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.     

Evidence for a partially folded intermediate in alpha-synuclein fibril formation

Intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) of alpha-synuclein are hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple systemic atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into such filamentous inclusions remain unknown. An intriguing aspect of this problem is that alpha-synuclein is a natively unfolded protein, with little or no ordered structure under physiological conditions. This raises the question of how an essentially disordered protein is transformed into highly organized fibrils. In the search for an answer to this question, we have investigated the effects of pH and temperature on the structural properties and fibrillation kinetics of human recombinant alpha-synuclein. Either a decrease in pH or an increase in temperature transformed alpha-synuclein into a partially folded conformation. The presence of this intermediate is strongly correlated with the enhanced formation of alpha-synuclein fibrils. We propose a model for the fibrillation of alpha-synuclein in which the first step is the conformational transformation of the natively unfolded protein into the aggregation-competent partially folded intermediate.     

Association of alpha-synuclein and mutants with lipid membranes: spin-label ESR and polarized IR

Alpha-synuclein is a presynaptic protein, the A53T and A30P mutants of which are linked independently to early-onset familial Parkinson's disease. The association of wild-type alpha-synuclein with lipid membranes was characterized previously by electron spin resonance (ESR) spectroscopy with spin-labeled lipids [Ramakrishnan, M., Jensen, P. H., and Marsh, D. (2003) Biochemistry 42, 12919-12926]. Here, we study the interaction of the A53T and A30P alpha-synuclein mutants and a truncated form that lacks the acidic C-terminal domain with phosphatidylglycerol bilayer membranes, using anionic phospholipid spin labels. The strength of the interaction with phosphatidylglycerol membranes lies in the order: wild type approximately truncated > A53T > A30P > fibrils approximately 0, and only the truncated form interacts with phosphatidylcholine membranes. The selectivity of the interaction of the mutant alpha-synucleins with different spin-labeled lipid species is reduced considerably, relative to the wild-type protein, whereas that of the truncated protein is increased. Polarized infrared (IR) spectroscopy is used to study the interactions of the wild-type and truncated proteins with aligned lipid membranes and additionally to characterize the fibrillar form. Wild-type alpha-synuclein is natively unfolded in solution and acquires secondary structure upon binding to membranes containing phosphatidylglycerol. Up to 30-40% of the amide I band intensity of the membrane-bound wild-type and truncated proteins is attributable to beta-sheet structure, at the surface densities used for IR spectroscopy. The remainder is alpha-helix and residual unordered structure. Fibrillar alpha-synuclein contains 62% antiparallel beta-sheet and is oriented on the substrate surface but does not interact with deposited lipid membranes. The beta-sheet secondary-structural elements of the wild-type and truncated proteins are partially oriented on the surface of membranes with which they interact.     

Interaction of alpha-synuclein and synphilin-1: effect of Parkinson's disease-associated mutations

alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1. A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1. The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1. However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1.     

Alpha-synuclein targets the plasma membrane via the secretory pathway and induces toxicity in yeast

A pathological feature of Parkinson's disease is the presence of Lewy bodies within selectively vulnerable neurons. These are ubiquitinated cytoplasmic inclusions containing alpha-synuclein, an abundant protein normally associated with presynaptic terminals. Point mutations in the alpha-synuclein gene (A30P and A53T), as well as triplication of the wild-type (WT) locus, have been linked to autosomal dominant Parkinson's. How these alterations might contribute to disease progression is unclear. Using the genetically tractable yeast Saccharomyces cerevisiae as a model system, we find that both the WT and the A53T isoforms of alpha-synuclein initially localize to the plasma membrane, to which they are delivered via the classical secretory pathway. In contrast, the A30P mutant protein disperses within the cytoplasm and does not associate with the plasma membrane, and its intracellular distribution is unaffected by mutations in the secretory pathway. When their expression is elevated, WT and A53T, but not A30P, are toxic to cells. At moderate levels of expression, WT and A53T induce the cellular stress (heat-shock) response and are toxic to cells bearing mutations in the 20S proteasome. Our results reveal a link between plasma membrane targeting of alpha-synuclein and its toxicity in yeast and suggest a role for the quality control (QC) system in the cell's effort to deal with this natively unfolded protein.     

Mutation E46K increases phospholipid binding and assembly into filaments of human alpha-synuclein

Missense mutations (A30P and A53T) in alpha-synuclein and the overproduction of the wild-type protein cause familial forms of Parkinson's disease and dementia with Lewy bodies. Alpha-synuclein is the major component of the filamentous Lewy bodies and Lewy neurites that define these diseases at a neuropathological level. Recently, a third missense mutation (E46K) in alpha-synuclein was described in an inherited form of dementia with Lewy bodies. Here, we have investigated the functional effects of this novel mutation on phospholipid binding and filament assembly of alpha-synuclein. When compared to the wild-type protein, the E46K mutation caused a significantly increased ability of alpha-synuclein to bind to negatively charged liposomes, unlike the previously described mutations. The E46K mutation increased the rate of filament assembly to the same extent as the A53T mutation. Filaments formed from E46K alpha-synuclein often had a twisted morphology with a cross-over spacing of 43 nm. The observed effects on lipid binding and filament assembly may explain the pathogenic nature of the E46K mutation in alpha-synuclein.     

Vesicle permeabilization by protofibrillar alpha-synuclein: implications for the pathogenesis and treatment of Parkinson's disease.

Fibrillar alpha-synuclein is a component of the Lewy body, the characteristic neuronal inclusion of the Parkinson's disease (PD) brain. Both alpha-synuclein mutations linked to autosomal dominant early-onset forms of PD promote the in vitro conversion of the natively unfolded protein into ordered prefibrillar oligomers, suggesting that these protofibrils, rather than the fibril itself, may induce cell death. We report here that protofibrils differ markedly from fibrils with respect to their interactions with synthetic membranes. Protofibrillar alpha-synuclein, in contrast to the monomeric and the fibrillar forms, binds synthetic vesicles very tightly via a beta-sheet-rich structure and transiently permeabilizes these vesicles. The destruction of vesicular membranes by protofibrillar alpha-synuclein was directly observed by atomic force microscopy. The possibility that the toxicity of alpha-synuclein fibrillization may derive from an oligomeric intermediate, rather than the fibril, has implications regarding the design of therapeutics for PD.     

Isomers of human alpha-synuclein stabilized by disulfide bonds exhibit distinct structural and aggregative properties

The discovery of three mutants in the -synuclein (alphaSyn) gene and the identification of alphaSyn as the major component of Lewy body have opened a new field for understanding the pathogenesis of Parkinson's disease (PD). AlphaSyn is a natively unfolded protein with unknown function and unspecified conformational heterogeneity. In this study, we introduce four Ser/Ala --> Cys mutations at positions 9, 42, 69, and 89 in human wild-type alphaSyn (wt-alphaSyn) and two PD-associated alphaSyn mutants, A30P-alphaSyn and A53T-alphaSyn. This allows expression of three alphaSyn mutants, wt-alphaSyn(4C), A30P-alphaSyn(4C), and A53T-Syn(4C). Subsequent oxidative folding enables each alphaSyn(4C) mutant to form three partially stabilized two-disulfide isomers, designated as alphaSyn(2SS), that are amenable to further isolation and characterization. These alphaSyn mutants exhibit the following properties. (a) A30P-alphaSyn(4C) exhibits a lower folding flexibility than wt-alphaSyn(4C) and A53T-alphaSyn(4C). (b) All three alphaSyn(4C) mutants, like wt-alphaSyn, exhibit a predominant structure of random coil. However, wt-alphaSyn(2SS) adopts an alpha-helical conformation, whereas A30P-alphaSyn(2SS) and A53T-alphaSyn(2SS) take on significant beta-sheet structure. (c) A30P-alphaSyn(2SS) shows a stronger tendency to aggregate than A53T-alphaSyn(2SS) and wt-alphaSyn(2SS). (d) Three isolated isomers of wt-alphaSyn(2SS) exhibit a propensity for forming oligomers different yet enhanced versus that for wt-alphaSyn. These data together substantiate the notion that under physiological conditions, human alphaSyn exists as diverse conformational isomers which exhibit distinct propensities for aggregation and fibril formation.     

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.     

A Raman optical activity study of rheomorphism in caseins, synucleins and tau. New insight into the structure and behaviour of natively unfolded proteins.

The casein milk proteins and the brain proteins alpha-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of alpha-synuclein and tau, have a propensity to form the fibrils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with differential scanning calorimetry, of bovine beta- and kappa-casein, recombinant human alpha-, beta- and gamma-synuclein, together with the A30P and A53T mutants of alpha-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau 46 together with the tau 46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at approximately 1318 cm(-1) that may be due to the poly(l-proline) II (PPII) helical conformation. There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated beta strand may be present. Differential scanning calorimetry revealed no thermal transitions for these proteins in the range 15-110 degrees C, suggesting that the structures are loose and noncooperative. As it is extended, flexible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (flowing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of alpha-synuclein and tau, result in a propensity for pathological fibril formation due to particular residue properties.     

Membrane association and protein conformation of alpha-synuclein in intact neurons. Effect of Parkinson's disease-linked mutations

Two missense mutations (Ala-30 --> Pro and Ala-53 --> Thr) in the gene encoding alpha-synuclein are associated with rare autosomal dominant forms of familial Parkinson's disease. In addition, alpha-synuclein is an abundant component of Lewy bodies in sporadic Parkinson's disease and diffuse Lewy body disease. However, the normal conformation of alpha-synuclein, its cellular localization in neurons, and the effects of the mutations remain to be determined. In the present study, we examine these questions using sensitive fluorescence resonance energy transfer techniques. Transient transfection of alpha-synuclein expression constructs into primary cortical neurons and counterstaining with the lipophilic fluorescent marker, DiI, demonstrates a close association between alpha-synuclein and cellular membranes. Both the N- and C-terminal regions of alpha-synuclein are tightly associated with membranes. A weak interaction also occurs between the N and C termini themselves. The Parkinson's disease-associated mutations have no effect on membrane interaction; however, the Ala-30 --> Pro mutation alters the three-dimensional conformation of alpha-synuclein, as measured by significantly increased fluorescence resonance energy transfer between the N and C termini.     

Enhanced oligomerization of the alpha-synuclein mutant by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

The alpha-synuclein is a major component of Lewy bodies that are found in the brains of patients with Parkinson's disease (PD). Also, two point mutations in this protein, A53T and A30P, are associated with rare familial forms of the disease. We investigated whether there are differences in the Cu,Zn-SOD and hydrogen peroxide system mediated-protein modification between the wild-type and mutant alpha-synucleins. When alpha-synuclein was incubated with both Cu,Zn-SOD and H2O2, then the amount of A53T mutant oligomerization increased relative to that of the wild-type protein. This process was inhibited by radical scavenger, spin-trapping agent, and copper chelator. These results suggest that the oligomerization of alpha-synuclein is mediated by the generation of the hydroxyl radical through the metal-catalyzed reaction. The dityrosine formation of the A53T mutant protein was enhanced relative to that of the wild-type protein. Antioxidant molecules, carnosine, and anserine effectively inhibited the wild-type and mutant proteins' oligomerization. Therefore, these compounds may be explored as potential therapeutic agents for PD patients. The present experiments, in part, may provide an explanation for the association between PD and the alpha-synuclein mutant.     

Rat alpha-synuclein interacts with Tat binding protein 1, a component of the 26S proteasomal complex

The alpha-synuclein gene, which encodes a brain presynaptic nerve terminal protein of unknown function, is linked to familial early-onset Parkinson's disease (PD). The finding that alpha-synuclein forms the major fibrillary component of Lewy bodies in brains of PD patients suggests that the two point mutations in alpha-synuclein (Ala(53)Thr, Ala(30)Pro) may promote the aggregation of alpha-synuclein into filaments. To address the role of alpha-synuclein in neurodegenerative diseases, we performed a yeast two-hybrid screen of a rat adult brain cDNA library using rat alpha-synuclein 2 (alphaSYN2). Here we report that alphaSYN2 interacts specifically with Tat binding protein 1, a subunit of the 700-kDa proteasome activator (PA700), the regulatory complex of the 26S proteasome and of the modulator complex, which enhances PA700 activation of the proteasome.     

Alpha-Synuclein is degraded by both autophagy and the proteasome

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of aggregates (Lewy bodies) in neurons. alpha-Synuclein is the major protein in Lewy bodies and rare mutations in alpha-synuclein cause early-onset PD. Consequently, alpha-synuclein is implicated in the pathogenesis of PD. Here, we have investigated the degradation pathways of alpha-synuclein, using a stable inducible PC12 cell model, where the expression of exogenous human wild-type, A30P, or A53T alpha-synuclein can be switched on and off. We have used a panel of inhibitors/stimulators of autophagy and proteasome function and followed alpha-synuclein degradation in these cells. We found that not only is alpha-synuclein degraded by the proteasome, but it is also degraded by autophagy. A role for autophagy was further supported by the presence of alpha-synuclein in organelles with the ultrastructural features of autophagic vesicles. Since rapamycin, a stimulator of autophagy, increased clearance of alpha-synuclein, it merits consideration as a potential therapeutic for Parkinsons disease, as it is designed for chronic use in humans.     

Parkinson's disease-associated alpha-synuclein is more fibrillogenic than beta- and gamma-synuclein and cannot cross-seed its homologs

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in alpha-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes beta- and gamma-synuclein. alpha-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for beta- or gamma-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between alpha- and beta-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of alpha-, beta-, and gamma- synuclein show the same natively unfolded structure. While over time alpha-synuclein forms the previously described fibrils, no fibrils could be detected for beta- and gamma-synuclein under the same conditions. Most importantly, beta- and gamma-synuclein could not be cross-seeded with alpha-synuclein fibrils. However, under conditions that drastically accelerate aggregation, gamma-synuclein can form fibrils with a lag phase roughly three times longer than alpha-synuclein. These results indicate that beta- and gamma-synuclein are intrinsically less fibrillogenic than alpha-synuclein and cannot form mixed fibrils with alpha-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to alpha-synuclein and are also abundant in brain.     

Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson''s disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.     

Effect of reparation of repeat sequences in the human alpha-synuclein on fibrillation ability

The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in the Parkinson's disease. The hexamer motif KTKEGV is found in each of the seven imperfect repeat sequences in the N-terminal half of alpha-synuclein. The motif is not fully conserved in the sixth and seventh repeats. We created mutants in which the motif was repaired to be fully conserved in either (Rep6 and Rep7) or both (Rep67) of these two repeats. The Rep6 and Rep67 mutants showed a greatly reduced propensity to aggregate and fibrillate while all three mutants showed greater resistance to HFIP-induced formation of the alpha-helix intermediate. Resistance to formation in the partially folded intermediate may repress the folding of alpha-synuclein, consequently interfering with the aggregation and fibril formation. These results demonstrated that KTKEGV repeats may have a significant role in keeping native unfolded status of alpha-synuclein.     

Temperature-induced reversible conformational change in the first 100 residues of alpha-synuclein

Natively disordered proteins are a growing class of anomalies to the structure-function paradigm. The natively disordered protein alpha-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. We noticed a dramatic difference in dilute solution 1H-15N Heteronuclear Single Quantum Coherence (HSQC) spectra of wild-type alpha-synuclein and two disease-related mutants (A30P and A53T), with spectra collected at 35 degrees C showing fewer cross-peaks than spectra acquired at 10 degrees C. Here, we show the change to be the result of a reversible conformational exchange linked to an increase in hydrodynamic radius and secondary structure as the temperature is raised. Combined with analytical ultracentrifugation data showing alpha-synuclein to be monomeric at both temperatures, we conclude that the poor quality of the 1H-15N HSQC spectra obtained at 35 degrees C is due to conformational fluctuations that occur on the proton chemical shift time scale. Using a truncated variant of alpha-synuclein, we show the conformational exchange occurs in the first 100 amino acids of the protein. Our data illustrate a key difference between globular and natively disordered proteins. The properties of globular proteins change little with solution conditions until they denature cooperatively, but the properties of natively disordered proteins can vary dramatically with solution conditions.     

Inhibiting aggregation of alpha-synuclein with human single chain antibody fragments

The alpha-synuclein protein has been strongly correlated with Parkinson's disease (PD) and is a major component of the hallmark Lewy body aggregates associated with PD. Two different mutations in the alpha-synuclein gene as well as increased gene dosage of wild-type alpha-synuclein all associate with early onset cases of PD; and transgenic animal models overexpressing alpha-synuclein develop PD symptoms. Alpha-synuclein, a natively unfolded protein, can adopt a number of different folded conformations including a beta-sheet form that facilitates formation of numerous aggregated morphologies, including long fibrils, spherical and linear protofibrils, and smaller aggregates or oligomers. The roles of the various morphologies of alpha-synuclein in the progression of PD are not known, and different species have been shown to be toxic. Here we show that single chain antibody fragments (scFv's) isolated from na?ve phage display antibody libraries can be used to control the aggregation of alpha-synuclein. We isolated an scFv with nanomolar affinity for monomeric alpha-synuclein (K(D) = 2.5 x 10(-8) M). When co-incubated with monomeric alpha-synuclein, the scFv decreased not only the rate of aggregation of alpha-synuclein, but also inhibited the formation of oligomeric and protofibrillar structures. The scFv binds the carboxyl terminal region of alpha-synuclein, suggesting that perturbation of this region can influence folding and aggregation of alpha-synuclein in vitro along with the previously identified hydrophobic core region of alpha-synuclein (residues 61-95, particularly residues 71-82). Since the scFv has been isolated from an antibody library based on human gene sequences, such scFv's can have potential therapeutic value in controlling aggregation of alpha-synuclein in vivo when expressed intracellularly as intrabodies in dopaminergic neurons.     

Pesticides directly accelerate the rate of alpha-synuclein fibril formation: a possible factor in Parkinson's disease.

Parkinson's disease involves intracellular deposits of alpha-synuclein in the form of Lewy bodies and Lewy neurites. The etiology of the disease is unknown, however, several epidemiological studies have implicated environmental factors, especially pesticides. Here we show that several pesticides, including rotenone, dieldrin and paraquat, induce a conformational change in alpha-synuclein and significantly accelerate the rate of formation of alpha-synuclein fibrils in vitro. We propose that the relatively hydrophobic pesticides preferentially bind to a partially folded intermediate conformation of alpha-synuclein, accounting for the observed conformational changes, and leading to association and subsequent fibrillation. These observations suggest one possible underlying molecular basis for Parkinson's disease.