Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro

Parkinson's disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha-synuclein (alpha-syn), the protein mutated in some patients with familial PD. The mechanisms of alpha-syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave alpha-syn in vitro. Calpain I cleaved wild-type alpha-syn predominantly after amino acid 57 and within the non-amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized alpha-syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild-type alpha-syn after amino acid 57, this did not occur in mutant A53T alpha-syn. This paucity of proteolysis could increase the stability of A53T alpha-syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild-type alpha-syn. However, once alpha-syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of alpha-syn by cleaving at aberrant sites within the C-terminal region. Elucidating the role of calpain I in the proteolytic processing of alpha-syn in normal and diseased brains may clarify mechanisms of neurodegenerative alpha-synucleinopathies.     

Sensitive fluorescence polarization technique for rapid screening of alpha-synuclein oligomerization/fibrillization inhibitors

Parkinson's disease (PD) is characterized by the accumulation of fibrillar alpha-synuclein (alpha-Syn) inclusions known as Lewy bodies (LBs) and Lewy neurites. Mutations in the alpha-Syn gene or extra copies thereof cause familial PD or dementia with LBs (DLB) in rare kindreds, but abnormal accumulations of wildtype alpha-Syn also are implicated in the pathogenesis of sporadic PD, the most common movement disorder. Insights into mechanisms underlying alpha-Syn mediated neurodegeneration link alpha-Syn oligomerization and fibrillization to the onset and progression of PD. Thus, inhibiting alpha-Syn oligomer or fibril formation is a compelling target for discovering disease modifying therapies for PD, DLB, and related synucleinopathies. Although amyloid dyes recognize alpha-Syn fibrils, efficient detection of soluble oligomers remains a challenge. Here, we report a novel fluorescence polarization (FP) technique for examining alpha-Syn assembly by monitoring changes in its relative molecular mass during progression of normal alpha-Syn from highly soluble monomers to higher order multimers and thence insoluble amyloid fibrils. We report that FP is more sensitive than conventional amyloid dye methods for the quantification of mature fibrils, and that FP is capable of detecting oligomeric alpha-Syn, allowing for rapid automated screening of potential inhibitors of alpha-Syn oligomerization and fibrillization. Furthermore, FP can be combined with an amyloid dye in a single assay that simultaneously provides two independent biophysical readouts for monitoring alpha-Syn fibrillization. Thus, this FP method holds potential to accelerate discovery of disease modifying therapies for LB PD, DLB, and related neurodegenerative synucleinopathies.     

Reversible inhibition of alpha-synuclein fibrillization by dopaminochrome-mediated conformational alterations

Previous studies demonstrated that alpha-synuclein (alpha-syn) fibrillization is inhibited by dopamine, and studies to understand the molecular basis of this process were conducted (Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T., Jr. (2001) Science 294, 1346-1349). Dopamine inhibition of alpha-syn fibrillization generated exclusively spherical oligomers that depended on dopamine autoxidation but not alpha-syn oxidation, because mutagenesis of Met, His, and Tyr residues in alpha-syn did not abrogate this inhibition. However, truncation of alpha-syn at residue 125 restored the ability of alpha-syn to fibrillize in the presence of dopamine. Mutagenesis and competition studies with specific synthetic peptides identified alpha-syn residues 125-129 (i.e. YEMPS) as an important region in the dopamine-induced inhibition of alpha-syn fibrillization. Significantly, the dopamine oxidation product dopaminochrome was identified as a specific inhibitor of alpha-syn fibrillization. Dopaminochrome promotes the formation of spherical oligomers by inducing conformational changes, as these oligomers regained the ability to fibrillize by simple denaturation/renaturation. Taken together, these data indicate that dopamine inhibits alpha-syn fibrillization by inducing structural changes in alpha-syn that can occur through the interaction of dopaminochrome with the 125YEMPS129 motif of alpha-syn. These results suggest that the dopamine autoxidation can prevent alpha-syn fibrillization in dopaminergic neurons through a novel mechanism. Thus, decreased dopamine levels in substantia nigra neurons might promote alpha-syn aggregation in Parkinson's disease.     

Functional consequences of alpha-synuclein tyrosine nitration: diminished binding to lipid vesicles and increased fibril formation

Previous studies have shown the presence of nitrated alpha-synuclein (alpha-syn) in human Lewy bodies and other alpha-syn inclusions. Herein, the effects of tyrosine nitration on alpha-syn fibril formation, lipid binding, chaperone-like function, and proteolytic degradation were systematically examined by employing chromatographically isolated nitrated monomeric, dimeric, and oligomeric alpha-syn. Nitrated alpha-syn monomers and dimers but not oligomers accelerated the rate of fibril formation of unmodified alpha-syn when present at low concentrations. Immunoelectron microscopy revealed that nitrated monomers and dimers are incorporated into the fibrils. However, the purified nitrated alpha-syn monomer by itself was unable to form fibrils. Nitration of the tyrosine residue at position 39 was largely responsible for decreased binding of nitrated monomeric alpha-syn to synthetic vesicles, which correlated with an impairment of the nitrated protein to adopt alpha-helical conformation in the presence of liposomes. The chaperone-like activity of alpha-syn was not inhibited by nitration or oxidation. Furthermore, the 20 S proteasome and calpain I degraded nitrated monomeric alpha-syn, although at a slower rate compared with control alpha-syn. Collectively, these data suggest that post-translational modification of alpha-syn by nitration can promote the formation of intracytoplasmic inclusions that constitute the hallmark of Parkinson disease and other synucleinopathies.     

Role of alpha-synuclein carboxy-terminus on fibril formation in vitro

Alpha-synuclein (alpha-syn) is the major component of intracellular inclusions in several neurodegenerative diseases, and the conversion of soluble alpha-syn into filamentous aggregates may contribute to disease pathogenesis. Since mechanisms leading to the formation of alpha-syn inclusions are unclear, in vitro models of alpha-syn aggregation may yield insights into this process. To that end, we examined the consequences on the progressive deletion of the carboxy-terminus of alpha-syn in regulating fibril formation, and we show here that carboxy-terminal truncated alpha-syn proteins aggregate faster than the full-length molecule. Protease digestion and immunoelectron microscopy indicate that the alpha-syn amino- and carboxy-termini are more solvent exposed than the central core and that filaments formed from carboxy-terminal truncated alpha-syn are narrower in diameter than the full-length molecule. Moreover, seeding experiments under conditions where full-length alpha-syn did not readily aggregate revealed that carboxy-truncated alpha-syn extending from amino acids 1-102 and 1-110 but not 1-120 were efficient in seeding full-length alpha-syn aggregation over a range of concentrations. Using site-directed mutagenesis, the negatively charged residues 104, 105 and 114, 115 in the carboxy-terminus were implicated in this reduced aggregation and the lack of seeding of full-length alpha-syn fibrillogenesis by 1-120. Our data support the view that the middle region of alpha-syn forms the core of alpha-syn filaments and that negative charges in the carboxy-terminus counteract alpha-syn aggregation. Thus, the carboxy-terminus of alpha-syn may regulate aggregation of full-length alpha-syn and determine the diameter of alpha-syn filaments.     

Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid

Two missense mutations in the gene encoding alpha-synuclein have been linked to rare, early-onset forms of Parkinson's disease (PD). These forms of PD, as well as the common idiopathic form, are characterized by the presence of cytoplasmic neuronal deposits, called Lewy bodies, in the affected region of the brain. Lewy bodies contain alpha-synuclein in a form that resembles fibrillar Abeta derived from Alzheimer's disease (AD) amyloid plaques. One of the mutant forms of alpha-synuclein (A53T) fibrillizes more rapidly in vitro than does the wild-type protein, suggesting that a correlation may exist between the rate of in vitro fibrillization and/or oligomerization and the progression of PD, analogous to the relationship between Abeta fibrillization in vitro and familial AD. In this paper, fibrils generated in vitro from alpha-synuclein, wild-type and both mutant forms, are shown to possess very similar features that are characteristic of amyloid fibrils, including a wound and predominantly unbranched morphology (demonstrated by atomic force and electron microscopies), distinctive dye-binding properties (Congo red and thioflavin T), and antiparallel beta-sheet structure (Fourier transform infrared spectroscopy and circular dichroism spectroscopy). alpha-Synuclein fibrils are relatively resistant to proteolysis, a property shared by fibrillar Abeta and the disease-associated fibrillar form of the prion protein. These data suggest that PD, like AD, is a brain amyloid disease that, unlike AD, is characterized by cytoplasmic amyloid (Lewy bodies). In addition to amyloid fibrils, a small oligomeric form of alpha-synuclein, which may be analogous to the Abeta protofibril, was observed prior to the appearance of fibrils. This species or a related one, rather than the fibril itself, may be responsible for neuronal death.     

Alpha-synuclein-induced aggregation of cytoplasmic vesicles in Saccharomyces cerevisiae

Aggregated alpha-synuclein (alpha-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson's disease (PD) and related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies have shown that when overexpressed in Saccharomyces cerevisiae, alpha-syn localizes to plasma membranes and forms cytoplasmic accumulations similar to human alpha-syn inclusions. However, the exact nature, composition, temporal evolution, and underlying mechanisms of yeast alpha-syn accumulations and their relevance to human synucleinopathies are unknown. Here we provide ultrastructural evidence that alpha-syn accumulations are not comprised of LB-like fibrils, but are associated with clusters of vesicles. Live-cell imaging showed alpha-syn initially localized to the plasma membrane and subsequently formed accumulations in association with vesicles. Imaging of truncated and mutant forms of alpha-syn revealed the molecular determinants and vesicular trafficking pathways underlying this pathological process. Because vesicular clustering is also found in LB-containing neurons of PD brains, alpha-syn-mediated vesicular accumulation in yeast represents a model system to study specific aspects of neurodegeneration in PD and related synucleinopathies.     

Biochemical and morphological classification of disease-associated alpha-synuclein mutants aggregates

Alpha-synuclein (a-syn) aggregation in brain is implicated in several synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Until date, at least six disease-associated mutations in a-syn (namely A30P, E46K, H50Q, G51D, A53T, and A53E) are known to cause dominantly inherited familial forms of synucleinopathies. Previous studies using recombinant proteins have reported that a subset of disease-associated mutants show higher aggregation propensities and form spectroscopically distinguishable aggregates compared to wild-type (WT). However, morphological and biochemical comparison of the aggregates for all disease-associated a-syn mutants have not yet been performed. In this study, we performed electron microscopic examination, guanidinium hydrochloride (GdnHCl) denaturation, and protease digestion to classify the aggregates from their respective point mutations. Using electron microscopy we observed variations of amyloid fibrillar morphologies among the aggregates of a-syn mutants, mainly categorized into two groups: twisted fibrils observed for both WT and E46K while straight fibrils for the other mutants. GdnHCl denaturation experiments revealed the a-syn mutants except for E46K were more resistant than WT against the denaturation. Mass spectrometry analysis of protease-treated aggregates showed a variety of protease-resistant cores, which may correspond to their morphological properties. The difference of their properties could be implicated in the clinicopathological difference of synucleinopathies with those mutations.     

Formation of a high affinity lipid-binding intermediate during the early aggregation phase of alpha-synuclein

The alpha-synuclein (alpha-syn) protein is clearly implicated in Parkinson's disease (PD). Mutations or triplication of the alpha-syn gene leads to early onset PD, possibly by accelerating alpha-syn oligomerization. alpha-syn interacts with lipids, and this membrane binding activity may relate to its toxic activity. To understand how the alpha-syn aggregation state affects its lipid binding activity we used surface plasmon resonance to study the interaction of wild-type and mutant alpha-syn with a charged phospholipid membrane, as a function of its aggregation state. Apparent dissociation constants for alpha-syn indicated that an intermediate species, present during the lag phase of amyloid formation, binds with an increased affinity to the membrane surface. Formation of this species was dependent upon the rate of fibril formation. Fluorescence anisotropy studies indicate that only upon the formation of amyloid material can alpha-syn perturb the acyl-chain region of the lipid bilayer. Circular dichroism spectroscopy showed that upon aging, both wild-type and mutant alpha-syn lose their ability to form lipid-bound alpha-helical species once they become fibrillar. These results indicate that alpha-syn forms a high affinity lipid binding intermediate species during fibril formation. Oligomeric alpha-syn is known to be toxic, and it is feasible that the high affinity binding species described here may correspond to a toxic species involved in PD.     

Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p

The [URE3] prion of Saccharomyces cerevisiae shares many features with mammalian prions and poly-glutamine related disorders and has become a model for studying amyloid diseases. The development of the [URE3] phenotype is thought to be caused by a structural switch in the Ure2p protein. In [URE3] cells, Ure2p is found predominantly in an aggregated state, while it is a soluble dimer in wild-type cells. In vitro, Ure2p forms fibrils with amyloid-like properties. Several studies suggest that the N-terminal domain of Ure2p is essential for prion formation. In this work, we investigated the fibril formation of Ure2p by isolating soluble oligomeric species, which are generated during fibrillization, and characterized them with respect to size and structure. Our data support the critical role of the N-terminal domain for fibril formation, as we observed fibrils in the presence of 5 M guanidinium chloride, conditions at which the C-terminal domain is completely unfolded. Based on fluorescence measurements, we conclude that the structure of the C-terminal domain is very similar in dimeric and fibrillar Ure2p. When studying the time course of fibrillization, we detected the formation of small, soluble oligomeric species during the early stages of the process. Their remarkable resistance against denaturants, their increased content of beta-structure, and their ability to 'seed' Ure2p fibrillization suggest that conversion to the amyloid-like conformation has already occurred. Thus, they likely represent critical intermediates in the fibrillization pathway of Ure2p.     

Inhibition of fibrillization and accumulation of prefibrillar oligomers in mixtures of human and mouse alpha-synuclein

Parkinson's disease (PD) is a neurodegenerative disorder attributed to the loss of dopaminergic neurons from the substantia nigra. Some surviving neurons are characterized by cytoplasmic Lewy bodies, which contain fibrillar alpha-synuclein. Two mutants of human alpha-synuclein (A53T and A30P) have been linked to early-onset, familial PD. Oligomeric forms of these mutants accumulate more rapidly and/or persist for longer periods of time than oligomeric, human wild-type alpha-synuclein (WT), suggesting a link between oligomerization and cell death. The amino acid sequences of the mouse protein and WT differ at seven positions. Mouse alpha-synuclein, like A53T, contains a threonine residue at position 53. We have assessed the conformational properties and fibrillogenicity of the murine protein. Like WT and the two PD mutants, mouse alpha-synuclein adopts a "natively unfolded" or disordered structure. However, at elevated concentrations, the mouse protein forms amyloid fibrils more rapidly than WT, A53T, or A30P. The fibrillization of mouse alpha-synuclein is slowed by WT and A53T. Inhibition of fibrillization leads to the accumulation of nonfibrillar, potentially toxic oligomers. The results are relevant to the interpretation of the phenotypes of transgenic animal models of PD and suggest a novel approach for testing the cause and effect relationship between fibrillization and neurodegeneration.     

PA700, the regulatory complex of the 26S proteasome, interferes with alpha-synuclein assembly

Parkinson's disease is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway accompanied by the presence of intracellular cytoplasmic inclusions, termed Lewy bodies. Fibrillized alpha-synuclein forms the major component of Lewy bodies. We reported a specific interaction between rat alpha-synuclein and tat binding protein 1, a subunit of PA700, the regulatory complex of the 26S proteasome. It has been demonstrated that PA700 prevents the aggregation of misfolded, nonubiquinated substrates. In this study, we examine the effect of PA700 on the aggregation of wild-type and A53T mutant alpha-synuclein. PA700 inhibits both wild-type and A53T alpha-synuclein fibril formation as measured by Thioflavin T fluorescence. Using size exclusion chromatography, we present evidence for a stable PA700-alpha-synuclein complex. Sedimentation analyses reveal that PA700 sequesters alpha-synuclein in an assembly incompetent form. Analysis of the morphology of wild-type and A53T alpha-synuclein aggregates during the course of fibrillization by electron microscopy demonstrate the formation of amyloid-like fibrils. Secondary structure analyses of wild-type and A53T alpha-synuclein assembled in the presence of PA700 revealed a decrease in the overall amount of assembled alpha-synuclein with no significant change in protein conformation. Thus, PA700 acts on alpha-synuclein assembly and not on the structure of fibrils. We hypothesize that PA700 sequesters alpha-synuclein oligomeric species that are the precursors of the fibrillar form of the protein, thus preventing its assembly into fibrils.     

Assembly of lysine 63-linked ubiquitin conjugates by phosphorylated alpha-synuclein implies Lewy body biogenesis

alpha-Synuclein (alpha-syn) and ubiquitin (Ub) are major protein components deposited in Lewy bodies (LBs) and Lewy neurites, which are pathologic hallmarks of idiopathic Parkinson disease (PD). Almost 90% of alpha-syn in LBs is phosphorylated at serine 129 (Ser(129)). However, the role of Ser(129)-phosphorylated alpha-syn in the biogenesis of LBs remains unclear. Here, we show that compared with coexpression of wild type (WT)alpha-syn and Ub, coexpression of phospho-mimic mutant alpha-syn (S129D) and Ub in neuro2a cells results in an increase of Ub-conjugates and the formation of ubiquitinated inclusions. Furthermore, S129D alpha-syn fails to increase the Ub-conjugates and form ubiquitinated inclusions in the presence of a K63R mutant Ub. In addition, as compared with WT alpha-syn, S129D alpha-syn increased cytoplasmic and neuritic aggregates of itself in neuro2a cells treated with H(2)O(2) and serum deprivation. These results suggest that the contribution of Ser(129)-phosphorylated alpha-syn to the Lys(63)-linked Ub-conjugates and aggregation of itself may be involved in the biogenesis of LBs in Parkinson disease and other related synucleinopathies.     

Cellular oligomerization of alpha-synuclein is determined by the interaction of oxidized catechols with a C-terminal sequence

The mechanisms that govern the formation of alpha-synuclein (alpha-syn) aggregates are not well understood but are considered a central event in the pathogenesis of Parkinson's disease (PD). A critically important modulator of alpha-syn aggregation in vitro is dopamine and other catechols, which can prevent the formation of alpha-syn aggregates in cell-free and cellular model systems. Despite the profound importance of this interaction for the pathogenesis of PD, the processes by which catechols alter alpha-syn aggregation are unclear. Molecular and biochemical approaches were employed to evaluate the mechanism of catechol-alpha-syn interactions and the effect on inclusion formation. The data show that the intracellular inhibition of alpha-syn aggregation requires the oxidation of catechols and the specific noncovalent interaction of the oxidized catechols with residues (125)YEMPS(129) in the C-terminal region of the protein. Cell-free studies using novel near infrared fluorescence methodology for the detection of covalent protein-ortho-quinone adducts showed that although covalent modification of alpha-syn occurs, this does not affect alpha-syn fibril formation. In addition, oxidized catechols are unable to prevent both thermal and acid-induced protein aggregation as well as fibrils formed from a protein that lacks a YEMPS amino acid sequence, suggesting a specific effect for alpha-syn. These results suggest that inappropriate C-terminal cleavage of alpha-syn, which is known to occur in vivo in PD brain or a decline of intracellular catechol levels might affect disease progression, resulting in accelerated alpha-syn inclusion formation and dopaminergic neurodegeneration.     

Acceleration of alpha-synuclein aggregation by homologous peptides

alpha-Synuclein (alpha-Syn), amyloid beta-protein and prion protein are among the amyloidogenic proteins that are associated with the neurodegenerative diseases. These three proteins share a homologous region with a consensus sequence mainly consisting of glycine, alanine and valine residues (accordingly named as the GAV motif), which was proposed to be the critical core for the fibrillization and cytotoxicity. To understand the role of the GAV motif in protein amyloidogenesis, we studied the effects of the homologous peptides corresponding to the sequence of GAV motif region (residues 66-74) on alpha-Syn aggregation. The result shows that these peptides can promote fibrillization of wild-type alpha-Syn and induce that of the charge-incorporated mutants but not the GAV-deficient alpha-Syn mutant. The acceleration of alpha-Syn aggregation by the homologous peptides is under a sequence-specific manner. The interplay between the GAV peptide and the core regions in alpha-Syn may accelerate the aggregation process and stabilize the fibrils. This finding provides clues for developing peptide mimics that could promote transforming the toxic oligomers or protofibrils into the inert mature fibrils.     

Observation of multiple intermediates in alpha-synuclein fibril formation by singular value decomposition analysis

One of the most well known characteristics for Parkinson's disease (PD) is a polymerization of wild-type or mutant alpha-synuclein into aggregates and fibrils, commonly observed as Lewy bodies and Lewy neuritis in PD patients. Although numerous studies on alpha-synuclein fibrillation have been reported, the molecular mechanisms of aggregation and fibrillation are not well understood yet. In the present study, structural properties and propensities to form fibrils of wild-type, A30P, E46K, and A53T alpha-synucleins were investigated using fluorescence and circular dichroism (CD) methods. The results from these studies were analyzed using singular value decomposition (SVD) method which estimates a number of conformationally independent species for a given process. The time-dependent CD spectra of the wild-type alpha-synuclein indicated a multi-step process in the fibril formation, and SVD analysis using the time-dependent CD spectra revealed that five or nine intermediates were formed at the early stage of fibrillation.     

A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly

Neuronal and oligodendrocytic aggregates of fibrillar alpha-synuclein define several diseases of the nervous system. It is likely that these inclusions impair vital metabolic processes and compromise viability of affected cells. Here, we report that a 12-amino acid stretch ((71)VTGVTAVAQKTV(82)) in the middle of the hydrophobic domain of human alpha-synuclein is necessary and sufficient for its fibrillization based on the following observations: 1) human beta-synuclein is highly homologous to alpha-synuclein but lacks these 12 residues, and it does not assemble into filaments in vitro; 2) the rate of alpha-synuclein polymerization in vitro decreases after the introduction of a single charged amino acid within these 12 residues, and a deletion within this region abrogates assembly; 3) this stretch of 12 amino acids appears to form the core of alpha-synuclein filaments, because it is resistant to proteolytic digestion in alpha-synuclein filaments; and 4) synthetic peptides corresponding to this 12-amino acid stretch self-polymerize to form filaments, and these peptides promote fibrillization of full-length human alpha-synuclein in vitro. Thus, we have identified key sequence elements necessary for the assembly of human alpha-synuclein into filaments, and these elements may be exploited as targets for the design of drugs that inhibit alpha-synuclein fibrillization and might arrest disease progression.     

In vitro tau fibrillization: mapping protein regions

We have investigated the propensity to form fibrillar aggregates of a variety of fragments and variants of the tau protein under the influence of a tau fibrillization inducer: coenzyme Q(0). To better identify fibrillization hotspots, we compare the polymerization propensity of tau fragments containing the sequence of putative hotspots with that of tau variants with that same sequence deleted. We also investigate the effects of biologically occurring modifications such as phosphorylation and deamidation. We found that residues 305 to 335 are essential for in vitro tau fibrillization. Residues 306 to 311 facilitate in vitro assembly, but are not sufficient to mimic the in vivo fibrillization of tau. Furthermore, the propensity of the 306-311 sequence to form fibrils is highly decreased by chemical modifications of tyrosine 310 that are commonly found in vivo.     

Dependence of alpha-synuclein aggregate morphology on solution conditions

Alpha-synuclein is the major component of Lewy bodies and Lewy neurites, which are granular and filamentous protein inclusions that are the defining pathological features of several neurodegenerative conditions such as Parkinson's disease. Fibrillar aggregates formed from alpha-synuclein in vitro resemble brain-derived material, but the role of such aggregates in the etiology of Parkinson's disease and their relation to the toxic molecular species remain unclear. In this study, we investigated the effects of pH and salt concentration on the in vitro assembly of human wild-type alpha-synuclein, particularly with regard to aggregation rate and aggregate morphology. Aggregates formed at pH 7.0 and pH 6.0 in the absence of NaCl and MgCl(2) were fibrillar; the pH 6.0 fibrils displayed a helical twist, as clearly evident by scanning force and electron microscopy. Incubations at pH 7.0 remained transparent during the process of aggregation and exhibited strong thioflavin-T and weak 8-anilino-1-naphthalenesulfonate (ANS) binding; furthermore, they were efficient in seeding fibrillization of fresh solutions. In contrast, incubating alpha-synuclein at low pH (pH 4.0 or pH 5.0) resulted in the rapid formation of turbid suspensions characterized by strong ANS binding, reduced thioflavin-T binding and reduced seeding efficiency. At pH 4.0, fibril formation was abrogated; instead, very large aggregates (dimensions approximately 100 microm) of amorphous appearance were visible by light microscopy. As with acidic conditions, addition of 0.2M NaCl or 10mM MgCl(2) to pH 7.0 incubations led to a shorter aggregation lag time and formation of large, amorphous aggregates. These results demonstrate that the morphology of alpha-synuclein aggregates is highly sensitive to solution conditions, implying that the fibrillar state does not necessarily represent the predominant or most functionally significant aggregated state under physiological conditions.     

A study of the regional effects of alpha-synuclein on the organization and stability of phospholipid bilayers

Associations between the protein alpha-synuclein (alpha-syn) and presynaptic vesicles have been implicated in synaptic plasticity and neurotransmitter release and may also affect how the protein aggregates into fibrils found in Lewy bodies, the cellular inclusions associated with neurodegenerative diseases. This work investigated how alpha-syn interacts with model phospholipid membranes and examined what effect protein binding has upon the physical properties of lipid bilayers. Wide line 2H and 31P NMR spectra of phospholipid vesicles revealed that alpha-syn associates with membranes containing lipids with anionic headgroups and can disrupt the integrity of the lipid bilayer, but the protein has little effect on membranes of zwitterionic phosphatidylcholine. A peptide, alpha-syn(10-48), which corresponds to the lysine-rich N-terminal region of alpha-syn, was found to associate with lipid headgroups with a preference for a negative membrane surface charge. Another peptide, alpha-syn(120-140), which corresponds to the glutamate-rich C-terminal region, also associates weakly with lipid headgroups but with a slightly higher affinity for membranes with no net surface charge than for negatively charged membrane surfaces. Binding of alpha-syn(10-48) and alpha-syn(120-140) to the lipid vesicles did not disrupt the lamellar structure of the membranes, but both peptides appeared to induce the lateral segregation of the lipids into clusters of acidic lipid-enriched and acidic lipid-deficient domains. From these findings, it is speculated that the N-terminal and C-terminal domains of full-length alpha-syn might act in concert to organize the membrane components during normal protein function and perhaps play a role in presynaptic vesicle synthesis, maintenance, and fusion.