Is there a cause-and-effect relationship between alpha-synuclein fibrillization and Parkinson's disease?

The first gene to be linked to Parkinson's disease encodes the neuronal protein alpha-synuclein. Recent mouse and Drosophila models of Parkinson's disease support a central role for the process of alpha-synuclein fibrillization in pathogenesis. However, some evidence indicates that the fibril itself may not be the pathogenic species. Our own biophysical studies suggest that a structured fibrillization intermediate or an alternatively assembled oligomer may be responsible for neuronal death. This speculation can now be experimentally tested in the animal models. Such experiments will have implications for the development of new therapies for Parkinson's disease and related neurodegenerative diseases.     

The significance of tau and alpha-synuclein inclusions in neurodegenerative diseases

Intracellular filamentous inclusions made of either the microtubule-associated protein tau or the protein alpha-synuclein define the majority of cases of neurodegenerative disease. Mutations in the tau gene in familial forms of frontotemporal dementia and in the alpha-synuclein gene in familial cases of Parkinson's disease have provided causal links between the dysfunction of these proteins and neurodegeneration. Over the past year, several novel tau gene mutations have been identified and more has been learned about possible mechanisms by which tau gene mutations lead to frontotemporal dementia. Experimental animal models have provided a link between tau filament formation and nerve cell degeneration. Along similar lines, animal models have been produced that result in the formation of alpha-synuclein filaments and the degeneration of dopaminergic nerve cells. Building on previous work, synthetic alpha-synuclein filaments have been shown to exhibit the characteristics of amyloid.     

Alpha-synuclein and Parkinson's disease: a proteomic view

In this review, we report how proteomic methodologies have been used to investigate cellular and animal models of Parkinson's disease (PD), with a special focus on alpha-synuclein. PD is a complex, multifactorial neurodegenerative disease affecting approximately 2% of the population over 65 years of age, pathologically characterized by alpha-synuclein intraneuronal inclusions. Etiopathogenetic mechanisms of PD are not fully understood, although a number of factors contributing to the selective degeneration of substantia nigra neurons have been identified. Therefore, cellular and animal models of the disease have been developed to investigate single factors contributing to disease pathogenesis; for example, alpha-synuclein aggregation and altered dopamine homeostasis. Proteomic studies on cellular and animal models have not only confirmed existing theories on PD pathogenesis (mitochondrial impairment, oxidative stress, failure of the ubiquitine-proteasome system), but also allowed the discovery of new important common features of presymptomatic (or premotor) stages of PD, such as dysregulation of cytoskeletal proteins that could be involved at the origin of the disorder.     

Cathepsin D is the main lysosomal enzyme involved in the degradation of alpha-synuclein and generation of its carboxy-terminally truncated species

Alpha-synuclein is likely to play a key role in the development of Parkinson's disease as well as other synucleinopathies. In animal models, overexpression of full-length or carboxy-terminally truncated alpha-synuclein has been shown to produce pathology. Although the proteosome and lysosome have been proposed to play a role in the degradation of alpha-synuclein, the enzyme(s) involved in alpha-synuclein clearance and generation of its carboxy-terminally truncated species have not been identified. In this study, the role of cathepsin D and calpain I in these processes was analyzed. In vitro experiments, using either recombinant or endogenous alpha-synuclein as substrates and purified cathepsin D or lysosomes, demonstrated that cathepsin D degraded alpha-synuclein very efficiently, and that limited proteolysis resulted in the generation of carboxy-terminally truncated species. Purified calpain I also cleaved alpha-synuclein, but carboxy-terminally truncated species were not the main cleavage products, and calpain I activity present in cellular lysates was not able to degrade the protein. Knockdown of cathepsin D in cells overexpressing wild-type alpha-synuclein increased total alpha-synuclein levels by 28% and lysosomal alpha-synuclein by 2-fold. In in vitro experiments, pepstatin A completely blocked the degradation of alpha-synuclein in purified lysosomes. Furthermore, lysosomes isolated from cathepsin D knockdown cells showed a marked reduction in alpha-synuclein degrading activity, indicating that cathepsin D is the main lysosomal enzyme involved in alpha-synuclein degradation. Our findings suggest that upregulation of cathepsin D could be an additional therapeutic strategy to lessen alpha-synuclein burden in synucleinopathies.     

Caught in the act: alpha-synuclein is the culprit in Parkinson's disease

Previous reports on Parkinson's disease indicate that genetic mutations in alpha-synuclein result in the aberrant accumulation of this protein, causing toxic gain of function leading to the development of Parkinson's. A recent report on the Iowan kindred, an extended pedigree with an autosomal dominant form of this disease, provides new mechanistic insight into Parkinson's disease by showing that an elevation in wild-type alpha-synuclein protein is sufficient to develop the early-onset form of the disorder. This review discusses how insights gained from these studies of alpha-synuclein may direct future research into Parkinson's disease.     

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.     

Overexpression of alpha-synuclein in rat substantia nigra results in loss of dopaminergic neurons, phosphorylation of alpha-synuclein and activation of caspase-9: resemblance to pathogenetic changes in Parkinson's disease

To elucidate the role of alpha-synuclein in the pathogenesis of Parkinson's disease, both human alpha-synuclein transgenic mice and targeted overexpression of human alpha-synuclein in rat substantia nigra using viral vector-based methods have been studied, however, little is known about the pathogenetic changes of dopaminergic neuron loss. Therefore, it is necessary to address whether the pathogenetic changes in brains with Parkinson's disease are recapitulated in these models. Here, we used the recombinant adeno-associated viral (rAAV) vector system for human alpha-synuclein gene transfer to rat substantia nigra and observed approximately 50% loss of dopaminergic neurons at 13 weeks after infection, which was comparably slower than the progression of neurodegeneration reported in other studies. In the slower progression of neurodegeneration, we identified several important features in common with the pathogenesis of Parkinson's disease, such as phosphorylation of alpha-synuclein at Ser129 and activation of caspase-9. Both findings were also evident in cortical tissues overexpressing alpha-synuclein via rAAV. Our results indicate that overexpression of alpha-synuclein via rAAV apparently recapitulates several important features of brains with Parkinson's disease and dementia with Lewy bodies, and thus alpha-synucleinopathy described here is likely to be an ideal model for the study of the pathogenesis of Parkinson's disease and dementia with Lewy bodies.     

Modelling synucleinopathies in genetically modified animals--successes and failures

The synuclein family and particularly alpha-synuclein takes a central part in etiology and pathogenesis of Parkinson's disease--one of the most common human neurodegenerative diseases. The pathological changes in certain other neurodegenerative diseases are also linked to changes in metabolism and function of alpha-synuclein, hence comprising a new group of diseases--synucleinopathies. The molecular and cellular mechanisms that are involved in the development of neurodegeneration in synucleinopathies are still largely unknown. As a result, the therapeutic approaches to the treatment of synucleinopathies are inadequately tampered. The development of models of neurodegenerative process in laboratory animals plays a crucial role in the study of these molecular mechanisms. Recently a special emphasis was placed on transgenic animal models with modified expression of genes, which mutations are associated with inherited forms of human neurodegenerative diseases. Current review is devoted to the analysis of different models of synucleinopathies as a result of genetic modifications of alpha-synuclein expression.     

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.     

Recent advances on alpha-synuclein cell biology: functions and dysfunctions

Alpha-synuclein is a recently discovered protein that was first identified as the major non amyloid component of senile plaques, the cerebral lesion likely responsible for Alzheimer's disease. The role of alpha-synuclein in another brain disease namely Parkinson's disease, has been more deeply documented. It appears that alpha-synuclein fills up the intracytoplasmic inclusions called Lewy bodies that likely contribute to the etiology of Parkinson's disease. Furthermore, rare familial forms of Parkinson's disease have been shown to be linked to autosomal dominant mutations of alpha-synucleins. Is alpha-synuclein a bridge between Alzheimer's and Parkinson's diseases? Could it be seen as a common denominator for these two neurodegenerative diseases? These issues could be better addressed by further delineating the physiological function of alpha-synuclein and, as a corollary, the dysfunction taking place along with the diseases. Here, I will review the recent advances concerning the physiology of alpha-synuclein and will particularly focus on the post-traductional events leading to drastic biophysical transformations. I will describe recent works suggesting that these modifications directly modulate the normal function of alpha-synuclein, likely accounting for the dysfunction associated with Parkinson's disease and perhaps contributing to Alzheimer's pathology.     

Synphilin-1 is developmentally localized to synaptic terminals, and its association with synaptic vesicles is modulated by alpha-synuclein

Alpha-synuclein is the major component of Lewy bodies in patients with Parkinson's disease, and mutations in the alpha-synuclein gene are responsible for some familial forms of the disease. alpha-Synuclein is enriched in the presynapse, but its synaptic targets are unknown. Synphilin-1 associates in vivo with alpha-synuclein promoting the formation of intracellular inclusions. Additionally synphilin-1 has been found to be an intrinsic component of Lewy bodies in patients with Parkinson's disease. To understand the role of synphilin-1 in Parkinson's disease, we sought to define its localization and function in the brain. We now report that, like alpha-synuclein, synphilin-1 was enriched in neurons. In young rats, synphilin-1 was prominent in neuronal cell bodies but gradually migrated to neuropil during development. Immunoelectron microscopy of adult rat cerebral cortex demonstrated that synphilin-1 was highly enriched in presynaptic nerve terminals. Synphilin-1 co-immunoprecipitated with synaptic vesicles, indicating a strong association with these structures. In vitro binding experiments demonstrated that the N terminus of synphilin-1 robustly associated with synaptic vesicles and that this association was resistant to high salt washing but was abolished by inclusion of alpha-synuclein in the incubation medium. Our data indicated that synphilin-1 is a synaptic partner of alpha-synuclein, and it may mediate synaptic roles attributed to alpha-synuclein.     

Selective detection, quantification, and subcellular location of alpha-synuclein aggregates with a protein aggregate filtration assay

Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy are caused by alpha-synuclein aggregates. At present, there is no good biochemical method defining alpha-synuclein aggregates formed in vivo versus oligomers as a means to investigate alpha-synuclein aggregation and its mechanisms of neurodegeneration. A simple method, therefore, for the selective and sensitive detection of alpha-synuclein aggregates suited for screening purposes would be useful. Since in contrast to prions a proper detection of alpha-synuclein aggregates by Western blot analysis is difficult, we developed a protein aggregate filtration (PAF) assay. It takes advantage of the inherent insolubility of aggregated alpha-synuclein using microfiltration to separate it from soluble isoforms. For the first time, this assay even makes quantitative comparisons possible. We describe how the PAF assay can be applied to human brain tissue and animal and cell culture models, as well as used as a screening method for the subcellular location of alpha-synuclein aggregates. Since it detects the pathological isoform instead of surrogate markers, the PAF assay may have also potential in diagnosis of PD and DLB.     

Climbing the scaffolds of Parkinson's disease pathogenesis

Several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases, are characterized neuropathologically by accumulation of misfolded proteins such as alpha-synuclein that disrupts scaffold molecules in the caveolae. A new study by Ihara et al. in this issue of Neuron shows that a novel scaffold protein, Sept4, may be an important player in modulating the pathological alterations of alpha-synuclein in models of Parkinson's disease, suggesting that gene therapies targeting scaffold proteins might be effective in the treatment of neurodegenerative disorders.     

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.     

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.     

Filamentous nerve cell inclusions in neurodegenerative diseases: tauopathies and alpha-synucleinopathies.

Alzheimer's disease and Parkinson's disease are the most common neurodegenerative diseases. They are characterized by the degeneration of selected populations of nerve cells that develop filamentous inclusions before degeneration. The neuronal inclusions of Alzheimer's disease are made of the microtubule-associated protein tau, in a hyperphosphorylated state. Recent work has shown that the filamentous inclusions of Parkinson's disease are made of the protein alpha-synuclein and that rare, familial forms of Parkinson's disease are caused by missense mutations in the alpha-synuclein gene. Besides Parkinson's disease, the filamentous inclusions of two additional neurodegenerative diseases, namely dementia with Lewy bodies and multiple system atrophy, have also been found to be made of alpha-synuclein. Abundant filamentous tau inclusions are not limited to Alzheimer's disease. They are the defining neuropathological characteristic of frontotemporal dementias such as Pick's disease, and of progressive supranuclear palsy and corticobasal degeneration. The recent discovery of mutations in the tau gene in familial forms of frontotemporal dementia has provided a direct link between tau dysfunction and dementing disease. The new work has established that tauopathies and alpha-synucleinopathies account for most late-onset neurodegenerative diseases in man. The formation of intracellular filamentous inclusions might be the gain of toxic function that leads to the demise of affected brain cells.     

Animal models of Parkinson's disease

Animal models are important tools in experimental medical science to better understand pathogenesis of human diseases. Once developed, these models can be exploited to test therapeutic approaches for treating functional disturbances observed in the disease of interest. On the basis of experimental and clinical findings, Parkinson's disease (PD) was the first neurological disease to be modeled and, subsequently, to be treated by neurotransmitter replacement therapy. Agents that selectively disrupt or destroy catecholaminergic systems, such as reserpine, methamphetamine, 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine have been used to develop PD models. Recently, it has been found that agricultural chemicals, such as rotenone and paraquat, when administered systemically, can reproduce specific features of PD in rodents, apparently via oxidative damage. Transgenic animals that over-express alpha-synuclein are used to study the role of this protein in dopaminergic degeneration. This review critically discusses animal models of PD and compares them with characteristics of the human disease.     

Challenges and complexities of alpha-synuclein toxicity: new postulates in unfolding the mystery associated with Parkinson's disease

The discovery of two missense mutations in alpha-synuclein gene and the identification of the alpha-synuclein as the major component of Lewy bodies and Lewy neurites have imparted a new direction in understanding Parkinson's disease. Now that alpha-synuclein has been implicated in several neurodegenerative disorders makes it increasingly clear that aggregation of alpha-synuclein is a hallmark feature in neurodegeneration. Although little has been learned about its normal function, alpha-synuclein appears to be associated with membrane phospholipids and may therefore participate in a number of cell signaling pathways. Here, we review the localization, structure, and function of alpha-synuclein and provide a new hypothesis on, (a) the disruption in the membrane binding ability of synuclein which may be the major culprit leading to the alpha-synuclein aggregation and (b) the complexity associated with nuclear localization of alpha-synuclein and its possible binding property to DNA. Further, we postulated the three possible mechanisms of synuclein induced neuronal degeneration in Parkinson's disease.     

Controlling the mass action of alpha-synuclein in Parkinson's disease

Parkinson's disease (PD) is an age-related neurodegenerative disease with unknown etiology. Growing evidence from genetic, pathologic, animal modeling, and biochemical studies strongly support the theory that abnormal aggregation of alpha-synuclein plays a critical role in the pathogenesis of PD. Protein aggregation is an alternative folding process that competes with the native folding pathway. Whether or not a protein is subject to the aggregation process is determined by the concentration of the protein as well as thermodynamic properties inherent to each polypeptide. An increase in cellular concentration of alpha-synuclein has been associated with the disease in both familial and sporadic forms of PD. Thus, maintenance of the intraneuronal steady state levels of alpha-synuclein below the critical concentration is a key challenge neuronal cells are facing. Expression of the alpha-synuclein gene is under the control of environmental factors and aging, the two best-established risk factors for PD. Studies also suggest that the degradation of this protein is mediated by proteasomal and autophagic pathways, which are two mechanisms that are related to the pathogenesis of PD. Recently, vesicle-mediated exocytosis has been suggested as a novel mechanism for disposal of neuronal alpha-synuclein. Relocalization of the protein to specific compartments may be another method for increasing its local concentration. Regulation of the neuronal steady state levels of alpha-synuclein has significant implications in the development of PD, and understanding the mechanism may disclose potential therapeutic targets for PD and other related diseases.     

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.