alpha-synuclein degradation by autophagic pathways: a potential key to Parkinson's disease pathogenesis

The neuronal protein alpha-synuclein is thought to be central in the pathogenesis of Parkinson's disease (PD). Excessive wild type alpha-synuclein levels can lead to PD in select familial cases and alpha-synuclein protein accumulation occurs in sporadic PD. Therefore, elucidation of the mechanisms that control alpha-synuclein levels is critical for PD pathogenesis and potential therapeutics. The subject of alpha-synuclein degradation has been controversial. Previous work shows that, in an assay with isolated liver lysosomes, purified wild type alpha-synuclein is degraded by the process of chaperone-mediated autophagy (CMA). Whether this actually occurs in a cellular context has been unclear. In our most recent work, we find that wild type alpha-synuclein, but not the closely related protein beta-synuclein, is indeed degraded by CMA in neuronal cells, including primary postnatal ventral midbrain neurons. Macroautophagy, but not the proteasome, also contributes to alpha-synuclein degradation. Therefore, two separate lysosomal pathways, CMA and macroautophagy, degrade wild type alpha-synuclein in neuronal cells. It is hypothesized that impairment of either of these two pathways, or of more general lysosomal function, may be an initiating factor in alpha-synuclein accumulation and sporadic PD pathogenesis.     

Alpha-synuclein degradation by autophagic pathways: A potential key to Parkinson’s Disease pathogenesis

The neuronal protein alpha-synuclein is thought to be central in the pathogenesis of Parkinson’s Disease (PD). Excessive wild type alpha-synuclein levels can lead to PD in select familial cases and alpha-synuclein protein accumulation occurs in sporadic PD. Therefore, elucidation of the mechanisms that control alpha-synuclein levels is critical for PD pathogenesis and potential therapeutics. The subject of alpha-synuclein degradation has been controversial. Previous work show that, in an assay with isolated liver lysosomes, purified wild type alpha-synuclein is degraded by the process of Chaperone Mediated Autophagy (CMA). Whether this actually occurs in a cellular context has been unclear. In our most recent work, we find that wild type alpha-synuclein, but not the closely related protein beta-synuclein, is indeed degraded by CMA in neuronal cells, including primary postnatal ventral midbrain neurons. Macroautophagy, but not the proteasome, also contributes to alpha-synuclein degradation. Therefore, two separate lysosomal pathways, CMA and macroautophagy, degrade wild type alpha-synuclein in neuronal cells. It is hypothesized that impairment of either of these two pathways, or of more general lysosomal function, may be an initiating factor in alpha-synuclein accumulation and sporadic PD pathogenesis.

Addendum to: Vogiatzi T, Xilouri M, Vekrellis K, Stefanis L. Wild type α-synuclein is degraded by chaperone mediated autophagy and macroautophagy in neuronal cells. J Biol Chem 2008; In press.     

DNA induces folding in alpha-synuclein: understanding the mechanism using chaperone property of osmolytes

Alpha-synuclein conformational modulation leading to fibrillation has been centrally implicated in Parkinson's disease. Previously, we have shown that alpha-synuclein has DNA binding property. In the present study, we have characterized the effect of DNA binding on the conformation and fibrillation kinetics of alpha-synuclein. It was observed that single-stranded circular DNA induce alpha-helix conformation in alpha-synuclein while plasmid supercoiled DNA has dual effect inducing a partially folded conformation and alpha-helix under different experimental conditions. Interestingly, alpha-synuclein showed a specificity for GC* nucleotide sequence in its binding ability to DNA. The aggregation kinetics data showed that DNA which induced partially folded conformation in alpha-synuclein promoted the fibrillation while DNA which induced alpha-helix delayed the fibrillation, indicating that the partially folded intermediate conformation is critical in the aggregation process. Further, the mechanism of DNA-induced folding/aggregation of alpha-synuclein was studied using effect of osmolytes on alpha-synuclein as a model system. Among the five osmolytes used, Glycerol, trimethylamine-N-oxide, Betaine, and Taurine induced partially folded conformation and in turn enhanced the aggregation of alpha-synuclein. The ability of DNA and osmolytes in inducing conformational transition in alpha-synuclein, indicates that two factors are critical in modulating alpha-synuclein folding: (i) electrostatic interaction as in the case of DNA, and (ii) hydrophobic interactions as in the case of osmolytes. The property of DNA inducing alpha-helical conformation in alpha-synuclein and inhibiting the fibrillation may be of significance in engineering DNA-chip based therapeutic approaches to PD and other amyloid disorders.     

Degradation of alpha-synuclein by proteasome

Mutations in alpha-synuclein are known to be associated with Parkinson's disease (PD). The coexistence of this neuronal protein with ubiquitin and proteasome subunits in Lewy bodies in sporadic disease suggests that alterations of alpha-synuclein catabolism may contribute to the pathogenesis of PD. The degradation pathway of alpha-synuclein has not been identified nor has the kinetics of this process been described. We investigated the degradation kinetics of both wild-type and A53T mutant 6XHis-tagged alpha-synuclein in transiently transfected SH-SY5Y cells. Degradation of both isoforms followed first-order kinetics over 24 h as monitored by the pulse-chase method. However, the t((1)/(2)) of mutant alpha-synuclein was 50% longer than that of the wild-type protein (p < 0.01). The degradation of both recombinant proteins and endogenous alpha-synuclein in these cells was blocked by the selective proteasome inhibitor beta-lactone (40 microM), indicating that both wild-type and A53T mutant alpha-synuclein are degraded by the ubiquitin-proteasome pathway. The slower degradation of mutant alpha-synuclein provides a kinetic basis for its intracellular accumulation, thus favoring its aggregation.     

Oxidative dimer formation is the critical rate-limiting step for Parkinson's disease alpha-synuclein fibrillogenesis

Intraneuronal deposition of alpha-synuclein as fibrils and oxidative stress are both implicated in the pathogenesis of Parkinson's disease. We found that the critical rate-limiting step in nucleation of alpha-synuclein fibrils under physiological conditions is the oxidative formation and accumulation of a dimeric, dityrosine cross-linked prenucleus. Dimer formation is accelerated for the pathogenic A30P and A53T mutant alpha-synucleins, because of their greater propensity to self-interact, which is reflected in the smaller values of the osmotic second virial coefficient compared to that of wild-type synuclein. Our finding that oxidation is an essential step in alpha-synuclein aggregation supports a mechanism of Parkinson's disease pathogenesis in which the separately studied pathogenic factors of oxidative stress and alpha-synuclein aggregation converge at the critical step of alpha-synuclein dimer formation.     

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.     

alpha-synuclein and Parkinson's disease: the first roadblock

alpha-synuclein gene mutations are major underlying genetic defects known in familial juvenile onset Parkinson's disease (PD), and alpha-synuclein is a major constituent of Lewy Bodies, the pathological hallmark of PD. The normal cellular function of alpha-synuclein has been elusive, and its exact etiological mechanism in causing dopaminergic neuronal death in PD is also not clearly understood. Very recent reports now indicate that mutant or simply over-expressed alpha- synuclein could cause damage by interfering with particular steps of neuronal membrane traffic. alpha-synuclein selectively blocks endoplamic reticulum-to-Golgi transport, thus causing ER stress. A screen in a yeast revealed that alpha- synuclein toxicity could be suppressed by over-expression of the small GTPase Ypt1/Rab1, and that over-expression of the latter rescues neuron loss in invertebrate and mammalian models of alpha-synuclein-induced neurodegeneration. alpha-synuclein may also serve a chaperone function for the proper folding of synaptic SNAREs that are important for neurotransmitter release. We discuss these recent results and the emerging pathophysiological interaction of alpha-synuclein with components of neuronal membrane traffic.     

alpha-synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD. alpha-Synuclein fibrils similar to the Lewy body filaments can be formed in vitro, and we have shown recently that both PD-linked mutations accelerate their formation. This study addresses the mechanism of alpha-synuclein aggregation: we show that (i) it is a nucleation-dependent process that can be seeded by aggregated alpha-synuclein functioning as nuclei, (ii) this fibril growth follows first-order kinetics with respect to alpha-synuclein concentration, and (iii) mutant alpha-synuclein can seed the aggregation of wild type alpha-synuclein, which leads us to predict that the Lewy bodies of familial PD patients with alpha-synuclein mutations will contain both, the mutant and the wild type protein. Finally (iv), we show that wild type and mutant forms of alpha-synuclein do not differ in their critical concentrations. These results suggest that differences in aggregation kinetics of alpha-synucleins cannot be explained by differences in solubility but are due to different nucleation rates. Consequently, alpha-synuclein nucleation may be the rate-limiting step for the formation of Lewy body alpha-synuclein fibrils in Parkinson's disease.     

Aggregation of alpha-synuclein induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

Alpha-synuclein is a major component of the abnormal protein aggregation in Lewy bodies of Parkinson's disease (PD) and senile plaques of Alzheimer's disease (AD). Previous studies have shown that the aggregation of alpha-synuclein was induced by copper (II) and H(2)O(2) system. Since copper ions could be released from oxidatively damaged Cu,Zn-superoxide dismutase (SOD), we investigated the role of Cu,Zn-SOD in the aggregation of alpha-synuclein. When alpha-synuclein was incubated with both Cu,Zn-SOD and H(2)O(2), alpha-synuclein was induced to be aggregated. This process was inhibited by radical scavengers and spin trapping agents such as 5,5'-dimethyl 1-pyrolline N-oxide and tert-butyl-alpha-phenylnitrone. Copper chelators, diethyldithiocarbamate and penicillamine, also inhibited the Cu,Zn-SOD/H(2)O(2) system-induced alpha-synuclein aggregation. These results suggest that the aggregation of alpha-synuclein is mediated by the Cu,Zn-SOD/H(2)O(2) system via the generation of hydroxyl radical by the free radical-generating function of the enzyme. The Cu,Zn-SOD/H(2)O(2)-induced alpha-synuclein aggregates displayed strong thioflavin-S reactivity, reminiscent of amyloid. These results suggest that the Cu,Zn-SOD/H(2)O(2) system might be related to abnormal aggregation of alpha-synuclein, which may be involved in the pathogenesis of PD and related disorders.     

Gene dosage and pathogenesis of Parkinson's disease

Four recent papers related specifically to the familial form of Parkinson's disease reinforce the idea that endogenous levels of alpha-synuclein can strongly influence disease phenotype. Two recent publications of alpha-synuclein-duplication mutations show that the severity of familial Parkinsonian phenotype is dependent upon SNCA gene dosage and corresponding protein levels. Familial point mutations in SNCA were found to impair the efficient lysosomal degradation of alpha-synuclein, potentially resulting in elevated levels of alpha-synuclein. Conversely, the complete knockout of SNCA has little effect on transgenic mice. It is now clear that the regulation of alpha-synuclein levels has potential significance in the pathogenesis and treatment of sporadic PD.     

Alpha-synuclein and Protein Degradation Systems: a Reciprocal Relationship

An increasing wealth of data indicates a close relationship between the presynaptic protein alpha-synuclein and Parkinson’s disease (PD) pathogenesis. Alpha-synuclein protein levels are considered as a major determinant of its neurotoxic potential, whereas secreted extracellular alpha-synuclein has emerged as an additional important factor in this regard. However, the manner of alpha-synuclein degradation in neurons remains contentious. Both the ubiquitin–proteasome system (UPS) and the autophagy–lysosome pathway (ALP)—mainly macroautophagy and chaperone-mediated autophagy—have been suggested to contribute to alpha-synuclein turnover. Additionally, other proteases such as calpains, neurosin, and metalloproteinases have been also proposed to have a role in intracellular and extracellular alpha-synuclein processing. Both UPS and ALP activity decline with aging and such decline may play a pivotal role in many neurodegenerative conditions. Alterations in these major proteolytic pathways may result in alpha-synuclein accumulation due to impaired clearance. Conversely, increased alpha-synuclein protein burden promotes the generation of aberrant species that may impair further UPS or ALP function, generating thus a bidirectional positive feedback loop leading to neuronal death. In the current review, we summarize the recent findings related to alpha-synuclein degradation, as well as to alpha-synuclein-mediated aberrant effects on protein degradation systems. Identifying the factors that regulate alpha-synuclein association to cellular proteolytic pathways may represent potential targets for therapeutic interventions in PD and related synucleinopathies.     

Impairment of redox state and dopamine level induced by alpha-synuclein aggregation and the prevention effect of hsp70

One hypothesis for the etiology of Parkinson's disease (PD) is that the formation of proteinaceous inclusion, which is mainly composed of alpha-synuclein, may contribute to the selective loss of dopaminergic neurons. To further explore the role of alpha-synuclein in neurodegeneration of PD, we examined the possible effects of aggregated alpha-synuclein on the intracellular redox state, dopamine level, and cell death of SK-N-SH cells. Our present studies show that alpha-synuclein aggregation gives rise to both elevated intracellular oxidative state and dopamine level in SK-N-SH cells. Moreover, alpha-synuclein aggregation results in a higher ratio of apoptosis population (55.8%+/-SEM) in cells overexpressing alpha-synuclein aggregation, compared to their normal control groups (8.0%+/-SEM). In contrast, coexpression of hsp70 with alpha-synuclein suppresses the oxidative state shift, restores the normal dopamine levels and blocks neuron cell loss. Therefore, our data provided one possible mechanism by which the alpha-synuclein aggregation may lead to the neurodegeneration in PD via regulating the level of cytoplasmic dopamine and then disturbing the intracellular redox homeostasis. On the other hand, hsp70 can mitigate the degenerative effect conferred by alpha-synuclein, acting as a protective factor in treatment of PD.     

Heat shock proteins reduce alpha-synuclein aggregation induced by MPP+ in SK-N-SH cells

Alpha-synuclein has been implicated in the pathogenesis of Parkinson's disease (PD). Heat shock proteins (HSPs) can reduce protein misfolding and accelerate the degradation of misfolded proteins. 1-methyl-4-phenylpyridinium ion (MPP+) is the compound responsible for the PD-like neurodegeneration caused by MPTP. In this study, we found that MPP+ could increase the expression of alpha-synuclein mRNA but could not elevate proteasome activity sufficiently, leading to alpha-synuclein protein accumulation followed by aggregation. Both HSPs and HDJ-1, a homologue of human Hsp40, can inhibit MPP+-induced alpha-synuclein mRNA expression, promote ubiquitination and elevate proteasome activity. These findings suggest that HSPs may inhibit the MPP+-induced alpha-synuclein expression, accelerate alpha-synuclein degradation, thereby reducing the amount of alpha-synuclein protein and accordingly preventing its aggregation.     

alpha-Synuclein implicated in Parkinson's disease catalyses the formation of hydrogen peroxide in vitro

Some rare inherited forms of Parkinson's disease (PD) are due to mutations in the gene encoding a 140-amino acid presynaptic protein called alpha-synuclein. In PD, and some other related disorders such as dementia with Lewy bodies, alpha-synuclein accumulates in the brain in the form of fibrillar aggregates, which are found inside the neuronal cytoplasmic inclusions known as Lewy bodies. By means of an electron spin resonance (ESR) spin trapping method, we show here that solutions of full-length alpha-synuclein, and a synthetic peptide fragment of alpha-synuclein corresponding to residues 61-95 (the so-called non-Abeta component or NAC), both liberate hydroxyl radicals upon incubation in vitro followed by the addition of Fe(II). We did not observe this property for the related beta- and gamma-synucleins, which are not found in Lewy bodies, and are not linked genetically to any neurodegenerative disorder. There is abundant evidence for the involvement of free radicals and oxidative stress in the pathogenesis of nigral damage in PD. Our new data suggest that the fundamental molecular mechanism underlying this pathological process could be the production of hydrogen peroxide by alpha-synuclein.     

The ceruloplasmin and hydrogen peroxide system induces alpha-synuclein aggregation in vitro

Alpha-synuclein is a key component of Lewy bodies in the brain of patients with Parkinson's disease (PD) and recent studies suggest that oxidative stress reactions might contribute to abnormal aggregation of this molecule. Since hydrogen peroxide-mediated ceruloplasmin (CP) modification can induce the formation of free radicals and release of copper ions, we investigated the role of CP in the aggregation of alpha-synuclein. When alpha-synuclein was incubated with both CP and H(2)O(2), alpha-synuclein concomitantly was induced to be aggregated. Thioflavin-S staining of alpha-synuclein aggregates showed that they displayed characteristic fibrillar structures. Hydroxyl radical scavengers and spin-trapping agent such as 5,5'-dimethyl 1-pyrolline N-oxide and tert-butyl-alpha-phenylnitrone significantly inhibited the aggregation of alpha-synuclein. Copper chelator, penicillamine also inhibited the CP/H(2)O(2) system-induced alpha-synuclein aggregation. This indicates that the aggregation of alpha-synuclein can be mediated by the CP/H(2)O(2) system via the generation of hydroxyl radical. The CP/H(2)O(2) system-induced alpha-synuclein aggregation resulted in the generation of protein carbonyl derivatives. Antioxidant molecules, carnosine, homocarnosine and anserine significantly inhibited the CP/H(2)O(2) system-induced aggregation of alpha-synuclein. These results suggest that the CP/H(2)O(2) system may be related to abnormal aggregation of alpha-synuclein which may be involved in the pathogenesis of PD and related disorders.     

Both familial Parkinson's disease mutations accelerate alpha-synuclein aggregation

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant alpha-synuclein form insoluble fibrillar aggregates with antiparallel beta-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of alpha-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.     

Protein aggregation in Huntington's and Parkinson's disease: implications for therapy

The accumulation of highly insoluble intracellular protein aggregates in neuronal inclusions is a hallmark of Huntington's disease (HD) and Parkinson's disease (PD) as well as several other late-onset neurodegenerative disorders. The aggregates formed in vitro and in vivo generally have a fibrillar morphology, consist of individual beta-strands and are resistant to proteolytic degradation. Although the causal relationship between aggregate formation and disease remains to be proven, the gradual deposition of mutant protein in neurons is consistent with the late-onset and progressive nature of symptoms. Recently, circumstantial evidence from mouse and Drosophila model systems suggests that abnormal protein folding and aggregation play a key role in the pathogenesis of both HD and PD. Therefore, a detailed understanding of the molecular mechanisms of protein aggregation and its effects on neuronal cell death could open new opportunities for therapy.     

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.     

NMR of alpha-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation

The aggregation of alpha-synuclein is characteristic of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. The 140-aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of alpha-synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with alpha-synuclein by NMR and assigned the binding site to C-terminal residues 109-140. Dissociation constants were derived from chemical shift perturbations. Greater polyamine charge (+2 --> +5) correlated with increased affinity and enhancement of fibrillation, for which we propose a simple kinetic mechanism involving a dimeric nucleation center. According to the analysis, polyamines increase the extent of nucleation by approximately 10(4) and the rate of monomer addition approximately 40-fold. Significant secondary structure is not induced in monomeric alpha-synuclein by polyamines at 15 degrees C. Instead, NMR reveals changes in a region (aa 22-93) far removed from the polyamine binding site and presumed to adopt the beta-sheet conformation characteristic of fibrillar alpha-synuclein. We conclude that the C-terminal domain acts as a regulator of alpha-synuclein aggregation.