形成

帕金森病是一种常见的老年神经退行性疾病,其致病机理复杂.其中α-synuclein基因是较早发现的与帕金森病相关的基因,其编码的α-synuclein蛋白是帕金森病神经元内出现的一种蛋白包涵体结构——路易体的主要组成成分.最近的研究结果显示,α-synuclein蛋白存在不同聚集状态间的转换,其中聚集过程中形成的寡聚体中间构象具有较强的细胞毒性,可能对帕金森病的发病过程有着重要作用;而且这种聚集状态的转换过程受到多种遗传学与细胞学因素的影响,从而在某种程度上反映了帕金森病发生形成的遗传学与细胞学机制.本文将对α-synuclein蛋白聚集状态转换特性及其在帕金森病发病过程中作用机制方面的研究进展作一综述．     

Alpha-synuclein sequesters Dnmt1 from the nucleus: a novel mechanism for epigenetic alterations in Lewy body diseases

DNA methylation is a major epigenetic modification that regulates gene expression. Dnmt1, the maintenance DNA methylation enzyme, is abundantly expressed in the adult brain and is mainly located in the nuclear compartment, where it has access to chromatin. Hypomethylation of CpG islands at intron 1 of the SNCA gene has recently been reported to result in overexpression of α-synuclein in Parkinson disease (PD) and related disorders. We therefore investigated the mechanisms underlying altered DNA methylation in PD and dementia with Lewy bodies (DLB). We present evidence of reduction of nuclear Dnmt1 levels in human postmortem brain samples from PD and DLB patients as well as in the brains of α-synuclein transgenic mice models. Furthermore, sequestration of Dnmt1 in the cytoplasm results in global DNA hypomethylation in human and mouse brains, involving CpG islands upstream of SNCA, SEPW1, and PRKAR2A genes. We report that association of Dnmt1 and α-synuclein might mediate aberrant subcellular localization of Dnmt1. Nuclear Dnmt1 levels were partially rescued by overexpression of Dnmt1 in neuronal cell cultures and in α-synuclein transgenic mice brains. Our results underscore a novel mechanism for epigenetic dysregulation in Lewy body diseases, which might underlie the decrease in DNA methylation reported for PD and DLB.     

Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.     

Effect of Pseudorepeat Rearrangement on α-Synuclein Misfolding, Vesicle Binding, and Micelle Binding

The pathological and physiological hallmarks of the protein α-synuclein (aS) are its misfolding into cytotoxic aggregates and its binding to synaptic vesicles, respectively. Both events are mediated by seven 11-residue amphiphilic pseudorepeats and, most generally, involve a transition from intrinsically unstructured conformations to structured conformations. Based on aS interactions with aggregation-inhibiting small molecules, an aS variant termed shuffled α-synuclein (SaS), wherein the first six pseudorepeats had been rearranged, was introduced. Here, the effects of this rearrangement on misfolding, vesicle binding, and micelle binding are examined in reference to aS and β-synuclein to study the sequence characteristics underlying these processes. Fibrillization correlates with the distinct clustering of residues with high β-sheet propensities, while vesicle affinities depend on the mode of pseudorepeat interchange and loss. In the presence of micelles, the pseudorepeat region of SaS adopts an essentially continuous helix, whereas aS and β-synuclein encounter a distinct helix break, indicating that a more homogeneous distribution of surfactant affinities in SaS prevented the formation of an extensive helix break in the micelle-bound state. By demonstrating the importance of the distribution of β-sheet propensities and by revealing inhomogeneous aS surfactant affinities, the present study provides novel insights into two central themes of synuclein biology.     

Quantifying Interactions of β-Synuclein and γ-Synuclein with Model Membranes

The synucleins are a family of proteins involved in numerous neurodegenerative pathologies [α-synuclein and β-synuclein (βS)], as well as in various types of cancers [γ-synuclein (γS)]. While the connection between α-synuclein and Parkinson's disease is well established, recent evidence links point mutants of βS to dementia with Lewy bodies. Overexpression of γS has been associated with enhanced metastasis and cancer drug resistance. Despite their prevalence in such a variety of diseases, the native functions of the synucleins remain unclear. They have a lipid-binding motif in their N-terminal region, which suggests interactions with biological membranes in vivo. In this study, we used fluorescence correlation spectroscopy to monitor the binding properties of βS and γS to model membranes and to determine the free energy of the interactions. Our results show that the interactions are most strongly affected by the presence of both anionic lipids and bilayer curvature, while membrane fluidity plays a very minor role. Quantifying the lipid-binding properties of βS and γS provides additional insights into the underlying factors governing the protein-membrane interactions. Such insights not only are relevant to the native functions of these proteins but also highlight their contributions to pathological conditions that are either mediated or characterized by perturbations of these interactions.     

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.     

Dopamine and the Dopamine Oxidation Product 5,6-Dihydroxylindole Promote Distinct On-Pathway and Off-Pathway Aggregation of α-Synuclein in a pH-Dependent Manner

The deposition of α-synuclein (α-syn) aggregates in dopaminergic neurons is a key feature of Parkinson's disease. While dopamine (DA) can modulate α-syn aggregation, it is unclear which other factors can regulate the actions of DA on α-syn. In this study, we investigated the effect of solution conditions (buffer, salt and pH) on the oligomerization of α-syn by DA. We show that α-syn oligomerization is dependent on the oxidation of DA into reactive intermediates. Under acidic pH conditions, DA is stable, and DA-mediated oligomerization of α-syn is inhibited. From pH 7.0 to pH 11.0, DA is unstable and undergoes redox reactions, promoting the formation of SDS-resistant soluble oligomers of α-syn. We show that the reactive intermediate 5,6-dihydroxylindole mediates the formation of α-syn soluble oligomers under physiological conditions (pH 7.4). In contrast, under acidic conditions (pH 4.0), 5,6-dihydroxylindole promotes the formation of SDS-resistant insoluble oligomers that further associate to form sheet-like fibrils with β-sheet structure that do not bind the dye thioflavin T. These results suggest that distinct reactive intermediates of DA, and not DA itself, interact with α-syn to generate the α-syn aggregates implicated in Parkinson's disease.