Alpha-synuclein adopts an alpha-helical conformation in the presence of polyunsaturated fatty acids to hinder micelle formation

Alpha-synuclein is a small cytosolic protein involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Recent studies suggested a lipid-related function for this brain-enriched protein. Since the brain carries a high level of docosahexaenoic acid (DHA) and since the extent of alpha-synuclein gene expression increases in response to DHA intake, we have investigated the interaction of alpha-synuclein with this essential omega-3 fatty acid. We show that alpha-synuclein allows DHA to be present in a soluble rather than micellar form. Upon interaction with DHA, the normally unstructured alpha-synuclein rapidly adopts an alpha-helical conformation. Prolonged exposure to DHA, however, gradually converts alpha-synuclein into amyloid-like fibrils. These results identify a potential biological function for alpha-synuclein and define an omega-3-linked pathway leading to alpha-synuclein aggregation.     

Structural Evidence for α-Synuclein Fibrils Using in Situ Atomic Force Microscopy

Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson‘s disease, dementia with Lewy bodies and Lewy body variant of Alzheimer‘s disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentous topography for over 1h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how 13-sheets assemble into α-synuclein fibrils on a nanometer scale.     

Down-regulation of alpha-synuclein expression can rescue dopaminergic cells from cell death in the substantia nigra of Parkinson's disease rat model

Fibrillization and aggregation of alpha-synuclein may play a critical role in neurodegenerative diseases like Parkinson's diseases. Adeno-associated virus (AAV) vector delivery of an alpha-synuclein ribozyme was tested for its silencing effect on degenerating nigrostriatal neurons in the MPP(+) model of Parkinson's disease. We designed alpha-synuclein ribozyme against human alpha-synuclein gene expression and constructed alpha-synuclein ribozymes-carrying rAAV vector (designated rAAV-SynRz). Co-transfection of rAAV-SynRz and rAAV-alpha-synuclein into HEK293 cells resulted in down-regulation of alpha-synuclein protein expression in vitro. Then, rAAV-SynRz was injected into the substantia nigra (SN) of MPP(+)-treated rats. Cell counts of TH-positive neurons in the SN revealed that rAAV-SynRz significantly protected TH-positive cells against apoptotic death, compared with those of rAAV-EGFP or no rAAV injected rats. Our results indicate that the use of rAAV-SynRz allowed the survival of higher number of TH-positive neurons in SN in the MPP(+) model. Down-regulation of alpha-synuclein expression could be potentially a suitable target for gene therapy of Parkinson's disease.     

Could a loss of α‐synuclein function put dopaminergic neurons at risk?

The alpha-synuclein gene is implicated in Parkinson's disease, the symptoms of which occur after a marked loss of substantia nigra dopamine neurons. While the function of alpha-synuclein is not entirely elucidated, one function appears to be as a normal regulatory protein that can bind to and inhibit tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Soluble alpha-synuclein levels may be diminished in Parkinson's disease substantia nigra dopamine neurons both by reduced expression and by alpha-synuclein aggregation as Lewy bodies and Lewy neurites form. The loss of functional alpha-synuclein may then result in dysregulation of tyrosine hydroxylase, dopamine transport and dopamine storage, resulting in excess cytosolic dopamine. Because dopamine and its metabolites are reactive molecules capable of generating highly reactive quinones and reactive oxygen species, a failure to package dopamine into vesicles could cause irreversible damage to cellular macromolecules and contribute to resultant neurotoxicity. This review focuses on how a loss of normal alpha-synuclein function may contribute to the dopamine-related loss of substantia nigra neurons during Parkinson's disease pathogenesis.     

Parkinson's disease: what have we learned from the genes responsible for familial forms?

Parkinson's disease is characterized by the progressive and selective loss of the dopaminergic neurons in the substantia nigra and the presence of ubiquitinated protein inclusions termed Lewy bodies. In the past six years, four genes involved in rare inherited forms of Parkinson's disease have been identified: mutations in the alpha-synuclein and ubiquitin carboxyterminal hydrolase L1 genes (UCH-L1) cause autosomal dominant forms, whereas mutations in the Parkin and DJ-1 genes are responsible for autosomal recessive forms of the disease. A toxic gain of function related to the ability of alpha-synuclein to assemble into insoluble amyloid fibrils may underlie neuronal cell death in parkinsonism due to alpha-synuclein gene mutations. In contrast, loss of protein function appears to be the cause of the disease in parkinsonism due to mutations in the genes encoding Parkin and UCH-L1, which are key enzymes of the ubiquitin-proteasome pathway. The presence of alpha-synuclein, Parkin and UCH-L1 in Lewy bodies suggests that dysfunction of pathways involved in protein folding and degradation is not only involved in the pathogenesis of familial Parkinson's disease, but could also play a role in the frequent sporadic form of the disease (idiopathic Parkinson's disease).     

alpha-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate-induced phospholipase D activation in human embryonic kidney-293 cells

alpha-Synuclein has been implicated in the pathogenesis of many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Although the function of alpha-synuclein remains largely unknown, recent studies have demonstrated that this protein can interact with phospholipids. To address the role of alpha-synuclein in neurodegenerative disease, we have investigated whether it binds phospholipase D (PLD) and affects PLD activity in human embryonic kidney (HEK)-293 cells overexpressing wild type alpha-synuclein or the mutant forms of alpha-synuclein (A53T, A30P) associated with Parkinson's disease. Tyrosine phosphorylation of alpha-synuclein appears to play a modulatory role in the inhibition of PLD, because mutation of Tyr(125) to Phe slightly increases inhibitory effect of alpha-synuclein on PLD activity. Treatment with pervanadate or phorbol myristate acetate inhibits PLD more in HEK 293 cells overexpressing alpha-synuclein than in control cells. Binding of alpha-synuclein to PLD requires phox and pleckstrin homology domain of PLD and the amphipathic repeat region and non-Abeta component of alpha-synuclein. Although biologically important, co-transfection studies indicate that the interaction of alpha-synuclein with PLD does not influence the tendency of alpha-synuclein to form pathological inclusions. These results suggest that the association of alpha-synuclein with PLD, and modulation of PLD activity, is biologically important, but PLD does not appear to play an essential role in the pathophysiology of alpha-synuclein.     

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.     

The flavonoid baicalein inhibits fibrillation of alpha-synuclein and disaggregates existing fibrils

The aggregation of alpha-synuclein has been implicated as a critical step in the development of Parkinson's disease. Parkinson's disease is a progressive neurodegenerative disorder caused by the loss of dopaminergic neurons from the substantia nigra; currently, no cure exists. Baicalein is a flavonoid with antioxidant properties; upon oxidation, it forms several products including quinones. We show here that low micromolar concentrations of baicalein, and especially its oxidized forms, inhibit the formation of alpha-synuclein fibrils. In addition, existing fibrils of alpha-synuclein are disaggregated by baicalein. The product of the inhibition reaction is predominantly a soluble oligomer of alpha-synuclein, in which the protein molecules have been covalently modified by baicalein quinone to form a Schiff base with a lysine side chain in alpha-synuclein. The binding of baicalein was abolished by conversion of the Tyr residues into Phe, demonstrating that Tyr is involved in the interaction of alpha-synuclein with baicalein. In disaggregation baicalein causes fragmentation throughout the length of the fibril. These observations suggest that baicalein and similar compounds may have potential as therapeutic leads in combating Parkinson's disease and that diets rich in flavonoids may be effective in preventing the disorder.     

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.     

Mechanisms of Suppression of {alpha}-Synuclein Neurotoxicity by Geldanamycin in Drosophila

Parkinson's disease is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of the protein alpha-synuclein into aggregates called Lewy bodies and Lewy neurites. Parkinson's disease can be modeled in Drosophila where directed expression of alpha-synuclein induces compromise of dopaminergic neurons and the formation of Lewy body-like aggregates. The molecular chaperone Hsp70 protects cells from the deleterious effects of alpha-synuclein, indicating a potential therapeutic approach to enhance neuron survival in Parkinson's disease. We have now investigated the molecular mechanisms by which the drug geldanamycin protects neurons against alpha-synuclein toxicity. Our studies show that geldanamycin sensitizes the stress response within normal physiological parameters to enhance chaperone activation, offering protection against alpha-synuclein neurotoxicity. Further, geldanamycin uncouples neuronal toxicity from Lewy body and Lewy neurite formation such that dopaminergic neurons are protected from the effects of alpha-synuclein expression despite the continued presence of (and even increase in) inclusion pathology. These studies indicate that compounds that modulate the stress response are a promising approach to treat Parkinson's disease.     

Prolyl oligopeptidase stimulates the aggregation of alpha-synuclein

Despite its thorough enzymological and biochemical characterization the exact function of prolyl oligopeptidase (PO, E.C. 3.4.21.26) remains unclear. The positive effect of PO inhibitors on learning and memory in animal models for amnesia, enzyme activity measurements in patient samples and (neuro)peptide degradation studies link the enzyme with neurodegenerative disorders. The brain protein alpha-synuclein currently attracts much attention because of its proposed role in the pathology of Parkinson's disease. A fundamental question concerns how the essentially disordered protein is transformed into the highly organized fibrils that are found in Lewy bodies, the hallmarks of Parkinson's disease. Using gel electrophoresis and MALDI TOF/TOF mass spectrometry we investigated the possibility of alpha-synuclein as a PO substrate. We found that in vitro incubation of the protein with PO did not result in truncation of full-length alpha-synuclein. Surprisingly, however, we found an acceleration of the aggregation process of alpha-synuclein using turbidity measurements that was reversed by specific inhibitors of PO enzymatic activity. If PO displays this activity also in vivo, PO inhibitors might have an effect on neurodegenerative disorders through a decrease in the aggregation of alpha-synuclein.     

Effect of familial Parkinson's disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human alpha-synuclein

Parkinson's disease involves the loss of dopaminergic neurons in the substantia nigra, leading to movement disorders. The pathological hallmark of Parkinson's disease is the presence of Lewy bodies and Lewy neurites, which are intracellular inclusions consisting primarily of alpha-synuclein. Although essentially all cases of sporadic and early-onset Parkinson's disease are of unknown etiology, two point mutations (A53T and A30P) in the alpha-synuclein gene have been identified in familial early-onset Parkinson's disease. Previous reports have shown that mutant alpha-synuclein may form fibrils more rapidly than wild-type protein. To determine the underlying molecular basis for the enhanced fibrillation of the mutants, the structural properties, responses to changes in the environment, and propensity to aggregate of wild-type, A30P, and A53T alpha-synucleins were systematically investigated. A variety of biophysical methods, including far-UV circular dichroism, FTIR, small-angle X-ray scattering, and light scattering, were employed. Neither the natively unfolded nor the partially folded intermediate conformations are affected by the familial Parkinson's disease point mutations. However, both mutants underwent self-association more readily than the wild type (i.e., at much lower protein concentration and more rapidly). We attribute this effect to the increased propensity of their partially folded intermediates to aggregate, rather than to any changes in the monomeric natively unfolded species. This increased propensity of these mutants to aggregate, relative to wild-type alpha-synuclein, would account for the correlation of these mutations with Parkinson's disease.     

Alpha-synuclein protects cerebellar granule neurons against 6-hydroxydopamine-induced death

The physiological role of alpha-synuclein, a protein found enriched in intraneuronal deposits characterizing Parkinson's disease, is debated. While its aggregation is usually considered linked to neuropathology, its normal function may be related to fundamental processes of synaptic transmission and plasticity. By using antisense oligonucleotide strategy, we report in this study that alpha-synuclein silencing in cultured cerebellar granule cells results in widespread death of these neurons, thus demonstrating an essential pro-survival role of the protein towards primary neurons. To study alpha-synuclein expression and processing in a Parkinson's disease model of neurotoxicity, we exposed differentiated cultures of cerebellar granule neurons to toxic concentrations of 6-hydroxydopamine (6-OHDA). This resulted in neuronal death accompanied by a decrease of the monomeric form of alpha-synuclein, which was due to both decreased synthesis of the protein and its increased mono-ubiquitination accompanied by nuclear translocation. The essential neuroprotective role of alpha-synuclein was confirmed by the fact that subchronic valproate treatment, which increases alpha-synuclein expression and prevents its nuclear translocation in cerebellar granule cells exposed to 6-OHDA, significantly protected these neurons from 6-OHDA insult. In agreement with the pro-survival role of alpha-synuclein in this model, subtoxic concentrations of alpha-synuclein antisense oligonucleotides, aggravated 6-OHDA toxicity towards granule neurons. Our results demonstrate that normal alpha-synuclein expression is essential for the viability of primary neurons and that its pro-survival role is abolished in 6-OHDA neurotoxic challenge. These results are relevant to more precisely define the role of alpha-synuclein in neuronal cells and to better understand its putative involvement in neurodegeneration.     

Residual structure, backbone dynamics, and interactions within the synuclein family

The human synuclein protein family includes alpha-synuclein, which has been linked to both familial and sporadic Parkinson's disease, and the highly homologous beta and gamma-synuclein. Mutations in alpha-synuclein cause autosomal dominant early onset Parkinson's, and the protein is found deposited in a fibrillar form in hereditary and idiopathic forms of the disease. No genetic link between beta and gamma-synuclein, and any neurodegenerative disease has been established, and it is generally considered that these proteins are not highly pathogenic. In addition, beta and gamma-synuclein are reported to aggregate less readily than alpha-synuclein in vitro. Indeed, beta-synuclein has been reported to protect against alpha-synuclein aggregation in vitro, as well as alpha-synuclein-mediated toxicity in vivo. Earlier, we compared the structural properties of the highly helical states adopted by all three synucleins in association with detergent micelles in an attempt to delineate the basis for functional differences between the three proteins. Here, we report a comparison of the structural and dynamic properties of the free states of all three proteins in order to shed light on differences that may help to explain their different propensities to aggregate, which in turn may underlie their differing contributions to the etiology of Parkinson's disease. We find that gamma-synuclein closely resembles alpha-synuclein in its free-state residual secondary structure, consistent with the more similar propensities of the two proteins to aggregate in vitro. beta-Synuclein, however, differs significantly from alpha-synuclein, exhibiting a lower predisposition towards helical structure in the second half of its lipid-binding domain, and a higher preference for extended structures in its C-terminal tail. Both beta and gamma-synuclein show less extensive transient long-range structure than that observed in alpha-synuclein. These results raise questions regarding the role of secondary structure propensities and transient long-range contacts in directing synuclein aggregation reactions.     

A protein-chameleon: conformational plasticity of alpha-synuclein,a disordered protein involved in neurodegenerative disorders

Under the physiological conditions in vitro, alpha-synuclein, a conservative presynaptic protein, the aggregation and fibrillation of which is assumed to be involved into the pathogenesis of Parkinson's disease and several other neurodegenerative disorders, known as synucleinopathies, is characterized by the lack of rigid well-defined structure; i.e., it belongs to the class of intrinsically unstructured proteins. Intriguingly, alpha-synuclein is characterized by a remarkable conformational plasticity, adopting a series of different conformations depending on the environment. For example, this protein may either stay substantially unfolded, or adopt an amyloidogenic partially folded conformation, or fold into alpha-helical or beta-structural species, both monomeric and oligomeric. Furthermore, it might form several morphologically different types of aggregates, including oligomers (spheres or doughnuts), amorphous aggregates, and or amyloid-like fibrils. The peculiarities of this astonishing conformational behavior are analyzed to shed light on structural plasticity of this protein-chameleon.     

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.     

Molecular mechanisms of alpha-synuclein neurodegeneration

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

Properties of NACP/alpha-synuclein and its role in Alzheimer's disease

The precursor of the non-amyloid beta/A4 protein (non-Abeta) component of Alzheimer's disease amyloid (NACP)/alpha-synuclein is the human homologue of alpha-synuclein, a member of a protein family which includes alpha-, beta- and gamma-synuclein. This protein is thought to be involved in neuronal plasticity because of its unique expression, mainly in the telencephalon during maturation. Consequently, disarrangement of NACP/alpha-synuclein might disrupt synaptic activity, resulting in memory disturbance. Previous studies have shown that damage to synaptic terminals is closely associated with global cognitive impairment and is an early event in the pathogenesis of Alzheimer's disease. Although the relationship between synaptic damage and amyloidogenesis is not clear, some proteins at the synaptic site have been implicated in both neuronal alteration and amyloid formation. Indeed, abnormal accumulation of both NACP/alpha-synuclein and Abeta precursor protein occurs at synapses of Alzheimer's patients. Other evidence suggests that NACP/alpha-synuclein is a component of the Lewy bodies found in patients with Parkinson's disease or dementia with Lewy bodies, and that a point mutation in this protein may be the cause of familial Parkinson's disease. Consequently, abnormal transport, metabolism or function of NACP/alpha-synuclein appears to impair synaptic function, which induces, at least in part, neuronal degeneration in several neurodegenerative diseases.     

Organization of the human synphilin-1 gene, a candidate for Parkinson's disease

We have recently identified a protein we called synphilin-1, which interacts in vivo with alpha-synuclein. Mutations in alpha-synuclein cause familial Parkinson's disease (PD). Alpha-synuclein protein is present in the pathologic lesions of familial and sporadic PD, and diffuse Lewy body disease, indicating an important pathogenic role for alpha-synuclein. Here we describe the structure of the human synphilin-1 gene (SNCAIP). The open reading frame of this gene is contained within ten exons. We have designed primers to amplify each SNCAIP exon, so these primers can now be used to screen for mutations or polymorphisms in patients with Parkinson's disease or related diseases. We found a highly polymorphic GT repeat within intron 5 of SNCAIP, suitable for linkage analysis of families with PD. We have mapped SNCAIP locus to Chromosome (Chr) 5q23.1-23.3 near markers WI-4673 and AFMB352XH5. In addition, using immunohistochemistry in human postmortem brain tissue, we found that synphilin-1 protein is present in neuropil, similar to alpha-synuclein protein. Because of its association with alpha-synuclein, synphilin-1 may be a candidate for involvement in Parkinson's disease or other related disorders. Received: 21 September 1999 / Accepted: 16 March 2000     

Interactions between metals and alpha-synuclein--function or artefact?

alpha-synuclein is one of a family of proteins whose function remains unknown. This protein has become linked to a number of neurodegenerative disease although its potential causative role in these diseases remains mysterious. In diseases such as Parkinson's disease and Lewy body dementias, alpha-synuclein becomes deposited in aggregates termed Lewy bodies. Also, some inherited forms of Parkinson's diseases are linked to mutations in the gene for alpha-synuclein. Studies have mostly focussed on what causes the aggregation of the protein but, like many amyloidogenic proteins associated with a neurodegenerative disorder, this protein has now been suggested to bind copper. This finding is currently controversial. This review examines the evidence that alpha-synuclein is a copper binding protein and discusses whether this has any significance in determining the function of the protein or whether copper binding is at all necessary for aggregation.